Patent Application
20180265911
Despite significant advances in modern medicine and drug discovery, there are still tremendous unmet medical needs for combating human diseases.
Nucleic acid junctions are ubiquitous in biological systems. Small molecule control of these structures would allow for regulation of a myriad of nucleic acid dependent biological processes. In addition, these probes will provide fundamental insight into biological systems. Small molecule nucleic acid junction binders could open new avenues for the discovery and development of therapeutics to address unmet medical needs.
However, targeting DNA and RNA structure for therapeutic use has been hampered by an incomplete understanding of the requirements needed for small molecule recognition in a structure and sequence specific manner. To date, the most popular strategies for targeting nucleic acid structures over the last few decades are to make use of known binding modes, utilizing fragments of known intercalating and groove binding ligands or structural modification of natural products such as the aminoglycoside antibiotics. Despite this progress, there are few classes of molecules with the ability to target DNA and RNA motifs in a structure and sequence specific manner.
Significant unmet medical needs necessitate the development of new strategies for combating infectious diseases and neurodegenerative diseases. Alternative strategies to combat life-threatening illness from infectious agents such as drug resistant bacteria and viruses are desperately needed as resistance builds and our current therapies begin to fail. Neurodegenerative disease will become a significant burden with our aging population. Small molecule nucleic acid regulation could have a significant impact in both of these diverse areas and provide new tools for elucidating the fundamental biology. Viral, bacterial, and parasitic diseases contribute to global health and economic problems of significant magnitude.
Nucleic acid and Nucleic acid-protein interactions represent examples of non-traditional drug targets with significant potential in nearly all areas of human medicine. This is primarily due to the central role of nucleic acids in a diverse array of biological processes. For example, RNA is involved in a multitude of vital biological processes ranging from information transfer (mRNA) and gene regulation (siRNA's and microRNA's) to catalysis (ribozymes and riboswitches). The siRNA pathway and the diverse world of non-coding RNA's have regulatory functions ranging from cellular differentiation and chromosomal organization to the regulation of gene expression. The ability to target RNA-dependent processes in bacterial and viral pathogens in addition to pathogenic RNAs implicated in neurological diseases and cancer represent important challenges. Currently, we lack the ability to design molecules to target important DNA and RNA structural motifs beyond B-form DNA with high affinity and specificity. A clear set of rules and guidelines for targeting specific, non-B-form DNA, nucleic acid structural motifs is non-existent and the ability to design small molecules from secondary structure prediction is in its infancy. Given the vital role of nucleic acids in biological processes, a guide to targeting specific structural elements could have far reaching impacts in areas ranging from fundamental biology to new strategies for combating human disease.
Nucleic acid junctions are ubiquitous structural elements present in prokaryotes and eukaryotes. Three-way junctions (3WJ or 3HSm junctions) are formed at the interface of three double helical nucleic acids, forming a Y-shape junction, with a hydrophobic cavity in the center. DNA 3WJs are present in important biological processes, including replication and recombination. They are also present in trinucleotide repeat expansions associated with neurodegenerative diseases and occur in viral genomes. RNA 3WJs are found in a number of important RNA targets including the IRES of the hepatitis C virus (HVC), the hammered ribozyme and in bacterial temperature sensors such as the mRNA of sigma32 (σ32) in E. Coli. Advances towards targeting nucleic acids in a structure-specific manner remain a challenge. The ability to selectively target these junctions would allow for the precise control of cellular processes at the nucleic acid level.
The development of nucleic acid binding small molecules is a major challenge with great potential. To date, there are just a handful of commonly recognized nucleic acid binding modes: minor groove binding, major groove binding, intercalation, and phosphate backbone recognition in addition to several hybrid binding manifolds that rely on simultaneous intercalation and groove binding. The threading intercalators are a prime example along with several natural products that utilize multiple simultaneous binding events to increase their overall affinity and sometimes specificity. The Py-Im polyamides are perhaps one of the most successful platforms for nucleic acid recognition although they are primarily limited to targeting dsDNA structure and have not been shown to be effective at targeting RNA structure. Polyamides are selective for dsDNA over dsRNA, primarily due to an absence of a deep narrow minor groove in RNA structure. Selective and sequence specific targeting of unique DNA structures beyond the double helix has not been demonstrated. Utilizing fragment-based approaches to target RNA structure is an area beginning to show promise. These approaches often build upon common nucleic acid binding scaffolds such as the aminoglycosides, Hoechst, polyamides, known intercalators, or polyamides in addition to polyvalent peptide, peptoid and polymeric scaffolds often bearing multiple cationic functional groups. Minor groove binding ligands have also been reported to bind nucleic acid junctions; however, there is a lack of specificity over binding double helical nucleic acid structures. Intercalators have been broadly utilized to target both DNA and RNA structure however this approach also suffers from a lack of specificity.
There is a minimal amount of precedent for targeting nucleic acid three-way junctions; however, to date all prior studies utilize molecular scaffolds that are already known to bind non-junction nucleic acid structures leading to little selectivity for their intended target. Nucleic acid junctions have been targeted with poly-intercalators with the major limitation again being non-specific intercalation events leading to promiscuity. Cationic peptides have been extensively utilized to target nucleic acid structure and a particularly interesting example by Segall demonstrated the use in modulating holiday junctions (four-way junctions) for the inhibition of junction processing enzymes. It should be noted that the approach of Segall is fundamentally different from the approach proposed in this invention. In addition, the holiday junctions previously targeted by Segall unique structures in their own right are distinct from the three-way junctions targeted in the present invention. Hannon, Coll, and co-workers have demonstrated that metal helicates bind to many nucleic acid structures including quadruplexes and other helical motifs. Different from the metal helicates in the literature, the triptycenes are a novel class of small molecule for targeting nucleic acid junctions.
In one aspect, the present disclosure provides a method of screening for triptycene derivative (TCD) compounds that stabilize a target nucleic acid three way junction (TWJ) structures comprising:
In one aspect, the present disclosure provides a method of screening for triptycene derivative (TCD) compounds that stabilize a target nucleic acid three way junction (TWJ) structures comprising:
In one aspect, the present disclosure provides a method of screening for triptycene derivative (TCD) compounds that stabilize nucleic acid three way junction (TWJ) structures comprising:
In one embodiment of the methods provided herein, the nucleic acid substrate is contacted with a plurality of TCDs.
In one embodiment of the methods provided herein, the TCDs have the structure:
wherein each of S1 to S14 is independently and optionally selected from the group consisting of a hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, halogen, CN, CF3, acyl, an amino acid analog, a peptide (including peptide analogs), a nucleotide (including nucleotide analogs) and an oligonucleotide (including oligonucleotide analogs), and wherein at least one S group is a non-hydrogen group.
In one embodiment of the methods provided herein, at least one of the S groups is an amino acid.
In one embodiment of the methods provided herein, at least one of the S groups is an amino acid analog.
In one embodiment of the methods provided herein, at least one of the S groups is a peptide.
In one embodiment of the methods provided herein, at least one of the S groups is a peptide analog.
In one embodiment of the methods provided herein, at least one of the S groups is a nucleotide.
In one embodiment of the methods provided herein, at least one of the S groups is a nucleotide analog.
In one embodiment of the methods provided herein, at least one of the S groups is an oligonucleotide.
In one embodiment of the methods provided herein, at least one of the S groups is an oligonucleotide analog.
In one embodiment of the methods provided herein, the TCD is covalently attached to a solid support.
In one embodiment of the methods provided herein, a plurality of different TCDs are attached at different sites to said solid support in an array pattern.
In one aspect, the present disclosure provides a method of screening for a cytotoxic TCD comprising contacting said TCD with a cell and determining the viability of said cell.
In one embodiment of the methods provided herein, the cell is a mammalian cell.
In one embodiment of the methods provided herein, the TCD is contacted with a healthy cell or a cancerous cell.
In one embodiment of the methods provided herein, the cell is a bacterial cell.
In one aspect, the present disclosure provides a method of screening for a TCD that inhibits viral replication comprising contacting a cell hosting a virus and determining the viability of the virus.
In one aspect, the present disclosure provides a composition comprising a solid support comprising an array of different TCDs.
In one embodiment of the composition provided herein, each TCD has the structure:
wherein each of S1 to S14 is independently and optionally selected from the group consisting of a hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, halogen, CN, CF3, acyl, an amino acid analog, a peptide (including peptide analogs), a nucleotide (including nucleotide analogs) and an oligonucleotide (including oligonucleotide analogs), and wherein at least one S group is used to covalently attached said TCD to said array.
In one embodiment of the composition provided herein, at least one of the S groups is attached via an amido group.
In one embodiment of the composition provided herein, at least one of the S groups is an amino acid.
In one embodiment of the composition provided herein, at least one of the S groups is an amino acid analog.
In one embodiment of the composition provided herein, at least one of the S groups is a peptide.
In one embodiment of the composition provided herein, at least one of the S groups is a peptide analog.
In one embodiment of the composition provided herein, at least one of the S groups is a nucleotide.
In one embodiment of the composition provided herein, at least one of the S groups is a nucleotide analog.
In one embodiment of the composition provided herein, at least one of the S groups is an oligonucleotide.
In one embodiment of the composition provided herein, at least one of the S groups is an oligonucleotide analog.
While the invention will be described in conjunction with the following figures in order to explain certain principles of the invention and their practical applications. It will be understood that the present description is not intended to limit the invention(s) to those Figures or examplary embodiments. On the contrary, the invention(s) is/are intended to cover not only the exemplary embodiments as represented by the figures, but also various alternatives, modifications, equivalents and other embodiments, which are included within the spirit and scope of the invention as defined by the appended claims.
This application expressly incorporates by reference in the entirety Barros et al., Angew Chem Int Ed Engl., 2014(53):13746, published on Sep. 24, 2014; Barros et al., Angew. Chem. Int. Ed., 2016(55):8258, published on May 30, 2016; Barros et al., Chemical Science, 2015(6):4752, published on Jun. 10, 2015; Yoon et al., Organic Letters, 2016(18):1096, published on Feb. 17, 2016; and Barros et al., Organic Letters, 2016(18):2423, published on May 12, 2016.
The present invention is directed to the recognition that triptycene, shown in
As outlined above, TWJs play important roles in biological processes. TWJs are found as transient intermediates during replication, recombination and DNA damage repair. Junctions are also present in several viral genomes, such as HIV-1, HCV and adeno-associated virus in addition to playing key roles in viral assembly. TWJs also occur in trinucleotide repeat expansions found in unstable genomic DNA associated with neurodegenerative diseases. In addition, TWJs are important in a number of bacterial processes, including the heat shock response (HSF).
Accordingly, the ability to stabilize such junctions, e.g. to “lock” a structure into a TWJ, can allow for the inhibition of certain biological processes that result in the treatment or amelioration of disease, including cancer and pathogen infections. For example, locking specific TWJs in place prevents the induction of the heat shock response in bacteria, thus leading to a new class of antibiotics. Similarly, the triptycene derivatives (TCDs) of the invention can be used to halt DNA replication, similar to the metal helicates such as cisplatin, and thus used as cytotoxic (including chemotherapeutic) agents. Furthermore, trinucleotide repeat nucleic acid sequences are associated with a large number (>30) of inherited human muscular and neurological diseases. The trinucleotide repeat tract length is dynamic and often correlates with disease severity, where short stable tracts are commonplace in the non-affected population. Longer unstable triplet repeat tracts are more prone to expansion as opposed to contraction, in addition to being predisposed to generational transmission. Trincleotide repeat repair outcomes are also affected by structural features present in slipped sequences, where the structure may determine which proteins are recruited for repair. Stabilization of a particular structure could lead to increased repair of these slipped-out junction. Addition of ligands that bind to these junctions can affect repair outcomes as well as recruitment of proteins.
It is important to note that the three strands of nucleic acid that make up a TWJ can come from one strand that folds into a junction, two strands that assemble to form a junction, or three separate strands that assemble to form a junction, all of which have important biological ramifications, and depend on whether the junctions form naturally or not. That is, in some cases, the TWJ occurs naturally, such as with the rho temperature system, and TCDs are added to disrupt the junction or lock it into place as needed. Alternatively, the TWJ can be induced to form using one or more exogeneous nucleic acid strands in combination with a TCD.
For example the oligonucleotides comprising a junction could come from multiple natural sources and unnatural sources. An example is using a triptycene to form a junction between one oligonucleotide sequence from a human source, one from a viral source, and one from a unnatural source. This is a heterotrimeric junction that binds the TCD. It should be noted that a junction of this kind may not form in the absence of the TCD but forms in the presence of the TCD or is stabilized to a greater extent.
In this way, TCDs are used for direct therapeutic benefit, augmentation of oligonucleotide therapeutics, augmentation of endogenous oligonucleotides, induction of cryptic junctions, allosteric modulation of junctions, use in oligonucleotide diagnostics, use in oligonucleotide sensors, in PCR applications, or in any other capacity where formation, modulation, induction, or perturbation of a junction exerts a desired effect. For example, a micro RNA that anneals to an endogenous oligonucleotide sequence to form a junction, the introduction of a triptycene simply binds this complex and potentiates the effect in come way. In this embodiment, the triptycene is not causing the direct effect but rather augmenting or enhancing the effect that is already there.
Thus, TCDs can be used to “lock” an existing, naturally occuring or endogeneous structure in a sequence specific manner.
Alternatively, TCDs can be used in conjunction with other introduced (or endogeneous) nucleic acids to both form and lock a structure in a sequence specific manner. That is, exogeneous TWJs can be introduced using the administration of nucleic acid strands that will participate in a TWJ in the presence of a TCD either in an intermolecular or intramolecular configuration. As will be appreciated by those in the art, the junction may not pre-exist prior to interaction with the TCD. In some cases, the TCD induces formation of a junction that was not present prior to interaction with a triptycene or other small molecule.
Furthermore, the inhibitor displacement assay outlined herein shows that the equilibrium between one strand can be shifted to a new structure comprised of an intramolecular junction with one strand. This is not limited to this case and can be any structure of any number of initial strands that upon interaction with a triptycene small molecule converts into a new junction structure comprised of any number of strands. The new structure can contain oligonucleotide strands from the initial structure in combination with new strands from both natural and synthetic sources to form a junction structure in complex with a shape selective binding molecule such as triptycene.
As alluded to herein, one or more of the strands forming the junction could be DNA, RNA, nucleic acid analogs or hybrids of both DNA and RNA from natural or synthetic sources. Additionally, the junctions could be formed by alternative synthetic mimicking oligonucleotide structures such as PNA, LNA, or other oligomeric nucleic acid mimicking and targeting technologies. Triptycenes could be used to target hybrid junctions created or formed from mixed strands in an intermolecular or intramolecular sense containing DNA, RNA, PNA, LNA, or any other oligonucleotide recognizing technology.
For example, if one strand forms a junction, the terminal loops can be any size, an example might be annealing regions separated by many base pairs that fold to form a junction. The synthetic oligonucleotides could be of therapeutic relevance such as siRNA or medicinal aptamers.
Furthermore, the invention provides methods of generating new TCDs, including libraries, both in solution as well as immobilized on solid supports. Bridgehead (S1, S6) or non-bridgehead positions (S2-S5, S7 to S14) can be used as possible attachment sites for attachment to a solid support (
Accordingly, the present invention is directed to compositions and methods relying on TCDs.
The present invention provides TCDs, as compositions and for use in methods. Triptycene, shown in
In some embodiments, the invention provides water soluble TCDs. The term “water-soluble” refers to moieties that have some detectable degree of solubility in water. Methods to detect and/or quantify water solubility are well known in the art. Thus, in general, at least one “R” position of
Substituent “R” groups fall into several categories, including traditional organic compounds such as alkyl groups (including heteroalkyl and substituted alkyl groups), aryl groups (including heteroaryl and substituted aryl groups) as described below, as well as amino acid side chains and analogs, as well as nucleic acids and analogs.
In some embodiments, the R groups are solubility conferring R groups. Suitable R groups include, but are not limited to, hydrogen, alkyl, alcohol, aromatic, amino, amido, nitro, ethers, esters, aldehydes, sulfonyl, silicon moieties, halogens, sulfur containing moieties, phosphorus containing moieties, and ethylene glycols. In the structures depicted herein, R is hydrogen when the position is unsubstituted. It should be noted that some positions may allow two substitution groups, R and R′, in which case the R and R′ groups may be either the same or different.
By “alkyl group” or grammatical equivalents herein is meant a straight or branched chain alkyl group, with straight chain alkyl groups being preferred. If branched, it may be branched at one or more positions, and unless specified, at any position. The alkyl group may range from about 1 to about 30 carbon atoms (C1-C30), with a preferred embodiment utilizing from about 1 to about 20 carbon atoms (C1-C20), with about C1 through about Cl2 to about C15 being preferred, and C1 to C5 being particularly preferred, although in some embodiments the alkyl group may be much larger. Also included within the definition of an alkyl group are cycloalkyl groups such as C5 and C6 rings, and heterocyclic rings with nitrogen, oxygen, sulfur or phosphorus. Alkyl also includes heteroalkyl, with heteroatoms of sulfur, oxygen, nitrogen, and silicone being preferred. Alkyl includes substituted alkyl groups. By “substituted alkyl group” herein is meant an alkyl group further comprising one or more substitution moieties “R”, as defined above. One preferred linkage of an alkyl group to the TC molecule is using amido groups.
By “amino groups” or grammatical equivalents herein is meant —NH2, —NHR and —NR2 groups, with R being as defined herein.
By “nitro group” herein is meant an —NO2 group.
By “sulfur containing moieties” herein is meant compounds containing sulfur atoms, including but not limited to, thia-, thio- and sulfo-compounds, thiols (—SH and —SR), and sulfides (—RSR—). By “phosphorus containing moieties” herein is meant compounds containing phosphorus, including, but not limited to, phosphines and phosphates. By “silicon containing moieties” herein is meant compounds containing silicon.
By “ether” herein is meant an —O—R group. Preferred ethers include alkoxy groups, with —O—(CH2)2CH3 and —O—(CH2)4CH3 being preferred.
By “ester” herein is meant a —COOR group.
By “halogen” herein is meant bromine, iodine, chlorine, or fluorine. Preferred substituted alkyls are partially or fully halogenated alkyls such as CF3, etc.
By “aldehyde” herein is meant —RCHO groups.
By “alcohol” herein is meant —OH groups, and alkyl alcohols —ROH.
By “amido” herein is meant —RCONH— or RCONR— groups.
By “ethylene glycol” or “(poly)ethylene glycol” (PEG) herein is meant a —(O-CH2—CH2)n— (heteroalkyl) group, although each carbon atom of the ethylene group may also be singly or doubly substituted, e.g. —(O—CR2—CR2)n—, with R as described herein. Ethylene glycol derivatives with other heteroatoms in place of oxygen (i.e. —(N—CH2—CH2)n— or —(S—CH2—CH2)n—, or with substitution groups) are also preferred.
Preferred substitution groups include, but are not limited to, methyl, ethyl, propyl, alkoxy groups such as —O—(CH2)2CH3 and —O—(CH2)4CH3 and ethylene glycol and derivatives thereof.
Preferred aromatic groups include, but are not limited to, phenyl, naphthyl, naphthalene, anthracene, phenanthroline, pyrole, pyridine, thiophene, porphyrins, and substituted derivatives of each of these, included fused ring derivatives.
In some embodiments, the “R” alkyl and/or aryl groups (including heteroalkyl and heteroaryl) can be further substituted with additional R groups, such as an amino substituted phenyl group or a hydroxy phenyl group.
In some embodiments, the R groups are proteinaceous in nature, generally including proteins, which includes peptides and amino acid side chains and side chain analogs, including both monomers (e.g. a single amino acid side chain or analog) as well as multimers (e.g. peptides and peptide analogs, including dimers (two amino acids), trimers (three), tetramers, etc.). By “proteins” or grammatical equivalents herein is meant proteins, oligopeptides and peptides, derivatives and analogs, including proteins containing non-naturally occurring amino acids and amino acid analogs, and peptidomimetic structures. The side chains may be in either the (R) or the (S) configuration. In a preferred embodiment, the amino acids are in the (S) or L-configuration. Some suitable amino acid side chains and analogs are shown in
In some embodiments, the R groups are nucleic acids and/or nucleic acid analogs. (For clarity, it should be noted that there are nucleic acid R groups, sometimes referred to herein as “R group nucleic acid radicals”, in addition to the nucleic acid substrates and inhibitors outlined herein, and this definition applies to both). The nucleic acids can be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence. The nucleic acid can be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xathanine hypoxathanine, isocytosine, isoguanine, etc. A preferred embodiment utilizes isocytosine and isoguanine in nucleic acids designed to be complementary to other probes, for example when the nucleic acids are part of the substrate or inhibitors as discussed herein, as this reduces non-specific hybridization, as is generally described in U.S. Pat. No. 5,681,702. As used herein, the term “nucleoside” includes nucleotides as well as nucleoside and nucleotide analogs, and modified nucleosides such as amino modified nucleosides. In addition, “nucleoside” includes non-naturally occurring analog structures. Thus for example the individual units of a peptide nucleic acid, each containing a base, are referred to herein as a nucleoside.
As for the amino acid R groups, nucleic acid analogs include both monomers (e.g. a single nucleotide or nucleoside, or analog) as well as multimers (e.g. oligonucleotides and analogs). “Oligonucleotide” or grammatical equivalents herein means at least two nucleotides covalently linked together. A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437 (1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al., J. Am. Chem. Soc. 111:2321 (1989)), O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), and peptide nucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature 380:207 (1996), all of which are incorporated by reference). Other analog nucleic acids include those with bicyclic structures including locked nucleic acids, (Koshkin et al., J. Am. Chem. Soc. 120:13252-3 (1998)); positive backbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3, ASC Symposium Series 580, “Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, “Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp 169-176). Several nucleic acid analogs are described in Rawls, C & E News Jun. 2, 1997 page 35. All of these references are hereby expressly incorporated by reference.
In some embodiments, peptide nucleic acids (PNA) find use, which includes peptide nucleic acid analogs. These backbones are substantially non-ionic under neutral conditions, in contrast to the highly charged phosphodiester backbone of naturally occurring nucleic acids. This results in several advantages. First, the PNA backbone exhibits improved hybridization kinetics. PNAs have larger changes in the melting temperature (Tm) for mismatched versus perfectly matched basepairs. DNA and RNA typically exhibit a 2-4° C. drop in Tm for an internal mismatch. With the non-ionic PNA backbone, the drop is closer to 7-9° C. Similarly, due to their non-ionic nature, hybridization of the bases attached to these backbones is relatively insensitive to salt concentration. Furthermore, due to their synthetic nature, PNAs are not digested by native enzymes, making them very stable in in vivo applications.
The number and orientation of the R groups (both R1 and R2 in
The inhibitor displacement assay in
In some embodiments, substitutions at the R2 positions are done for immobilization of the TCD onto a solid support. This may be done for several reasons, including, but not limited to, for chemical synthesis of additional derivatives as well as for immobilization of TCDs for screening against a variety of TWJs, particularly for use in identifying TCDs that are sequence specific, e.g. will bind one particular TWJ preferentially over those of different sequences (although as outlined herein, that may not be required in all applications, particularly in chemotherapeutic applications). In some embodiments, the R2 positions are used for additional substituent substitution for the purposes of gaining sequence selectivity and specificity.
In some cases, the TCD may have an inherent fluorescence built in by addition of R groups that fluoresce (although will generally only be done when other R groups in the TCD confer significant solubility, as many fluorophores are also quite hydrophobic and water insoluble). This may be done to measure TWJ binding directly, if the fluorescent R group changes it's fluorescent profile in the hydrophobic pocket of the junction; that is, a change in fluorescence of the TCD when in solution versus bound in the junction can be used to assay binding.
Arrays of TCDs
As discussed herein, the present invention provides solid supports comprising arrays of TCD generally at least a first substrate with a surface comprising a plurality of assay locations. By “array” herein is meant a plurality of TCDs in an array format; the size of the array will depend on the composition and end use of the array. Arrays containing from about 2 different TCDs to many millions can be made, with many embodiments using microtiter plate arrays.
By “substrate” or “solid support” or other grammatical equivalents herein is meant any material that can be modified to contain discrete individual sites appropriate for the attachment, association or synthesis of TCDs and is amenable to at least one detection method. As will be appreciated by those in the art, the number of possible substrates is very large. Possible substrates include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TeflonJ, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, optical fiber bundles, and a variety of other polymers. In general, the substrates allow optical detection and do not themselves appreciably fluorescese.
Generally the substrate is flat (planar), although as will be appreciated by those in the art, other configurations of substrates may be used as well; for example, three dimensional configurations can be used. Preferred substrates include flat planar substrates such as glass, polystyrene and other plastics and acrylics, with microtiter plate formats finding use in many embodiments. In some embodiments, silicon wafer substrates can be used.
The solid support comprises a surface comprising a plurality of assay locations, i.e. the location where the TCD will be placed or synthesized. The assay locations are generally physically separated from each other, for example as assay wells in a microtiter plate, although other configurations (hydrophobicity/hydrophilicity, etc.) can be used to separate the assay locations.
The sites may be a pattern, i.e. a regular design or configuration, or randomly distributed. A preferred embodiment utilizes a regular pattern of sites such that the sites may be addressed in the X-Y coordinate plane. “Pattern” in this sense includes a repeating unit cell.
In some embodiments, the surface of the substrate is modified to contain modified sites, particularly chemically modified sites, that can be used to attach, either covalently or non-covalently, the TCDs of the invention to the discrete sites or locations on the substrate. “Chemically modified sites” in this context includes, but is not limited to, the addition of a pattern of chemical functional groups including amino groups, carboxy groups, oxo groups and thiol groups, that can be used to covalently attach TCDs, which generally also contain corresponding reactive functional groups.
In some embodiments, the TCDs may be synthesized or attached to beads (which can be magnetic, in some cases), and then put down on a second support in an array pattern; for example; the addition of a pattern of adhesive that can be used to bind the microspheres with TCDs (either by prior chemical functionalization for the addition of the adhesive or direct addition of the adhesive); the addition of a pattern of charged groups (similar to the chemical functionalities) for the electrostatic attachment of the microspheres, i.e. when the microspheres comprise charged groups opposite to the sites; the addition of a pattern of chemical functional groups that renders the sites differentially hydrophobic or hydrophilic, such that the addition of similarly hydrophobic or hydrophilic microspheres under suitable experimental conditions will result in association of the microspheres to the sites on the basis of hydroaffinity. For example, the use of hydrophobic sites with hydrophobic beads, in an aqueous system, drives the association of the beads preferentially onto the sites.
In some embodiments, libraries are made of different TCDs for screening. “Library” in this context means a plurality of different TCD compounds, from 2 to millions, depending on the synthetic options.
The TCDs of the invention are used to stabilize TWJs, sometimes also referred to as nucleic acid target substrates, as the structure that is acted upon by the TWJ to stabilize it thermodynamically, thus preventing normal biological processes from occurring.
In general, preferred TWJs are those that are found in such normal biological processes, including disease processes such as pathogen infection and replication as well as cancer. As shown in
All strands forming the junction can be DNA, RNA, or hybrids of both DNA and RNA from natural or synthetic sources. The DNA and RNA strands comprising the junction can be a combination of natural and synthetic oligonucleotides. The synthetic oligonucleotides can be of therapeutic relevance such as siRNA or medicinal aptamers. In some embodiments, the oligonucleotides comprising a junction come from multiple natural sources and unnatural sources, as shown in an example using a triptycene to form a junction between one oligonucleotide sequence from a human source, one from a viral source, and one from an unnatural source. This would be a heterotrimeric junction binding a small molecule such as a triptycene. In some embodiments, the junctions are formed by alternative synthetic mimicking oligonucleotide structures such as peptide nucleic acid (PNA), locked nucleic acid (LNA), or other oligomeric nucleic acid mimicking and targeting technologies.
In some embodiments, a junction may not pre-exist prior to interaction with a triptycene or its derivative or other small molecule. A junction may form or be stabilized to a greater extent in the presense of a small molecule, such as triptycene or or its derivative.
The TWJ substrates of the invention take on a number of formats, and can be a variety of lengths and sequences, and can be naturally occurring nucleic acids (e.g. the sequences are both naturally occurring as well as made of standard nucleotides). In some cases, particularly for screening applications, the nucleic acid TWJs can comprise nucleic acid analogs as needed, as described above.
As shown in
Triptycenes can be used to target hybrid junctions created or formed from mixed strands in an intermolecular or intramolecular sense containing DNA, RNA, PNA, LNA, or any other oligonucleotide recognizing technology. Triptycenes can also be used for direct therapeutic benefit, augmentation of oligonucleotide therapeutics, augmentation of endogenous oligonucleotides, induction of cryptic junctions, allosteric modulation of junctions, use in oligonucleotide diagnostics, use in oligonucleotide sensors, in PCR applications, or in any other capacity where formation, modulation, induction, or perturbation of a junction might exert a desired effect. For example, a micro RNA is annealed to an endogenous oligonucleotide sequence to form a complex and a triptycene or its derivative binds this complex and augment this effect.
The assays of the invention generally include an inhibitor, which, again, with reference to
The invention includes a number of different assay formats, depending on the goal of the assay; three different formats are shown in
In some embodiments, assays are run to find TCDs that preferentially bind to one of RNA and DNA over the other, that is, TCDs that bind preferentially to RNA over DNA or to DNA over RNA.
In general, the assay methods are designed to find and/or determine the sequence specificity of different TCDs and/or find TCDs that specifically bind to a particular TWJ of therapeutic relevance. The assays generally rely on adding one or more TCDs to one or more TWJs and then determining the change in FRET status. That is, as outlined in
As will be appreciated by those in the art, when done in an array format, the extra substrates can be washed away, leaving only the complex of TCD:TWJ at the array site. The identity and structure of the TCD is known as it was placed and/or synthesized on the support. The identity and structure of the TWJ can be done in a number of ways, for example by using an extra label (e.g. a distinct fluorophore, preferably one who's emission and/or excitation spectra is distinguishable from the FRET pairs), or by heating the sample to disassociate the TCD from the TWJ, removing the TWJ, and sequencing it. As will be appreciated by those in the art, the sequencing can be done in any number of ways, including nucleic acid sequencing. Alternatively, the extra label can actually be a unique nucleic acid tag that is part of the TWJ substrate sequence, that can be hybridized to a secondary array for identification purposes.
In some embodiments, the screening techniques are done on solid supports, generally in array formats as discussed herein, using multimode plate readers to rapidly screen libraries of compounds. The initial concept has been tried, the results of which are shown in the Figures. This assay can be run in small volumes, for example volumes as low as 12 microliters, and is amenable to high throughput screening with fluorescence plate readers. As shown in Example 2, good results have been achieved from studies using a sigma32 mRNA with this assay and full-length sigma32 will be done.
In some embodiments, the present invention finds use in screening methods for TCDs that induce cytotoxicity in cells, including mammalian and bacterial cells. Mammalian cells, whether primary cells or cell lines, may be screened with different TCDs for cytotoxic TCDs. In general, any mammalian cells can be used with rodent, monkey and human cells being of particular use in some embodiments. As shown in the examples, TCDs can be cytotoxic even against resistant cancerous cell lines.
In some embodiments, for example similar to the heat shock response experiments outlined in Example 2, bacterial cells can be screened for TCDs that can preferentially bind bacterial TWJs (including RNA TWJs), as these are used in many bacterial strains as regulatory structures. These TCDs can then also be run against mammalian cells (particular of a human host) to determine preferential binding activity.
In some embodiments, the methods and compositions of the invention can be used to screen for TCDs that preferentially bind viral TWJs. In this embodiment, the TCDs are contacted with host cells (generally mammalian) that harbor a viral strain, and the effect of the TCD on the viral viability is measured.
New triptycene core molecules (TCDs) are made with functionality in either the 3, 9, 12 or 2, 10, 11 positions as shown in
New trisubstituted triptycenes are synthesized to probe the influence of triptycene substitution pattern on structure and sequence specificity. Starting with commercially available 1,8 dichloroanthraquinone as reported in the literature, 1,8-bis(methoxycarbonyl)-anthracene has been previously accessed in 4 steps and 38% yield. 2-amino-6-methoxycarbonyl benzoic acid can be synthesized from commercially available 3-nitrophthalic acid via a Fischer esterification, followed by a palladium on carbon hydrogenation to reduce the nitro group. The central step in this scheme is a Diels-Alder reaction between the anthracene derivative and a benzyne equivalent, which is generated in situ by diazotization of anthranilic acid derivatives. In some embodiments, a modified, more efficient synthesis by utilizing a combined Heck coupling/benzyne Diels-Alder strategy is used. The new triptycene building block is further diversified on solid phase with short di- and tripeptides.
In some embodiments, the invention provides one or more sets of versatile orthogonally protected triptycene building blocks as shown in the Examples 4 and 5. These building blocks based on bridgehead-substituted triptycenes are used to immobilize our triptycene core structures on solid substrate spot arrays to create large immobilized libraries for diversification and screening against biologically relevant RNA targets that are either fluorophore or radio labeled.
While the invention will be described in conjunction with the following exemplary embodiments/Examples in order to explain certain principles of the invention and their practical application, thereby enabling those skilled in the art to make and utilize various exemplary embodiments of the present invention. It will be understood that the present description is not intended to limit the invention(s) to those Examples. On the contrary, the invention(s) is/are intended to cover not only the exemplary embodiments, but also various alternatives, modifications, equivalents and other embodiments, which are included within the spirit and scope of the invention as defined by the appended claims.
Figures and references are as published in Barros et al., Angew Chem Int Ed Engl., 2014(53):13746, published on Sep. 24, 2014.
Nucleic acid modulation by small molecules is an essential process across the kingdoms of life. Targeting nucleic acids with small molecules represents a significant challenge at the forefront of chemical biology. Nucleic acid junctions are ubiquitous structural motifs in nature and in designed materials. Herein, we describe a new class of structure specific nucleic acid junction stabilizers based on a triptycene scaffold. Triptycenes provide significant stabilization of DNA and RNA three-way junctions, providing a new scaffold for building nucleic acid junction binders with enhanced recognition properties. Additionally, cytotoxicity and cell uptake data in two human ovarian carcinoma cell lines are reported.
Nucleic acid junctions are ubiquitous structural motifs, occurring in both DNA and RNA. Three-way junctions have been extensively studied by many biophysical techniques and represent important and sometimes transient structures in biological processes, such as replication and recombination while also occurring in triplet repeat expansions, which are associated with a number of neurodegenerative diseases. Nucleic acid junctions are ubiquitous in viral genomes and represent important structural motifs in riboswitches where small molecule modulation holds great potential. Three-way junctions are key building blocks present in many nanostructures, soft materials, multichromophore assemblies, and aptamer-based sensors. In the case of aptamer based sensors, DNA three-way junctions serve as an important structural motif. The ability to modulate aptamers using specific small molecules represents an important challenge for designing nucleic acid sensors, switches and devices.
Pioneering discoveries by Hannon, Coll and co-workers have demonstrated that metal helicates can bind nucleic acid junctions in addition to quadruplexes and helical motifs. Inspiring structural studies have yielded high-resolution details of nucleic acid junctions complexed with proteins and metal helicates, providing a starting point for rational structure-based design efforts. Given the ubiquity of nucleic acid junctions and the potential for many diverse applications, a better understanding of junction molecular recognition is needed in addition to an expanded toolbox of small molecule probes. Despite the vast number of important nucleic acid targets, we still lack the ability to selectively modulate predetermined nucleic acid structures using small molecules with high specificity and affinity beyond a few well-established recognition modes such as groove binding. Small molecule targeting of non-canonical nucleic acid motifs and higher-order structures represent an important challenge at the forefront of chemistry and chemical biology.
Herein, we report a new structure selective triptycene-based scaffold for targeting nucleic acid junctions. UV-Vis, circular dichroism (CD), Gel shift, and fluorescence quenching experiments were used to assess junction recognition properties. We find that triptycene-based junction binders exhibit a significant stabilization of perfectly paired DNA and RNA three-way junctions. Further, we report initial cytotoxicity and cell uptake data compared to cisplatin, in two human ovarian carcinoma cell lines.
Our molecular design began with the recognition that triptycene possessed a 3-fold symmetric architecture with dimensions similar to those of the central helical interface of a perfectly base-paired nucleic acid three-way junction. Despite the occurrence of triptycene in materials applications, there is a paucity of examples where it has successfully been used for biomolecular recognition. From our analysis of the three-way junction binding site dimensions, we envisioned that the trigonal symmetry and non-planar n-surface of the aromatic rings in triptycene could potentially form stacking or buckled base pair interactions with nucleobases at the junction interface, allowing for a shape selective fit. Important to our design criteria was the choice of a non-intercalative scaffold to minimize non-specific nucleic acid binding and the triptycene structure satisfied this requirement. In previous studies, we have demonstrated that the introduction of geometric and macrocyclic constraints in nucleic acid binding small molecules can be used as a strategy to lock out common binding modes such as intercalation and groove binding. Recently, we reported that dimeric azaxanthone (diazaxanthylidene) structures lack the ability to bind B-form DNA due to their preference for an anti-folded conformation, despite the DNA binding ability of well-known monomeric xanthone intercalators. In our previous studies we attributed the lack of B-form DNA binding to the discontinuous pi-system, caused by the overcrowded anti-folded molecular architecture. This hypothesis is consistent with the architectural requirements for classical intercalator scaffolds, requiring more than one ring of continuous planar n-surface area. By a similar structural analysis, we expected the discontinuous n-surface area of triptycene to rule out classical intercalative binding modes. In addition to the shape complementarity and three-dimensional architecture of the triptycene scaffold, we were attracted to its modularity and potential for diversity. The triptycene scaffold provides up to 14 positions for diversification, three rings with four positions each in addition to two bridgehead positions. Triptycene can also be thought of as having three buckled n-faces and two three-fold symmetric edge faces with a bridgehead located at the center of each. These structural attributes of triptycene will become important for future studies as we consider topological differentiation of nucleic acid junction faces for achieving sequence specificity and distinguishing DNA from RNA junctions. Size comparison of the triptycene core relative to previously reported DNA and RNA three-way junction crystal structures confirmed our initial hypothesis regarding triptycene shape complementarity, prompting us to initiate synthetic efforts toward the first rationally designed nucleic acid junction binders based on triptycene (
UV thermal melting experiments were performed to determine the degree of stabilization of Trip 1-3 toward a DNA 3WJ versus dsDNA (
Circular dichroism (CD) was used to further explore the interaction of Trip 1 with DNA 3WJ (
Gel shift experiments were performed on DNA 3WJ2 to further support junction binding by 1 (
A fluorescence quenching experiment was used to verify binding to the three-way junction. The 5′- and 3′-ends of a DNA 3WJ forming oligonucleotide were labeled with a fluorophore (FAM) and a quencher (BHQ-1), respectively (
Initial studies of the cytotoxicity and cell uptake for Trip 1-3 were conducted using human ovarian carcinoma cell lines. Previous studies of metallohelicates show diverse biological activity and cytotoxic effects in cisplatin resistant A2780cis cell lines. This promising result for the metallohelicates led us to explore initial cytotoxicity studies for triptycenes 1-3 in these cell lines. Their activity was investigated in human ovarian carcinoma A2780 cells and the cisplatin-resistant A2780cis cell line using an Alamar blue assay. Significant differences in sensitivity were observed for each of the triptycenes in the two cell lines compared to cisplatin (
In summary, we have rationally designed a new class of non-intercalative triptycene-based nucleic acid junction binders. We have demonstrated that triptycene-based molecules have the ability to recognize both DNA and RNA three-way junctions, providing a new versatile scaffold for targeting higher-order nucleic acid structure. Initial biological studies show promising cytotoxicity in cisplatin resistant human ovarian carcinoma cell lines and positive cellular uptake. Ongoing efforts in our laboratory are directed toward the recognition of both DNA and RNA junctions in addition to the development of new classes of structure specific nucleic acid modulators. Junctions are one of the most ubiquitous structural motifs and many exciting opportunities exist for developing small molecule modulators of higher-order structures such three-way and four-way junctions. Seminal studies on four-way junctions have paved the way for recent important contributions where selective small molecule modulators are being developed. Small molecule targeting of non-canonical nucleic acid motifs and higher-order structures represent an important challenge at the forefront of chemistry and chemical biology with many exciting opportunities.
General Methods:
DNA 3WJ (5′-CGA CAA AAT GCA AAA GCA TTA CTT CAA AAG AAG TTT GTC G-3′), duplex DNA (5′-CCAGTACTGG-3′), DNA 3WJ2 (5′-GGG AGA CAA GGA AAA TCC TTC AAT GAA GTG GGT CGA CA-3′), and DNA hairpin (5′-CAA AAT GCA AAA GCA TTT TG-3′) were purchased from Integrated DNA Technologies (IDT). HPLC-purified DNA 3WJ2 oligo modified with a 5′-FAM and a 3′-BHQ-1 was purchased from IDT. The DNA 3WJ was predicted to have a cooperative single inflection melting curve using NUPACK. Trans-Dichlorobis(triphenylphospine)palladium(II) was purchased from Strem Chemicals, Inc. (Newburyport, Mass., USA). Sodium hydroxide was purchased from Fisher Scientific (Pittsburgh, Pa., USA). (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate) (HATU) was purchased from GenScript (Piscataway, N.J., USA). Benzoic acid was purchased from Acros Organics. All other reagents were purchased from Sigma Aldrich (St. Louis, Mo., USA) and used without further purification. Reactions requiring anhydrous conditions were run under argon with solvents purchased from Fisher dried via an alumina column. Silicycle silica gel (55-65 A pore diameter) was used for silica chromatography. Thin-layer chromatography was done using Sorbent Technologies (Norcross, Ga., USA) silica plates (250 μm thickness). Milli-Q (18 MΩ) water was used for all solutions (Millipore; Billerica, Mass., USA).
1H and 13C NMR were recorded on a Bruker UNI 500 NMR at 500 and 125 MHz, respectively. Matrix-assisted laser desorption ionization (MALDI) mass spectra were obtained on a Bruker Ultraflex III MALDI-TOF-TOF mass spectrometer (Billerica, Mass., USA) using a-cyano-4-hydroxycinnamic acid (CHCA). Low resolution electrospray ionization (ESI) mass spectra (LRMS) were collected on a Waters Acquity Ultra Performance LC (Milford, Mass., USA). High resolution mass spectra were obtained at the Univeristy of Pennsylvania Mass Spectrometry Center on a Micromass AutoSpec electrospray/chemical ionization spectrometer. Ultraviolet absorption spectroscopy and thermal denaturation experiments were performed on a JASCO V-650 spectrophotometer (JASCO Analytical Instruments; Easton, Md., USA) equipped with a JASCO PC-734R multichannel Peltier using quartz cells with 1 cm path lengths. High-performance liquid chromatography was performed on a JASCO HPLC (Easton, Md., USA) equipped with a Phenomenx (Torrance, Calif., USA) column (Luna 5μ C18(2) 100 A; 250×4.60 mm, 5 μm) using aqueous (H2O+0.1% CF3CO2H) and organic (CH3CN) phases. Circular dichroism experiments were performed on an Aviv 410 CD spectrometer (Aviv Biomedical; Lakewood, N.J., USA) using a 0.1 cm path length quartz cuvette. Fluorescence measurements were collected on a Tecan M1000 plate reader (Mannedorf, Switzerland).
HPLC chromatograms were obtained at all wavelengths from 200 to 800 nm (bottom plot). 254 and 214 nm were chosen as virtual channels to show the absorbances at those two specific wavelengths (top plot). The blue line corresponds to 254 nm and the red line corresponds to 214 nm (top plot). The lamps used were D2+W with a slit width of 4 nm. A flow rate of 1.00 mL/min was used over 30 minutes. The method began at 10% acetonitrile and 90% water+0.1% TFA. The gradient was slowly increased to 100% acetonitrile.
Synthesis:
2,7,15-triiodo-9,10-dihydro-9,10-[1,2]benzenoanthracene (5) was synthesized according to previously described method, C. Zhang, C. F. Chen, J. Org. Chem. 2006, 71, 6626-6629, hereby expressly incorporated herein by reference for the methods, figures and legends herein. To a solution of 5 (23.6 mg, 0.037 mmol) in anhydrous DMF (0.5 mL) was added PdCl2(PPh3)2 (1 mg, 0.0014 mmol), Et3N (27.2 mg, 0.27 mmol) and CH3OH (0.2 mL). The solution was stirred under CO (150 psi) in a Parr apparatus at 60° C. for 14 hours. The reaction mixture was concentrated under vacuum and purified by column chromatography on silica gel with 30% dichlromethane/hexanes. The product was isolated as a yellow solid (15.9 mg, 0.037 mmol, 60%). 1H NMR (500 MHz, CDCl3) δ 8.07 (s, 3H), 7.77-7.75 (m, 3H), 7.48-7.46 (d, 3H), 5.62 (s, 1H), 5.59 (s, 1H), 3.87 (s, 9H); 13C NMR (500 MHz, CDCl3) δ 166.77, 148.52, 144.80, 128.01, 127.96, 124.98, 124.12, 54.29, 53.74, 52.26; HRMS m/z calcd for C26H20O6 [M−H] 427.1259, observed [M−H] 427.1201.
To a solution of 6 (9.6 mg, 0.019 mmol) in dioxane (0.2 mL) was added 1 M NaOH (0.2 mL) and heated to 45° C. for 2 hours. Dioxane was removed under vacuum, diluted with water and extracted with CH2Cl2. The aqueous phase was acidified with 1 M HCl to pH 2 and the solution was extracted with EtOAc. The organic phase was dried over Na2SO4, and concentrated under vacuum to leave 7 (7.1 mg, 0.018 mmol, 95%) as a white solid. 1H NMR (500 MHz, MeOD) δ 8.10 (d, 3H), 7.77-7.75 (m, 3H), 7.56-7.55 (d, 3H), 5.82 (s, 1H), 5.80 (s, 1H). 13C NMR (500 MHz, MeOD) δ 169.58, 150.37, 146.61, 129.61, 128.97, 125.85, 125.01, 55.11, 54.57; HRMS m/z calcd for C23H14O6 [M−H] 385.079, observed 385.0725.
To a solution of 7 (7 mg, 0.018 mmol) in DMF (0.2 mL) was added HATU (24 mg, 0.063 mmol) and DIEA (15.5 mg, 0.12 mmol) and was stirred at room temperature for 5 min. Tert-butyl (3-((3-aminopropyl)(methyl)amino)propyl)carbamate (15.5 mg, 0.063 mmol) was added and stirred overnight. The reaction mixture was concentrated under vacuum, then diluted with water and extracted with EtOAc. The organic phase was dried over Na2SO3 and concentrated. Crude product was suspended in 4 M HCl in dioxane and stirred for 2 hours. The mixture was concentrated, redissolved in acidic water (0.1% TFA) and washed with CH2Cl2. Product was purified on a JASCO High-Performance Liquid Chromatography (HPLC) instrument on a C18 column using aqueous (H2O+0.1% TFA) and organic (CH3CN) phases. 1H NMR (500 MHz, D2O) δ 7.87 (s, 3H), 7.60 (d, 6H), 5.92 (s, 1H), 5.89 (s, 1H), 3.45-3.44 (m, 6H), 3.31-3.17 (m, 12H), 3.09-3.06 (t, 6H), 2.89 (s, 9H), 2.16-2.03 (m, 12H); 13C NMR (500 MHz, D2O) δ 170.55, 147.95, 145.03, 131.01, 125.19, 124.34, 122.54, 117.46, 115.14, 112.82, 53.97, 52.86, 52.69, 39.54, 36.59, 36.38, 23.83, 21.87; HRMS m/z calcd for C44H65N9O3 [M+H] 768.521, observed 768.5291.
To a solution of 7 (4.5 mg, 0.012 mmol) in DMF (0.2 mL) was added HATU (14.2 mg, 0.037 mmol) and DIEA (9.0 mg, 0.070 mmol) and was stirred at room temperature for 5 min. tert-butyl (7-aminoheptyl)carbamate (8.5 mg, 0.037 mmol) was added and stirred overnight. The reaction mixture was concentrated under vacuum, then diluted with water and extracted with EtOAc. The organic phase was dried over Na2SO3 and concentrated. Crude product was suspended in 4 M HCl in dioxane and stirred for 2 hours. The mixture was concentrated, redissolved in acidic water (0.1% TFA) and washed with CH2Cl2. Product was purified on a JASCO High-Performance Liquid Chromatography (HPLC) instrument on a C18 column using aqueous (H2O+0.1% TFA) and organic (CH3CN) phases. 1H NMR (500 MHz, D2O) δ 7.80 (s, 3H), 7.61-7.60 (d, 3H) 7.47-7.46 (d, 3H), 5.90 (s, 1H), 5.79 (s, 1H), 3.36-3.33 (t, 6H), 2.92-2.89 (t, 6H), 1.61-1.58 (m, 12H), 1.34 (m, 18H); 13C NMR (500 MHz, D2O) δ 169.20, 147.55, 144.77, 131.30, 124.15, 117.56, 115.24, 39.91, 39.362, 28.62, 27.91, 26.65, 26.00, 25.48; HRMS m/z calcd for C44H62N6O3 [M+H] 723.4848, observed 723.4964.
To a solution of 7 (4.2 mg, 0.011 mmol) in DMF (0.2 mL) was added HATU (13.2 mg, 0.035 mmol) and DIEA (8.7 mg, 0.068 mmol) and was stirred at room temperature for 5 min. N1,N1-dimethylpropane-1,3-diamine (8.7 mg, 0.035 mmol) was added and stirred overnight. The reaction mixture was concentrated under vacuum, then diluted with water and extracted with EtOAc. Organic phase was dried over Na2SO3 and concentrated. Crude product was suspended in 4 M HCl in dioxane and stirred for 2 hours. The mixture was concentrated, redissolved in acidic water (0.1% TFA) and washed with CH2Cl2. Product was purified on a JASCO High-Performance Liquid Chromatography (HPLC) instrument on a C18 column using aqueous (H2O+0.1% TFA) and organic (CH3CN) phases. 1H NMR (500 MHz, D2O) δ 7.77 (s, 3H), 7.48 (d, 3H), 7.43-7.41 (m, 3H), 5.77 (s, 1H), 5.73 (s, 1H), 3.42-3.39 (t, 6H), 3.13-3.10 (m, 6H), 2.82 (s, 18H), 2.00-1.94 (m, 6H); 13C NMR (500 MHz, D2O) δ 170.22, 147.81, 144.91, 130.92, 125.1, 124.26, 122.52, 117.47, 55.26, 42.63, 36.48, 24.16; MALDI m/z calcd for C38H50N6O3 [M+H] 639.3946, observed 639.326.
To a solution of benzoic acid (10 mg, 0.082 mmol) in DMF (0.2 mL) was added HATU (37.4 mg, 0.098 mmol) and DIEA (23.3 mg, 0.18 mmol) and was stirred at room temperature for 5 min. Tert-butyl (3-((3-aminopropyl)(methyl)amino)propyl)carbamate (24.1 mg, 0.098 mmol) was added and stirred overnight. The reaction mixture was concentrated under vacuum, then diluted with water and extracted with EtOAc. Organic phase was dried over Na2SO3 and concentrated. Crude product was suspended in 4 M HCl in dioxane and stirred for 2 hours. The mixture was concentrated, redissolved in acidic water (0.1% TFA) and washed with CH2Cl2. Product was purified on a JASCO High-Performance Liquid Chromatography (HPLC) instrument on a C18 column using aqueous (H2O+0.1% TFA) and organic (CH3CN) phases. 1H NMR (500 MHz, D2O) δ 7.83-7.82 (d, 2H), 7.70-7.67 (m, 1H), 7.61-7.58 (m, 2H), 3.58-3.56 (m, 2H), 3.40-3.37 (m, 2H), 3.29-3.27 (m, 2H), 3.16-3.13 (t, 2H), 2.97 (s, 3H), 2.20-2.13 (m, 4H); 13C NMR (500 MHz, CDCl3) δ 171.35, 133.29, 132.38, 128.89, 127.05, 54.03, 52.97, 39.68, 36.57, 36.48, 23.94, 21.94. HRMS m/z calcd for C14H23N3O [M+H] 250.184, observed 250.1923
HPLC:
HPLC chromatograms were obtained at all wavelengths from 200 to 800 nm. 254 and 214 nm were chosen as virtual channels to show the absorbances at those two specific wavelengths. The blue line corresponds to 254 nm and the red line corresponds to 214 nm (top plot). The lamps used were D2+W with a slit width of 4 nm. A flow rate of 1.00 mL/min was used over 30 minutes. The method began at 10% acetonitrile and 90% water+0.1% TFA. The gradient was slowly increased to 100% acetonitrile.
Melting Temperature Analysis:
An aqueous solution of 10 mM potassium cacodylate (CacoK), pH 7.2 was used as analysis buffer. DNA 3WJ and dsDNA were brought to a final concentration of 1 1.1.1\4 and 2 μM, respectively. Samples were heated to 90° C. for 5 min, cooled to room temperature slowly, then to 4° C. Samples were incubated for 1 hour at room temperature with 3 μL of ligand at a final concentration of 4 μM for DNA 3WJ. The concentration of the ligand was 8 μM for dsDNA. Denaturation was recorded at 260 nm from 5° C. to 90° C. with a heating rate of 0.5° C. min−1. Upper and lower baselines were used to plot the fraction folded.
Circular Dichroism:
DNA was suspended at 20 μM in 10 mM CacoK, pH 7.2 and annealed by heating to 90° C. for 5 min, cooled to room temperature slowly, then to 4° C. Spectra were measured every 0.5 nm between 350 nm and 190 nm with a 5 s averaging time. Samples were incubated at each temperature for 20 minutes prior to scan.
A 50 μM solution of DNA was prepared in 10 mM CacoK, pH 7.2 and annealed by heating to 90° C. for 5 min, cooled to room temperature slowly, then to 4° C., for CD melting. Samples were incubated with ligand (200 μM) at room temperature for 1 hour. The CD melting experiment was measured at 255 nm using a 1° C. step and a 2 min equilibration time. The averaging time was 15 s. All CD spectra were buffer corrected and converted to molar ellipticity.
Fluorescence Quenching Experiments:
All binding experiments were conducted in 50 mM sodium phosphate buffer, pH 7.0. Fluorescence measurements were recorded with excitation at 495 nm and emission at 520 nm using 5 nm bandwidths. Inhibitor strand binding curves were obtained by adding 1 μL of increasing concentrations of inhibitor strand to 19 μL of 120 nM aptamer. Samples were incubated for 1 hour and ran in triplicate in a 384-well plate. Inhibitor strand displacement curves were obtained by incubating 120 nM aptamer with 1 μM inhibitor 12 for 1 hour, followed by addition of increasing concentrations of 1. Samples were incubated for 1 hour and measured in triplicate.
Gel Shift Assay:
The inhibitor strand titration gel was run by incubating aptamer (0.25 μM) with increasing concentrations of inhibitor strand in a 20 μL solution in 50 mM sodium phosphate buffer, pH 7.0 at room temperature for 1 hour. Samples for compound titration were prepared by incubating aptamer (0.25 μM) with inhibitor strand (0.25 μM) for 1 hour followed by titration of 1 and incubated at room temperature for 1 hour. Samples were run on a 15% non-denaturing polyacrylamide gel (19:1 monomer:bis) at 50V in 1×TBE buffer at 4° C. for 5 hours. Gels were stained with SYBR Gold for 10 minutes and visualized using a BioRad GelDoc XR+ imager.
Cell Culture and Cytotoxicity:
All cell lines were maintained in a humidified incubator at 37° C. in 5% CO2. A2780 and A2780cis cells were cultured in RPMI 1640 (Corning Cellgro) supplemented with 10% fetal bovine serum (Giboc, Life Technologies), 1% L-glutamine (Corning Cellgro), penicillin and streptomycin (Corning Cellgro). Cells were seeded at a density of 2,000 cells/well in culture media (50 pt) in 385-well plates 24 hours prior to treatment. Cells were treated with DMSO (vehicle control), doxorubicin (positive control, 10 μM final), cisplatin (50 μM to 25 nM), or triptycenes (50 μM to 25 nM) for 72 hours at 37° C. with 5% CO2. DMSO concentrations were kept at 1%. Alamar blue (5 μL) was added and incubated for 4 hours. Fluorescence was measured at 590 nm. The vehicle control was taken as 100% cell viability and doxorubicin was taken as 0% viability.
Cellular Uptake by MALDI:
A2780 cells were grown as described above. Cells were diluted to 250,000 cells/mL in fresh media. Cells (2 mL) were treated with DMSO (vehicle control) or triptycenes (50 μM final) for 2 hours at 37° C. with 5% CO2. DMSO concentrations were kept at 1%. The cells were centrifuged at 640 g for 2 minutes. The supernatant was removed and the cells were washed with 500 μL of 50 mM Tris-HCl, pH 7.4 three times. The supernatant was placed in a clean microcentrifuge tube for MALDI analysis. The cell pellet was suspended in lysis buffer (0.3% Triton-X-100, 100 mM NaCl) and heated to 100° C. for 15 minutes. The lysate was centrifuged at 7080 g for 5 minutes, the supernant was transferred to a clean microcentrifuge tube. MALDI was used to analyze the washes and lysate using a-cyano-4-hydroxycinnamic acid as the matrix.
Modulation of the rpoH Temperature Sensor in E. coli
Figures and references are as published in Barros et al., Angew. Chem. Int. Ed., 2016(55):8258, published on May 30, 2016.
Regulation of the heat shock response (HSR) is essential in all living systems. In E. coli, the HSR is regulated by an alternative σ factor, σ32, which is encoded by the rpoH gene. The mRNA of rpoH adopts a complex secondary structure that is critical for the proper translation of the σ32 protein. At low temperatures, the rpoH gene transcript forms a highly structured mRNA containing several three-way junctions, including a rare perfectly paired three-way junction (3WJ). This complex secondary structure serves as a primitive but highly effective strategy for the thermal control of gene expression. In this work, the first small-molecule modulators of the E. coli σ32 mRNA temperature sensor are reported.
Temperature is a universal stress factor for all living organisms, and a rapid response to temperature fluctuations is essential for cell survival. The heat shock response (HSR) is a cellular process characterized by the increased synthesis of a set of heat shock proteins (HSPs) in response to stress, such as temperature. The Escherichia coli (E. coli) HSR is regulated by an alternative σ factor, σ32, encoded by the rpoH gene. An increase in temperature from 30° C. to ≥37° C. results in the increased synthesis and stability of σ32, leading to the transcription of σ32-dependent genes involved in the HSR. Translational control is a common strategy for the modulation of the HSR in both eukaryotes and prokaryotes. It is found that the σ32 mRNA secondary structure acts as a thermosensor, crucial for the induction of σ32, in the E. coli HSR pathway (
Chemical and enzymatic probing of the 5′-end of the σ32 mRNA secondary structure reveals that the regulatory regions (regions A and B) within the RNA structure form a perfectly paired three-way junction (3WJ). Recently, we developed a new class of nucleic acid junction binders based on the triptycene scaffold. Herein, we report the first triptycene-based small molecules that are able to modulate the stability of the σ32 mRNA. We determined the ability of these ligands to modulate the structure of σ32 RNA by UV thermal melting, circular dichroism (CD), and fluorescence quenching experiments. Furthermore, we demonstrate the in vivo modulation of the heat shock response using a σ32-GFP fusion protein reporter system in E. coli.
We initiated our studies with a model system corresponding to the regulatory junction present in the rpoH mRNA (
A fluorescence quenching experiment was used to further support the modulation of the σ32 RNA (
Having characterized the interactions of Trip 1 and 2 with the model system by fluorescence quenching, we turned our attention to the full 5′-region of the σ32 mRNA (−19 to +229). The 5′-end of the σ32 mRNA was transcribed in vitro for characterization by temperature-dependent UV and CD techniques. UV thermal melting experiments in the absence of the triptycenes showed a double inflection, indicating that portions of the RNAmelt at different temperatures, with the most critical structural changes occurring below 42° C. The first inflection, with an initial onset below 42° C., is consistent with the temperature-dependent translation previously observed for rpoH mRNA. In the presence of Trip 1 and Trip 2, thermal stabilization of the full-length σ32 mRNA (−19 to +229,
A reporter assay based on a σ32 GFP fusion protein was developed and used to monitor the responses to cellular stress in E. coli (
Non-specific inhibition of translation was evaluated using a control GFP-only plasmid in the presence of Trip 1 and Trip 2. Interestingly, we observed an approximately fourfold increase in translation going from the GFP control plasmid at 42° C. to the σ32-GFP plasmid at 42° C. (
In summary, we have described triptycene-based molecules that modulate the 5′-region of the σ32 mRNA temperature sensor from E. coli. Trip 1 and Trip 2 thermally stabilize a model system consisting of the critical central three-way junction that is present in the σ32 mRNA and responsible for regulation of the heat shock response as determined by UV thermal melting experiments and temperature-dependent CD spectroscopy. UV thermal melting experiments on the full 5′-region of the σ32 mRNA also show thermal stabilization. This stabilization was corroborated by temperature-dependent CD spectroscopy in the presence of ligands. To determine the effect of the triptycenes on the heat shock response in E. coli, a σ32-GFP fusion protein assay was utilized. In the absence of the triptycenes, an increase in fluorescence was observed when the cells were heat-shocked at 42° C., indicating σ32 protein translation. However, the addition of Trip 1 or Trip 2 suppresses the fluorescence, which is consistent with a decrease in σ32 protein translation. This new class of small molecules may be useful for studying the effects of the heat shock response in E. coli. Furthermore, modulation of the temperature-sensing RNA regulatory elements in bacteria could lead to the development of novel methods for targeting pathogens or potentiating current antibiotics.
General Methods:
The σ32 mRNA model system (5′-GGCACAAACGCAACACUGCAUUACCAUGCGGUUGUGCC-3′), Inhibitor 16 (I16) (5′-GTGTTGCATTTGTGCC-3′) and all other oligonucleotides were purchased from Integrated DNA Technologies (IDT). HPLC-purified σ32 mRNA model system modified with a 5′-FAM and a 3′-IowaBlack was also purchased from IDT. E. coli genomic DNA was purchased from Addgene (Cambridge, Mass. USA). Restriction enzymes and T4 DNA ligase were purchased from New England BioLabs. T7 RNA polymerase was purchased from Promega (Madison, Wis. USA). Milli-Q (18 MS2) water was used for all solutions (Millipore; Billerica, Mass., USA).
Ultraviolet absorption spectroscopy and thermal denaturation experiments were performed on a JASCO V-650 spectrophotometer (JASCO Analytical Instruments; Easton, Md., USA) equipped with a JASCO PC-734R multichannel Peltier using quartz cells with 1 cm path lengths. Circular dichroism experiments were performed on a JASCO J-1500 CD Spectrometer (Easton, Md., USA) using a 0.1 cm path length quartz cuvette. Fluorescence measurements were collected on a Tecan M1000 plate reader (Mannedorf, Switzerland). Polymerase chain reaction (PCR) was run on a BioRad C1000 Touch Thermocycler.
Synthesis:
Trip 1 and Trip 2 were synthesized according to previously described methods, S. A. Barros, D. M. Chenoweth, Chem. Sci. 2015, 6, 4752-4755, hereby expressly incorporated herein by reference for the methods, figures and legends herein.
Synthesis of rpoH mRNA −19 to +229:
Plasmid pRSET-EmGFP (HA EmGFP ABC2 V94F) was used for cloning into the XbaI and EcoRI restriction sites. The rpoH (−19 to 229) gene was obtained by PCR amplification from genomic DNA from E. coli K-12. The forward and reverse primers used were 5′-GATCTAGAATCGATTGAGAGGATTTGAATG-3′ and 5′-GAGAATTCCCGCCTGTGGCAGGCCATAGC-3′, respectively. The pRSET-EmGFP plasmid was digested with XbaI and EcoRI then gel purified to isolate the linear vector. The σ32 (−19 to 229) PCR product was also digested and inserted into the plasmid using T4 DNA ligase. The resulting plasmid was verified by DNA sequencing using a T7 primer.
The DNA template was prepared for transcription by linearization with EcoRI then gel purified. The RNA was transcribed in vitro by T7 RNA polymerase (Promega). In vitro transcription reactions were set up using the protocol supplied by Promega with 1× transcription buffer, 10 mM DTT, 0.5 mM each rNTP, and 2-5 μg DNA.
UV Thermal Denaturation: model system RNA was suspended at 1 μM in 10 mM sodium phosphate buffer, pH 7.2 and annealed by heating to 90° C. for 5 min, cooled to room temperature slowly, then to 4° C. Samples were incubated for 1 hour at room temperature with 1 μL of ligand at a final concentration of 2 μM. σ32 RNA (−19 to +229) was suspended at 0.25 μM in 10 mM sodium phosphate buffer, pH 7.2 and annealed by heating to 65° C. for 5 min, cooled to room temperature slowly, then to 4° C. Samples were incubated for 1 hour at room temperature with 1 μL of ligand at a final concentration of 2.5 μM. Denaturation was recorded at 260 nm from 20° C. to 90° C. with a heating rate of 0.5° C. min-1.
Fluorescence quenching experiments: all binding experiments were conducted in 50 mM sodium phosphate buffer, pH 7.2. Fluorescence measurements were recorded with excitation at 495 nm and emission at 520 nm using 5 nm bandwidths. Inhibitor strand binding curves were obtained by adding 1 μL of increasing concentrations of inhibitor strand to 19 μL of 120 nM RNA. Samples were incubated for 2 hours and ran in triplicate in a 384-well plate. Inhibitor strand displacement curves were obtained by incubating 120 nM RNA with 1.4 μM inhibitor 16 for 2 hours, followed by addition of increasing concentrations of Trip 1 or Trip 2. Samples were incubated for 2 hours and measured in triplicate.
Circular Dichroism:
Model system RNA was suspended at 5 μM in 10 mM sodium phosphate buffer, pH 7.2 and annealed by heating to 90° C. for 5 min, cooled to room temperature slowly, then to 4° C. Spectra were measured every 1 nm between 350 nm and 200 nm with a 16 s averaging time. Samples containing ligand were incubated with Trip 1 or Trip 2 (10 μM) at room temperature for 1 hour. Samples were incubated at each temperature for 20 minutes prior to scan. All CD spectra were buffer corrected and converted to molar ellipticity.
σ32 mRNA (−19 to +229) was suspended at 0.5 μM in 10 mM sodium phosphate buffer, pH 7.2 and annealed by heating to 65° C. for 5 min, cooled to room temperature slowly, then to 4° C. Spectra were measured every 1 nm between 350 nm and 200 nm with a 16 s averaging time. Samples containing ligand were incubated with Trip 1 or Trip 2 (5 μM) at room temperature for 1 hour. Samples were incubated at each temperature for 20 minutes prior to scan All CD spectra were buffer corrected and converted to molar ellipticity.
σ32-EmGFP Plasmid Construction: Plasmid pRSET-EmGFP (HA EmGFP ABC2 V94F) was used for cloning into the XbaI and EcoRI restriction sites. The rpoH gene was obtained by PCR amplification from genomic DNA from E. coli K-12. This included four rpoH promoters (p2, p3, p4, and p5). The forward and reverse primers used were 5′-GATCTAGAGAACTTGTGGATAAAATCACG-3′ and 5′-GAGAATTCGGATCCTTACGCTTCAATGGCAGCAC-3′, respectively. The pRSET-EmGFP plasmid was digested with XbaI and EcoRI then gel purified to isolate the linear vector. The rpoH PCR product was also digested and inserted into the plasmid using T4 DNA ligase. The resulting plasmid was verified by DNA sequencing using a T7 primer.
σ32-EmGFP Assay: E. coli DHSa cells transformed with the 632-EmGFP plasmid were grown overnight at 30° C. in Luria broth (LB) supplemented with 50 μg/mL ampicillin. Overnight cultures were diluted 1:100 in LB. Triptycenes were added at a final concentration of 25 μM. Samples were allowed to grow at 30° C. for 3 hours. Cultures were kept at 30° C. or heat shocked at 42° C. for 18 hours. Optical density was measured at 600 nm. Fluorescence was measured by excitation at 486 nm and emission at 535 nm. Measurements were made in triplicate for each sample. The raw fluorescence intensity was divided by the optical density at 600 nm, which was then normalized.
qRT-PCR Experiment: E. coli DH5a cells transformed with the 632-EmGFP plasmid were grown as described in σ32-EmGFP Assay. Triptycenes were added at a final concentration of 25 or 12.5 μM. Samples were allowed to grow at 30° C. for 3 hours. Cultures were then kept at 30° C. for 18 hours. Total RNA from E. coli was extracted and purified using RNAprotect Bacteria Reagent (QIAGEN, catalog #: 76506) and RNeasy Mini Kit (QIAGEN, catalog #: 74104). The user manual provided by QIAGEN was followed. The expression was quantified in quadruplicate by qRT-PCR using Custom TaqMan™ Gene Expression Assays (Applied Biosystems by Life Technologies, Foster City, Calif., USA) at the University of Pennsylvania Perelman School of Medicine Molecular Profiling Core. The reverse transcription reaction was carried out with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) in 100 μl containing 1.0 μg RNA in 30.0 μl nuclease free water, 4 μl of 25× (100 mM) dNTPs, 5 μl of multiscribe reverse transcriptase (50 U/μl), 10 μl of 10× reverse transcription buffer, 10 μl 10× random primer. For synthesis of cDNA, the reaction mixtures were incubated at 25° C. for 10 min, at 37° C. for 120 min, at 85° C. for 5 min and then held at 4° C. Then, 4.5 μl of 1:5 diluted cDNA solution was amplified using 5.0 μl TaqMan 2× Fast Universal PCR Master Mix with no AmpErase UNG (Applied Biosystems), 0.5 μl of assay in a final volume of 10.0 μl. Quantitative PCR was run on a QuantStudio 12K Flex Real-Time PCR system (Applied Biosystems) and the reaction mixtures were incubated at 95° C. for 10 min, followed by 40 cycles of 95° C. for 15 s and 60° C. for 1 min. The cycle threshold (Ct) values were calculated with QuanStudio Software version 1.2.2 (Applied Biosystems). The mRNA levels of rpoH were normalized to two housekeeping genes, rrsG (16S ribosomal RNA of rrnG operon) and arcA (response regulator in two-component regulatory system with ArcB or CpxA), using the methods described below.
Method A: Ct (rpoH)/Ct (rrsG or arcA)
Method B2:
ΔCt1=Ct(rpoH_Trip 1 or 2)−Ct(rrsG or arcA_Trip 1 or 2)
ΔCt2=Ct(rpoH No compound)−Ct(rrsG or arcA_No compound)
ΔΔCt=Ct1−Ct2
Normalized rpoH gene expression level=2ΔΔCt
The figures and references are as published in Barros et al., Chemical Science, 2015 (6):4752, published on Jun. 10, 2015.
Nucleic acid three-way junctions (3WJs) play key roles in biological processes such as nucleic acid replication in addition to being implicated as dynamic transient intermediates in trinucleotide repeat sequences. Structural modulation of specific nucleic acid junctions could allow for control of biological processes and disease states at the nucleic acid level. Trinucleotide repeat expansions are associated with several neurodegenerative diseases where dynamic slippage is thought to occur during replication, forming transient 3WJ intermediates with the complementary strand. Here, we report triptycene-based molecules that bind to a d(CAG)⋅(CTG) repeat using a gel shift assay, fluorescence-quenching and circular dichroism
Nucleic acid junctions play important roles in biological processes and serve as key structural motifs in nanotechnology and aptamer-based sensing applications. In biology, three-way junctions (3WJs) are found as transient intermediates during replication, recombination, and DNA damage repair. Junctions are also present in several viral genomes, such as HIV-1, HCV, and adeno-associated virus in addition to playing key roles in viral assembly. Nucleic acid junctions are also prevalent in the emerging field of DNA and RNA nanotechnology where the bacteriophage phi29 pRNA containing RNA three-way junctions provide a particularly impressive example. Furthermore, they occur in trinucleotide repeat expansions found in unstable genomic DNA associated with neurodegenerative diseases. The development of structure and sequence-specific nucleic acid binding molecules remains an important challenge in chemical biology. The ability to target specific motifs using small molecules would allow for the precise control of biological processes and the possibility of modulating disease states.
DNA trinucleotide repeats are present throughout the genome. Expansions of these repeats, however, are associated with a number of neurodegenerative diseases, including Huntington's disease, spinobulbar muscular atrophy, and mytonic dystrophy. Current models of triplet repeat expansion disease suggest slippage during DNA synthesis by the formation of dynamic DNA hairpin structures. As the length of the repeat increases, the growing hairpin structure gains thermodynamic stability, with repeat length providing an important positively correlated diagnostic for disease severity. Slipped-out (CAG)n⋅(CTG)n repeats have been implicated in the pathogenesis of triplet repeat expansion diseases such as Huntington's disease and several others. These “slipped-out” regions are dynamic and occur along the duplex, forming three-way junctions. Current models suggest that one arm of the junction contains the excess repeats while the other arms maximize complementary pairing between adjacent strands. A dynamic ensemble of conformations are possible at the slipped junction interface, where base pairing interactions differ with each state. The slipped-out (CAG)n repeat in
Recently, we reported a new class of triptycene-based three-way junction (3WJ) binders. Here, we apply the triptycene scaffold as a first step toward developing new tools to recognize trinucleotide repeat junctions. We assessed the ability of our new triptycene based molecules to modulate the structure of d(CAG)n repeats using gel shift assays, a fluorescence quenching assay, and circular dichroism (CD).
Results and Discussion
Utilizing a well-studied (CAG)⋅(CTG) repeat sequence known to form slipped-DNA three-way junctions, we developed a competitive inhibitor-based gel shift assay. This assay was inspired by previous work from our laboratory and seminal studies from the Plaxco and Ricci laboratories. We utilized the junction forming (CAG)⋅(CTG) repeat sequence (TNR) and an inhibitor strand (I10) shown in
Inspired by nucleic acid binding proteins, we synthesized a new class of triptycene molecules containing amino acids commonly found at the protein-nucleic acid interface. Analysis of these interfaces reveals an abundance of positively charged amino acid residues such as arginine, lysine, and histidine. We synthesized triptycene derivatives substituted with arginine, lysine, and histidine (Trip 2-4) as shown in
Next, we tested the ability of Trip 3 and 4 to induce fluorescence-quenching upon junction formation using a double labelled (CAG)⋅(CTG) repeat oligonucleotide. The TNR oligonucleotide was labelled with a FAM fluorophore on the 5′-end and an IowaBlack quencher on the 3′-end (TNR*) (
Temperature-dependent circular dichroism (CD) was used to further characterize the interaction of Trip 3 and Trip 4 with the TNR junction. The CD spectra are indicative of B-DNA, showing positive signals at 280 nm due to base stacking and negative signals at 250 nm due to the right-handed helicity (
In summary, we have described triptycene-based molecules that binds to d(CAG)⋅(CTG) trinucleotide repeats. Trip 3 and Trip 4 bind to a model (CAG)⋅(CTG) repeat as determined by gel shift and fluorescence-quenching experiments. The CD spectra are also consistent with enhanced helicity of the slipped out junction upon addition of Trip 3 and 4. This new class of nucleic acid binding small molecule may serve as inspiration for creating valuable probes for diseases associated with trinucleotide repeat expansions. Trinucleotide repeat nucleic acid sequences are associated with a large number (>30) of inherited human muscular and neurological diseases. The trinucleotide repeat tract length is dynamic and often correlates with disease severity, where short stable tracts are commonplace in the non-affected population. Longer unstable triplet repeat tracts are more prone to expansion as opposed to contraction, in addition to being predisposed to generational transmission. Trincleotide repeat repair outcomes are also affected by structural features present in slipped sequences, where the structure may determine which proteins are recruited for repair. Stabilization of a particular structure could lead to increased repair of these slipped-out junction. Addition of ligands that bind to these junctions may affect repair outcomes as well as recruitment of proteins. Small molecule probes will provide valuable tools to study these processes. Small molecules binding and stabilization or modulation of these dynamic structures could lead to the development of therapeutic agents for their associated diseases.
General Methods:
TNR DNA 3WJ (5′-GCGGAGCAGCCCTTGGGCAGCACCTTGGTGCTGCTCCGC-3′) and DNA inhibitor 10 (5′-GCTGCTCCGC-3′) were purchased from Integrated DNA Technologies (IDT). HPLC-purified TNR DNA 3WJ oligo modified with a 5′-FAM and a 3′-IowaBlack was purchased from IDT. Amino acids (Boc-Arg(Mtr)-OH, Boc-Lys(Boc)-OH, and Boc-His-OH were purchased from Merck Millipore Novabiochem (Billerica, Mass., USA). (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate) (HATU) was purchased from GenScript (Piscataway, N.J., USA). All other reagents were purchased from Sigma Aldrich (St. Louis, Mo., USA) and used without further purification. Reactions requiring anhydrous conditions were run under argon with solvents purchased from Fisher dried via an alumina column. Thin-layer chromatography was done using Sorbent Technologies (Norcross, Ga., USA) silica plates (250 μm thickness). Milli-Q (18 MS2) water was used for all solutions (Millipore; Billerica, Mass., USA).
1H and 13C NMR were recorded on a Bruker UNI 500 NMR at 500 and 125 MHz, respectively. Matrix-assisted laser desorption ionization (MALDI) mass spectra were obtained on a Bruker Ultraflex III MALDI-TOF-TOF mass spectrometer (Billerica, Mass., USA) using α-cyano-4-hydroxycinnamic acid (CHCA). Low resolution electrospray ionization (ESI) mass spectra (LRMS) were collected on a Waters Acquity Ultra Performance LC (Milford, Mass., USA). High resolution mass spectra were obtained at the University of Pennsylvania Mass Spectrometry Center on a Waters LC-TOF mass spectrometer (model LCT-XE Premier) using electrospray ionization in positive or negative mode, depending on the analyte. High-performance liquid chromatography was performed on a JASCO HPLC (Easton, Md., USA) equipped with a Phenomenx (Torrance, Calif., USA) column (Luna 5μ C18(2) 100 A; 250×4.60 mm, 5 μm) using aqueous (H2O+0.1% CF3CO2H) and organic (CH3CN) phases. Circular dichroism experiments were performed on a JASCO J-1500 CD Spectrometer (Easton, Md., USA) using a 0.1 cm path length quartz cuvette. Fluorescence measurements were collected on a Tecan M1000 plate reader (Mannedorf, Switzerland). HPLC chromatograms were obtained at all wavelengths from 200 to 800 nm (bottom plot). 254 and 214 nm were chosen as virtual channels to show the absorbances at those two specific wavelengths (top plot). The blue line corresponds to 254 nm and the red line corresponds to 214 nm (top plot). The lamps used were D2+W with a slit width of 4 nm. A flow rate of 1.00 mL/min was used over 35 minutes. The method began at 10% acetonitrile and 90% water+0.1% TFA. The gradient was increased to 25% acetonitrile over 25 minutes and then increased to 100% acetonitrile.
Synthesis:
General Procedure:
To a solution of 5 (0.08 mmol) in DMF (1 mL) was added HATU (0.256 mmol) and DIEA (0.496 mmol) and was stirred at room temperature for 5 minutes. The corresponding amino acid (0.256 mmol) was added and stirred overnight. The reaction mixture was concentrated under vacuum, then diluted with water and extracted with EtOAc. The crude product was suspended in 4M HCl in dioxane and stirred for 2 hours. The mixture was concentrated, dissolved in acidic water (0.1% TFA) and washed with CH2Cl2. The product was purified on a JASCO High-Performance Liquid Chromatography (HPLC) instrument on a C18 column using aqueous (H2O+0.1% TFA) and organic (CH3CN) phases.
1H NMR (500 MHz, D2O) δ 7.65 (s, 3H), 7.51-7.50 (d, 3H) 7.12-7.10 (d, 3H), 5.72 (s, 1H), 5.66 (s, 1H), 4.13-4.10 (t, 3H), 3.23-3.20 (t, 3H), 2.03-1.98 (m, 6H), 1.72-1.66 (m, 6H); HRMS m/z calcd for C38H56H15O33+ [M+3H]3+ 256.8225, observed 256.8231.
1H NMR (500 MHz, D2O) δ 7.65 (s, 3H), 7.52-7.50 (d, 3H) 7.13-7.11 (d, 3H), 5.73 (s, 1H), 5.67 (s, 1H), 4.11-4.09 (t, 3H), 2.99-2.96 (t, 6H), 2.01-1.96 (m, 6H), 1.17-1.67 (m, 6H), 1.51 (m, 6H); HRMS m/z calcd for C38H54N9O3+ [M+H]+ 684.4344, observed 684.4357.
1H NMR (500 MHz, D2O) δ 8.58 (s, 3H), 7.65 (s, 3H) 7.54-7.52 (d, 3H), 7.40 (s, 3H), 7.07-7.05 (d, 3H), 5.75 (s, 1H), 5.69 (s, 1H), 4.40-4.36 (t, 3H), 3.47-3.45 (d, 6H); 13C NMR (500 MHz, D2O) δ 166.63, 145.73, 142.70, 134.65, 133.19, 126.72, 124.29, 118.65, 118.15, 117.52, 115.20, 52.93, 51.60, 26.58; MALDI-TOF m/z calcd for C38H38H12O3 [M+H] 710.80, observed 711.397, HRMS m/z calcd for C38H39H12O3+ [M+H]+ 711.3263, observed 711.3274.
Gel Shift Assay:
All gel shift experiments were conducted in 50 mM sodium phosphate buffer, pH 7.2. The screening gel was run by incubating TNR 3WJ (0.5 μM) with inhibitor strand 10 bases long (1.5 μM) in a 20 μL solution at room temperature for 2 hours. Triptycenes were then added at a final concentration of 10 μM and incubated for 2 hours. Samples were run on a 20% non-denaturing polyacrylamide gel (19:1 monomer:bis) at 50V in 1×TBE buffer at 4° C. for 10 hours. Gels were stained with SYBR Gold for 10 minutes and visualized using a BioRad GelDoc XR+ imager.
Inhibitor strand titration gel was run by incubating TNR 3WJ (0.5 μM) with increasing concentrations of inhibitor strand in a 20 μL solution at room temperature for 2 hours. Samples for compound titration were prepared by incubating TNR 3WJ (0.5 μM) with inhibitor strand (1.5 μM) for 2 hours followed by titration of Trip 4 and incubation at room temperature for 2 hours. Samples were run on a gel as described above.
Fluorescence Quenching Experiments:
All binding experiments were conducted in 50 mM sodium phosphate buffer, pH 7.2. Fluorescence measurements were recorded with excitation at 495 nm and emission at 520 nm using 5 nm bandwidths. Inhibitor strand binding curves were obtained by adding 1 μL of increasing concentrations of inhibitor strand to 19 μL of 120 nM TNR 3WJ. Samples were incubated for 2 hours and ran in triplicate in a 384-well plate. Inhibitor strand displacement curves were obtained by incubating 120 nM aptamer with 1 μM inhibitor 10 for 2 hours, followed by addition of increasing concentrations of 4. Samples were incubated for 2 hours and measured in triplicate.
Circular Dichroism:
DNA was suspended at 6 μM in 50 mM sodium phosphate buffer, pH 7.2 and annealed by heating to 90° C. for 5 min, cooled to room temperature slowly, then to 4° C. Spectra were measured every 0.5 nm between 350 nm and 200 nm with an 8 s averaging time. Samples were incubated at each temperature for 20 minutes prior to scan. Samples were incubated with ligand (24 μM) at room temperature for 1 hour. All CD spectra were buffer corrected and converted to molar ellipticity.
Figures, tables and references are as published in Yoon et al., Organic Letters, 2016(18):1096, published on Feb. 17, 2016.
Triptycenes have been shown to bind nucleic acid three-way junctions, but rapid and efficient methods to diversify the triptycene core are lacking. An efficient synthesis of a 9-substituted triptycene scaffold is reported that can be used as a building block for solid-phase peptide synthesis and rapid diversification. The triptycene building block was diversified to produce a new class of tripeptide-triptycenes, and their binding abilities toward d(CAG)⋅(CTG) repeat junctions were investigated.
Nucleic acid junctions play important roles in many biological events. Three-way junctions (3WJs) have diverse architectures and are found in DNA and RNA, where they often serve as important structural elements. Several small molecules are known to bind to nucleic acid junctions. However, these molecules often lack specificity, leading to binding of various structures.
Efficient strategies for triptycene diversification are needed to accelerate the discovery of new nucleic acid junction binders with enhanced specificity and binding properties. Triptycene building blocks that are amenable to immobilization on a solid support would allow for rapid diversification and compound library construction (
A carboxylic acid was chosen for functionalization at the C9 tertiary carbon of triptycene due to its versatility of conversion into other functional groups, such as aldehyde, haloalkane, ester, and amide. The carboxylic acid group may also be removed via decarboxylation at a later stage. More importantly, the carboxylic acid group has been extensively employed for directed C-H bond functionalization reactions, which could prove valuable during future triptycene diversification efforts.
Our synthetic plan (
Commercially available anthracene-9-carbaldehyde 1 was employed as a starting material. Reduction of 1 using sodium borohydride afforded anthracen-9-ylmethanol 2 in 96% yield within 1 h (
Treatment of 4 with nitric acid resulted in nitration of the aromatic rings. During the nitration reaction, the protecting group on the alcohol was simultaneously deprotected and oxidized to the carboxylic acid, providing 6a along with two other isomers 6b and 6c. The nitrated triptycene isomers proved inseparable by silica gel chromatography. Acid-catalyzed esterification of the crude mixture provided ester isomers 5a-c, which were separated via silica gel chromatography. The structures of ester isomers 5a-c were confirmed by twodimensional NMR spectroscopy, HMBC, and HSQC. Single crystals of 5a were grown in CHCl3/CH2Cl2/CH3OH, and the structure was determined by X-ray crystallography (
To investigate the O-directing effect observed during nitration and to further reduce the number of undesired side products, compound 8, containing a carboxylic acid at the C9 position, was prepared by deprotection of 4 followed by KMnO4 oxidation (
Interestingly, nitration of 7 produced little of desired products 6a-c. The composition of 6a and 6c significantly changed compared to that from the nitration of 4 and 8, and the overall yield increased for the nitration of 8. Attempts were made to increase the proportion of 6a over that of the other isomers. The highest ratio of 6a to 6b achieved using this nitration method was 0.33. The introduction of a carboxylic acid at the C9 position of triptycene significantly increased the ratio of 6a to 6b to 0.81. These observations are consistent with the carboxylic acid functioning as a directing group during the nitration reaction.
Isomer 6a was chosen for further elaboration due to its 3-fold symmetry, which is complementary to that of nucleic acid 3WJs. To extend the length of the linker at the 9-position, several standard reaction conditions for amide bond formation were examined. However, the amidation reaction proved recalcitrant, and all attempted conditions resulted in unreacted starting material (
Pd/C-catalyzed hydrogenation of 9 led to reduction of the three nitro groups to afford triaminotriptycene 10. Next, the free amines were protected with Fmoc groups by treatment with Fmoc chloride and pyridine. The linker ester group was hydrolyzed in the presence of sulfuric acid and water to produce acid 12. The free carboxylate of fully protected building block 12 allowed for attachment to 2-chlorotrityl chloride resin, which is compatible with Fmoc deprotection chemistry (
Triptycenes 17-19 were evaluated for binding toward a d(CAG)⋅(CTG) trinucleotide repeat junction using a previously developed fluorescence-quenching experiment. The binding of triptycenes 17-19 were compared to a previously reported triptycene that binds to the junction. The previously reported junction binder (20) is analogous to 17 but lacks the linker at the 9-position. A d(CAG)⋅(CTG) repeat junction was labeled with a fluorophore (FAM) and a quencher (IowaBlk). This labeled 3WJ was preincubated with a 10 bp inhibitor (I10) strand that is complementary to the junction. Hybridization of the inhibitor strand to the junction results in an open form, leading to an increase in fluorescence (
In summary, we have developed a synthetic approach for preparing new 9-substituted triptycene building blocks. This approach enables solid-phase diversification of triptycene. During the synthesis, O-directed nitration was observed from the MOM protected primary alcohol (4), primary alcohol (7), and carboxylic acid (8) at the C9 position of triptycene. These results indicated that the carboxylic group increased the ratio of nitration on β-carbons toward the linker position, pointing to a possible carboxylic acid directing effect. In addition, a key amide bond formation was achieved on a sterically hindered and geometrically fixed tertiary carboxylic acid using a unique MsCl activation strategy. This may be regarded as a general strategy toward functionalization of extremely sterically encumbered tertiary carboxylic acids. For diversification of the new triptycene building block, three amino acids were utilized including histidine, lysine, and asparagine to produce trisubstituted triptycenes 17-19. The binding ability of the synthesized triptycene derivatives toward a d(CAG)⋅(CTG) trinucleotide repeat junction was evaluated, and triptycenes 18 and 19 exhibited better binding affinity to the junction compared to that of a previously reported triptycene with no linker (20). This new synthetic strategy provides rapid and efficient access to triptycene building blocks, enabling high throughput diversification for rapid evaluation of potential junction binders and other medicinal chemistry targets.
General Methods:
All commercial reagents and solvents were used as received. 9-anthracenecarboxaldehyde, sodium borohydride, N,N-diisopropylethylamine, chloromethyl methyl ether, beta-Alanine ethyl ester hydrochloride, Fmoc chloride, palladium on activated carbon, cesium fluoride, 2-(trimethylsilyl)phenyl trifluoromethanesulfonate, and nitric acid from Aldrich, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxide hexafluorophosphate (HATU) from Oakwood Products, Inc., 2-chlorotrityl chloride resin from Advanced ChemTech, chloroform-d, methylene chloride-d2, dimethylsulfoxide-d6, and acetone-d6 from Cambridge Isotope Laboratories Inc. were purchased. HPLC-purified TNR DNA 3WJ oligo modified with a 5′-FAM and a 3′-IowaBlack (5′-(FAM)-GCGGAGCAGCCCTTGGGCAGCACCTTGGTGCTGCTCCGC-(IowaBlk)-3′) and DNA inhibitor 10 (5′-GCTGCTCCGC-3′) were purchased from Integrated DNA Technologies (IDT).
Flash column chromatography was performed using Silicycle silica gel (55-65 Å pore diameter). Thinlayer chromatography was performed on Sorbent Technologies silica plates (250 μm thickness). Proton nuclear magnetic resonance spectra (1H NMR) and Carbon nuclear magnetic resonance (13C NMR) spectra were recorded on a Bruker DMX 500. High-resolution mass spectrometry analysis was obtained by Dr. Rakesh Kohli at the University of Pennsylvania's Mass Spectrometry Service Center on a Waters LC-TOF mass spectrometer (model LCT-XE Premier) using electrospray ionization. High-performance liquid chromatography (HPLC) chromatograms were recorded and triptycenes 17-19 was purified on JASCO HPLC (Easton, Md.) equipped with a Phenomenx (Torrance, Calif.) column (Analytical: Luna 5μ C18(2) 100 A; 250×4.60 mm, 5 μm Semi-prep: 5μ C18(2) 100 A; 250×10.00 mm, 5 μm) using aqueous (H2O+0.1% CF3CO2H) and organic (CH3CN) phases. Matrix-assisted laser desorption ionization (MALDI) mass spectra were recorded on a Bruker Ultraflex III MALDITOF-TOF mass spectrometer (Billerica, Mass.) using α-cyano-4-hydroxycinnamic acid (CHCA). Fluorescence measurements were obtained on a Tecan M1000 plate reader (Mannedorf, Switzerland).
To 4.9 g (23.76 mmol) of anthracene-9-carbaldehyde (1) in THF (50 mL) was added 1.35 g (35.64 mmol) of NaBH4. The mixture was stirred for 1 h at 25° C. The mixture was poured into water (400 mL) resulting in a yellow precipitate. The yellow solid was filtered off, washed thoroughly with water, and dried. (4.7 g, 96% isolated yield).1
1H NMR (500 MHz, CDCl3) δ 8.46 (s, 1H), 8.40 (d, 2H, J=8.8 Hz), 8.02 (d, 2H, J=8.4 Hz), 7.59-7.53 (m, 2H), 7.52-7.46 (m, 2H), 5.65 (s, 2H).
To 354 mg (1.7 mmol) of anthracen-9-ylmethanol 2 in CH2Cl2 was added 1.76 mL (10.2 mmol) of N,N-diisopropylethylamine at 0° C. After stirring for 30 min, 0.4 mL (5.1 mmol) of chloromethylmethyl ether was added to this solution at 0° C. The mixture was stirred for 10 min, warmed to 25° C., and stirred for 18 h. Saturated NH4Cl (aq) solution was added to the reaction. The organic layer was extracted from the solution, dried with anhydrous sodium sulfate, concentrated in vacuo, and purified by column chromatography using ethyl acetate/hexanes (4%) as the eluent to give 391 mg of 3 (391 mg, 91% isolated yield). Physical Property: Pale yellow solid, m.p.=80-81° C.
TLC: Rf=0.52 (silica gel, 25% ethyl acetate/hexanes).
1H NMR (500 MHz, CDCl3) δ 8.53 (d, 2H, J=8.8 Hz), 8.47 (s, 1H), 8.04 (d, 2H, J=8.4 Hz), 7.68-7.62 (m, 2H), 7.58-7.51 (m, 2H), 5.67 (s, 2H), 4.90 (s, 2H), 3.61 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 131.6, 131.3, 129.2, 128.7, 128.4, 126.4, 125.1, 124.4, 95.7, 61.1, 55.8.
IR (neat): 1733, 1446, 1265, 1147, 1093, 1061, 1029, 934, 914, 891, 731, 703, 640 cm−1.
HRMS (ESI) calculated for C17H16NaO2+ [M+Na]+ 275.1043, found 275.1055.
To a round bottom flask was added 3.97 g (12.1 mmol) of 4 and 50 mL of concentrated nitric acid at 25° C. The solution was heated to 80° C. and stirred for 24 h. After the reaction was complete, water was added to the solution. The solution was neutralized with K2CO3 and re-acidified with 1M HCl. Ethyl acetate was added to the solution and the organic layer was extracted from the solution. The combined organic solution was dried with anhydrous sodium sulfate, and concentrated in vacuo. To the crude mixture was added 40 mg of H2SO4 and 100 mL of anhydrous methanol. The solution was stirred under reflux for 24 h. After the reaction was completed, the solution was cooled, extracted with ethyl acetate, dried with anhydrous sodium sulfate, and then concentrated in vacuo. The crude mixture of 5a-5c was then purified using column chromatography. The composition of each isomer was determined by HPLC analysis.
Physical Property: White solid, m.p.=282-283° C.
TLC: Rf=0.41 (silica gel, 50% ethyl acetate/hexanes).
1H NMR (500 MHz, CDCl3) δ 8.64 (d, 3H, J=2.1 Hz), 8.08 (dd, 3H, J=8.2, 2.2 Hz), 7.64 (d, 3H, J=8.2 Hz), 5.77 (s, 1H), 4.43 (s, 3H).
13C NMR (125 MHz, CDCl3) δ 167.9, 148.9, 146.3, 143.1, 124.9, 122.9, 120.1, 61.4, 53.8, 53.6.
IR (neat): 2924, 1746, 1522, 1455, 1340, 1301, 1274, 1250, 1214, 1166, 1025, 903 cm−1.
HRMS (ESI) calculated for C22H14N3O8+ [M+H]+, no peak matched the calculated exact mass. Hydrolysis of the ester to carboxylic acid 6a was required to obtain the HRMS. See next page for HRMS data on acid 6a.
Physical Property: White Solid, m.p.=163-164° C.
TLC: Rf=0.73 (silica gel, 50% ethyl acetate/hexanes).
1H NMR (500 MHz, CDCl3) δ 8.63 (d, 2H, J=2.0 Hz), 8.33 (d, 1H, J=2.0 Hz), 8.04 (dd, 2H, J=8.1, 2.0 Hz), 7.99 (dd, 1H, J=8.5, 2.0 Hz), 7.89 (d, 1H, J=8.5 Hz), 7.70 (d, 2H, J=8.1 Hz), 5.89 (s, 1H), 4.40 (s, 3H).
13C NMR (125 MHz, CDCl3) δ 168.1, 149.6, 147.9, 146.2, 144.4, 142.7, 125.4, 125.1, 122.9, 122.1, 120.1, 119.4, 61.6, 53.5, 53.4.
IR (neat): 2924, 1743, 1519, 1455, 1341, 1297, 1214, 1165, 1071, 1024, 892 cm−1.
HRMS (ESI) calculated for C22H14N3O8+ [M+H]+, no peak matched the calculated exact mass. Hydrolysis of the ester to carboxylic acid 6b was required to obtain the HRMS. See next page for HRMS data on acid 6b.
Physical Property: White Solid, m.p.=161-162° C.
TLC: Rf=0.81 (silica gel, 50% ethyl acetate/hexanes).
1H NMR (500 MHz, CDCl3) δ 8.65 (d, 1H, J=2.2 Hz), 8.33 (d, 2H, J=2.3 Hz), 8.09 (dd, 1H, J=8.2, 2.2 Hz), 8.05 (dd, 2H, J=8.6, 2.3 Hz), 7.93 (d, 2H, J=8.6 Hz), 7.67 (d, 1H, J=8.2 Hz), 5.80 (s, 1H), 4.37 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 168.1, 149.8, 147.4, 146.24, 146.21, 144.7, 142.3, 125.5, 124.9, 122.9, 122.1, 120.3, 119.3, 61.9, 53.4, 53.3.
IR (neat): 2924, 1744, 1598, 1458, 1438, 1342, 1254, 1165, 1023 cm−1.
HRMS (ESI) calculated for C22H14N3O8+ [M+H]+, no peak matched the calculated exact mass. Hydrolysis of the ester to carboxylic acid 6c was required to obtain the HRMS. See next page for HRMS data on acid 6c.
To 1 eq of 5a (or 5b, 5c) dissolved in p-dioxane was added 3 eq of 1M NaOH (aq) and heated to 60° C. for 24 h. After the reaction was completed, the solution was neutralized and acidified with 1N HCl. Ethyl acetate was added to the solution and the organic layer was extracted from the solution. The combined organic layer was washed with NH4Cl (aq) and brine. The organic layer was dried with anhydrous sodium sulfate, and concentrated in vacuo to give 6a (or 6b, 6c) in quantitative yield.
Physical Property: Pale yellow solid, m.p.=358-359° C.
TLC: Rf=0.43 (silica gel, 100% ethyl acetate).
1H NMR (500 MHz, (CD3)2CO) δ 9.23 (s, 3H), 7.96 (dd, 3H, J=8.1, 1.9 Hz), 7.74 (d, 3H, 8.1 Hz), 6.21 (s, 1H).
13C NMR (125 MHz, (CD3)2CO) δ 171.3, 151.3, 147.5, 146.1, 124.6, 121.8, 121.4, 63.7, 53.4.
IR (neat): 2924, 1592, 1518, 1341, 1262, 1092, 1069, 1023, 903 cm−1.
HRMS (ESI) calculated for C21H10N3O8 −[M−H]− 432.0473, found 432.0457.
Physical Property: Pale yellow solid, m.p.=265-266° C.
TLC: Rf=0.23 (silica gel, 100% ethyl acetate).
1H NMR (500 MHz, (CD3)2CO) δ 9.29 (d, 2H, J=2.2 Hz), 8.54 (d, 1H, J=8.3 Hz), 8.36 (d, 1H, J=2.2 Hz), 8.01 (dd, 2H, J=8.1, 2.2 Hz), 7.88 (d, 1H, J=8.1 Hz), 7.81 (d, 2H, J=8.1 Hz), 6.28 (s, 1H).
13C NMR (125 MHz, (CD3)2CO) δ 171.2, 152.5, 151.7, 146.8, 146.0, 145.9, 145.5, 127.3, 124.6, 121.8, 121.4, 121.1, 118.6, 63.7, 53.1.
IR (neat): 2921, 1737, 1593, 1524, 1462, 1377, 1344, 1260, 1093 cm−1.
HRMS (ESI) calculated for C21H10N3O8 [M−H]− 432.0473, found 432.0462.
Physical Property: White solid, m.p.=202-203° C.
TLC: Rf=0.03 (silica gel, 100% ethyl acetate).
1H NMR (500 MHz, (CD3)2CO) δ 8.92 (s, 1H), 8.49 (s, 2H), 8.23 (d, 2H, J=8.6 Hz), 8.15-8.06 (m, 3H), 7.95 (d, 1H, 8.2 Hz), 6.47 (s, 1H).
13C NMR (125 MHz, (CD3)2CO) δ 168.6, 151.1, 148.2, 146.2, 146.1, 145.7, 142.9, 125.8, 125.53, 122.51, 121.7, 120.1, 119.5, 61.8, 52.5.
IR (neat): 2927, 1720, 1598, 1518, 1458, 1418, 1341, 1260, 1179, 1090, 902 cm−1.
HRMS (ESI) calculated for C21H10N3O8 [M−H]− 432.0473, found 432.0466.
To a vial was added 1.95 g (5.94 mmol) of 4, 70 mL of 1M HCl, and 100 mL of THF. The solution was stirred at 25° C. After 1 h, ethyl acetate was added to the solution. The organic layer was extracted from the solution, dried with anhydrous sodium sulfate, and concentrated in vacuo to give 7 (1.68 g, 99% isolated yield).
Physical Property: Pale yellow solid.
TLC: Rf=0.31 (silica gel, 25% ethyl acetate/hexanes).
1H NMR (500 MHz, (CD3)2CO) δ 7.65-7.44 (m, 6H), 7.04-6.97 (m, 6H), 5.58 (s, 1H), 5.30 (d, 2H, J=3.6 Hz), 4.60 (t, 1H, J=3.6 Hz).
13C NMR (125 MHz, (CD3)2CO) δ 147.2, 145.6, 124.71, 124.66, 123.3, 122.9, 60.0, 54.2, 54.0.
IR (neat): 3308, 2962, 2918, 1579, 1458, 1261, 1071, 1035, 798, 740, 648, 629, 611, 481 cm−1.
HRMS (ESI) calculated for C21H17O+ [M+H]+ 285.1274, found 285.1288.
To a vial, 31.9 mg (0.11 mmol) of 7 was dissolved in acetone and heated to 50° C. 88.6 mg (0.56 mmol) of KMnO4 was added to the solution. Whenever the solution turned to black or brown, an additional 88.6 mg (0.56 mmol) of KMnO4 was added to the solution. After 3 days, sodium sulfite solution (aq) was added to the crude mixture and then extracted with ethyl acetate. The combined organic layer was dried with anhydrous sodium sulfate, and concentrated in vacuo to yield 8 (22.4 mg, 67% isolated yield).
Physical Property: Pale yellow solid.
TLC: Rf=0.28 (silica gel, 100% ethyl acetate).
1H NMR (500 MHz, (CD3)2CO) δ 8.09-8.01 (m, 3H), 7.46-7.42 (m, 3H), 7.02-6.97 (m, 6H), 5.57 (s, 1H)
13C NMR (125 MHz, (CD3)2CO) δ 172.3, 146.4, 144.6, 125.1, 124.6, 124.6, 123.2, 62.4, 54.2.
IR (neat): 2925, 1712, 1458, 1448, 1386, 1261, 1213, 1171, 1085, 1032, 867, 801, 748, 735, 703, 685, 645, 624, 609, 478 cm−1.
HRMS (ESI) calculated for C21H13O2− [M−H]− 297.0921, found 297.0914.
To a round bottom flask was added 213.2 mg (0.49 mmol) of 6a, 97.3 mg (1.23 mmol) of pyridine, and 10 mL of CH2Cl2. 140.9 mg (1.23 mmol) of MsCl was added to the solution at 0° C. After 30 minutes, 188.9 mg (1.23 mmol) of betaalanine ethyl ester hydrochloride and 97.3 mg (1.23 mmol) of pyridine in 10 mL of CH2Cl2 was added to the solution and warmed to 25° C. After 1 h, the solution was dried in vacuo and triturated with ethyl acetate several times to give 9 (240 mg, 91% isolated yield).
Physical Property: White solid, m.p.=304-305° C.
TLC: Rf=0.23 (silica gel, 50% ethyl acetate/hexanes).
1H NMR (500 MHz, (CD3)2CO) δ 8.92 (d, 3H, J=2.2 Hz), 8.19-8.12 (bs, 1H), 8.09 (dd, 3H, J=8.2, 2.2 Hz), 7.90 (d, 3H, J=8.2 Hz), 6.40 (s, 1H), 4.19 (q, 2H, J=7.2 Hz), 4.14-4.08 (m, 2H), 2.99 (t, 2H, J=6.6 Hz), 1.25 (t, 3H, J=7.2 Hz).
13C NMR (125 MHz, (CD3)2CO) δ 171.3, 166.6, 150.5, 146.1, 144.5, 125.5, 122.4, 120.4, 60.2, 60.0, 53.1, 35.9, 33.6, 13.6.
IR (neat): 3301, 2924, 1722, 1668, 1521, 1342, 1261, 1203, 1031, 800 cm−1.
HRMS (ESI) calculated for C26H20N4NaO9+ [M+Na]+ 555.1122, found 555.1127.
To a vial was charged 180 mg (0.338 mmol) of 9, 3.6 mg (0.034 mmol) of Pd/C, 2 mL of MeOH under H2 gas. After 24 h stirring at 25° C., the solution was filtered and concentrated in vacuo to yield crude of 10. To a round flask was added 10, 0.27 mL (3.38 mmol) of pyridine, and 30 mL of CH2Cl2. The solution was cooled and stirred for 30 minutes at 0° C. 612.1 mg (2.37 mmol) of Fmoc-Cl in 3 mL of CH2Cl2 was added to the solution. The crude mixture was warmed to 25° C. and stirred for 16 h. The solution was washed with saturated NH4Cl(aq), dried, and purified by column chromatography using ethyl acetate (100%) as the eluent to give 11 (310 mg, 83% isolated yield).
Physical Property: White solid, m.p.=157-158.3° C.
TLC: Rf=0.81 (silica gel, 50% ethyl acetate/hexanes).
1H NMR (500 MHz, CD2Cl2) δ 7.98 (s, 3H), 7.78 (d, 6H, J=7.5 Hz), 7.59 (d, 6H, J=6.3 Hz), 7.39 (t, 6H, J=7.5 Hz), 7.32-7.20 (m, 12H), 7.15 (s, 3H), 6.75 (bs, 1H), 5.28 (s, 1H), 4.40 (d, 6H, J=6.7 Hz), 4.19 (t, 3H, J=6.7 Hz), 4.06 (q, 2H, J=7.1 Hz), 3.89 (q, 2H, J=5.7 Hz), 2.80 (t, 2H, J=5.7 Hz), 1.11 (t, 3H, J=7.1 Hz).
13C NMR (125 MHz, CD2Cl2) δ 173.1, 168.8, 153.4, 144.3, 143.9, 141.4, 141.3, 135.2, 127.7, 127.0, 125.0, 123.6, 119.9, 115.9, 66.7, 60.9, 60.5, 52.4, 47.1, 35.8, 34.1, 13.9.
IR (neat): 1715, 1604, 1526, 1464, 1450, 1409, 1322, 1297, 1260, 1213, 1155, 1055, 985, 804, 758, 737, 702, 621, 531, 501 cm−1.
HRMS (ESI) calculated for C7,H56N4NaO9+[M+Na]+ 1131.3940, found 1131.3949.
To a vial was charged 30.0 mg (0.027 mmol) of 11, 0.1 mL of H2SO4, 4 mL of 1,4-dioxane, and 2 mL of water. The solution was stirred at 80° C. After 24 h, the solution was concentrated in vacuo and purified by column chromatography using ethyl acetate (100%) as the eluent to give 12 in quantitative yield.
Physical Property: White solid, m.p.=188-189° C.
TLC: Rf=0.67 (silica gel, 100% ethyl acetate).
1H NMR (500 MHz, (CD3)2SO) δ 9.83-9.53 (bs, 3H), 8.12-7.94 (bs, 4H), 7.87 (d, 6H, J=7.5 Hz), 7.71 (d, 6H, J=7.5 Hz), 7.39 (t, 6H, J=7.5 Hz), 7.30 (t, 6H, J=7.5 Hz), 7.28-7.07 (m, 6H), 5.36 (s, 1H), 4.39 (d, 6H, J=6.8 Hz), 4.25 (t, 3H, J=6.8 Hz), 3.78-3.68 (m, 2H), 2.78-2.62 (m, 2H).
IR (neat): 3324, 2924, 1719, 1604, 1536, 1464, 1299, 1216, 1052, 985 cm−1.
HRMS (ESI) calculated for C69H51N4O9− [M−H]− 1079.3662, found 1079.3630.
To a SPPS reaction vessel was added 1 eq of 2-chlorotrityl chloride resin (100-200 mesh, substitution: 1.4 mmol/g). The resin was stirred in dry CH2Cl2 for 30 min and the solvent was removed by vacuum. 1.2 eq of 12 dissolved in dimethylformamide:CH2Cl2 (1:5 volume ratio) and 5 eq of N,N-diisopropylethylamine (DIPEA) were added to the resin. After stirring for 10 min, an additional 1.5 eq of DIPEA was added to the resin and stirred overnight (12 hours) at 25° C. HPLC grade methanol was added and stirred for 20 min to cap the remaining reactive functional group on the resin. The solution was removed by vacuum and the resin was washed with CH2Cl2 (1 min, 3 times) and dimethylformamide (1 min, 3 times). 20% (v/v) piperidine in dimethylformamide was added to the resin, stirred for 1 h, and then the solution was drained. The resin was washed with dimethylformamide (1 min, 3 times), CH2Cl2 (1 min, 3 times) and dimethylformamide (1 min, 3 times). 9.5 eq of corresponding Fmoc-protected amino acid (Fmoc-His(trt)-OH, Fmoc-Lys(boc)-OH, or Fmoc-Asn(trt)-OH) was pre-activated with 9 eq of HATU and 18 eq of DIPEA in dimethylformamide. The pre-activated solution was then added to the reaction vessel and stirred for ˜12 hours overnight. The solution was removed by vacuum and the resin was washed with dimethylformamide (1 min, 3 times), CH2Cl2 (1 min, 3 times) and dimethylformamide (1 min, 3 times). 20% (v/v) piperidine in dimethylformamide was added to the resin, stirred for 1 h, and then the solution was drained. The process of washing the resin and the amino acid coupling was repeated until the desired sequence of peptide was achieved. When the peptide coupling is completed, the resin was washed with dimethylformamide (1 min, 4 times), CH2Cl2 (1 min, 4 times). The desired product was cleaved from the resin by treating a mixture of trifluoroacetic acid (TFA), 2,2,2-trifluoroethanol (TFE), and CH2Cl2 (9:1:1 volume ratio) for 30 min twice. For compound 19, cleavage took 12h. The cleavage solution was then collected and concentrated in vacuo. The crude mixture was dissolved in MilliQ water and purified by reverse-phase HPLC. Purified products (17-19) were analyzed by MALDI-MS and analytical reverse-phase HPLC for the purity.
HPLC analysis of compound 5a-5c:
After the nitration reaction of 4, 7, and 8 with nitric acid for 24 h at 80° C., the crude mixture was cooled, neutralized with K2CO3, and re-acidified with 1M HCl. Ethyl acetate was added to the solution and the organic layer was extracted from the solution. The combined organic solution was dried with anhydrous sodium sulfate, and concentrated in vacuo. Crude mixtures were dissolved in acetonitrile. For quantitative analysis, 9,10-diphenylanthracene (internal standard) dissolved in acetonitrile was added to the crude mixture. All samples were then analyzed by reverse-phase HPLC. Solvent gradient method used is shown below. (A: 0.1% CF3CO2H in MilliQ water, B: Methanol).
HPLC analysis of compound 12 and 17-19: the purified samples were dissolved in acetonitrile (for compound 12) or in MilliQ water (for compounds 17-19) and then analyzed by reverse-phase HPLC to confirm the purity of samples. Two different gradients were used as shown below (left: compound 12, right: compound 17-19, A: 0.1% CF3CO2H in MilliQ water, B: Acetonitrile).
MALDI-MS analysis: MALDI-MS data of compounds 12, 17, 18 and 19 are shown in
Fluorescence-quenching experiments were conducted in 50 mM sodium phosphate buffer, pH 7.2. Inhibitor (I10) strand binding curves were obtained by adding 1 μL of increasing concentrations of I10 to 19 μL of 120 nM FQTNR 3WJ. Samples were incubated for 2 hours and ran in triplicate. Inhibitor strand displacement curves with tripycenes were obtained by incubating 14 μL of 120 nM FQ-TNR 3WJ with 1 μL of 150 μM I10 for 2 hours a room temperature. To this complex, 1 μL of increasing concentrations of triptycene was added and incubated for 2 hours. Fluorescence measurements were conducted in a 384-well plate and were recorded with an excitation at 495 nm and emission at 520 nm using 5 nm bandwidths.
Figures, tables and references are as published in Barros et al., Organic Letters, 2016(18):2423, published on May 12, 2016.
Recently, the utility of triptycene as a scaffold for targeting nucleic acid three-way junctions was demonstrated. A rapid, efficient route for the synthesis of bridgehead-substituted triptycenes is reported, in addition to solid-phase diversification to a new class of triptycene peptides. The triptycene peptides were evaluated for binding to a d(CAG)n(CTG) repeat DNA junction exhibiting potent affinities. The bridgehead-substituted triptycenes provide new building blocks for rapid access to diverse triptycene ligands with novel architectures.
Nucleic acid junctions are important structural intermediates in biology. Junctions are present in important biological processes including replication. These junctions also occur in viral genomes in addition to trinucleotide repeat expansions associated with numerous neurodegenerative diseases. These structures are also present in nanostructures and aptamer-based sensors. The ability to selectively modulate a subset of nucleic acid structures using small molecules would allow for the chemical control of cellular processes as well as the reprogramming of cellular events. The ability to differentially stabilize predefined nucleic acid structures or to reprogram and bias the equilibrium distribution of an ensemble of structures in a precise manner could have a profound impact not only in biology but also in nucleic acid nanotechnology and materials applications.
We previously demonstrated that triptycene-based molecules can bind to three-way junctions (3WJs). Additionally, we have shown that these molecules bind to d(CAG)⋅(CTG) repeats implicated in triplet repeat expansion diseases. The ability to synthesize libraries of triptycene derivatives on solid supports will accelerate efforts to identify biologically relevant nucleic acid junction binders and provide further insight into the molecular recognition properties of triptycenes toward diverse junction sequences and topologies. To facilitate solidphase immobilization, a point of attachment on triptycene is required. The bridgehead position provided a strategic location, as it is equidistant from the three amino groups that serve as sites of diversification (
Similar to our previous route, our synthetic plan relied on the reduction of nitrated triptycene, a key intermediate, to install the three key amine functional groups that serve as points of future diversification (
We initiated our synthesis with a Heck reaction between 9-bromoanthracene 1 and methyl acrylate in the presence of palladium (II) acetate, tri-o-tolylphosphine, and triethylamine in a sealed tube. The Heck reaction proceeded cleanly and resulted in the desired product 2 in 84% yield (
Next, olefin 2 was reduced under mild conditions using palladium (II) acetate as the catalyst and potassium formate as the hydrogen source, producing 3 in 85% yield. The key Diels-Alder reaction with anthracene 3 and benzyne, generated in situ from 2-(trimethylsilyl)phenyl trifluoromethanesulfonate and cesium fluoride, proceeded smoothly to yield bridgeheadsubstituted triptycene 4 in 95% yield. Nitration of triptycene resulted in hydrolysis of the bridgehead ester and four major nitrated regioisomers that proved inseparable by standard chromatographic techniques. Esterification of the crude reaction greatly facilitated the separation of the regioisomeric mixture (5a-d) using standard silica gel column chromatography. The nitrated triptycene regioisomers were characterized by HMBC and HSQC. A crystal of triptycene 5d was obtained in chloroform to confirm its structure by X-ray crystallography (
Next, isomer 5d was utilized in subsequent transformations that were described in the previous publication. Pd/C-catalyzed hydrogenation, Fmoc protection, and acid-catalyzed hydrolysis of the ester were performed to yield protected triptycene acid 7 in 78% yield over three steps. A key building block 7 was immobilized on 2-chlorotrityl chloride resin in preparation for solid-phase diversification (
Binding of the amino acid substituted triptycenes was evaluated against a slipped-out d(CAG)n(CTG) repeat nucleic acid junction. Lysine and histidine containing triptycenes were synthesized due to their large presence in nucleic acid-protein interfacial interactions. Among the molecules previously tested, TripNL-(Lys)3 and TripNL-(His)3 exhibited the highest affinity toward the junction. Several dimeric and trimeric amino acid substituents were synthesized for comparison (
In summary, we have developed a shorter, more efficient synthetic strategy toward a bridgehead-substituted triptycene building block. This new synthetic route is improved in terms of solubility, enabling large-scale reactions. Moreover, this route provides an interesting new regioisomer that was not observed through the previous route. A building block with an attachment point at the bridgehead provided rapid access to new triptycene peptide derivatives using solid-phase synthesis methods. The triptycene peptides were evaluated for nucleic acid junction binding to a triplet repeat expansion oligonucleotide using a fluorescence-based assay, which revealed the most potent binder to this junction to date. New triptycene building blocks that are amenable to solid-phase diversification provide a path for the discovery of new junction binders with superior properties. This new class of bridgehead-substituted triptycenes may allow for the generation of one-bead-one-compound combinatorial libraries for the rapid discovery of new junction binders using fluorescently labeled junctions. Additionally, this new class of bridgehead-substituted triptycenes opens the door for the creation of pull-down probes to identify cellular targets in future studies.
General Materials:
All commercial reagents and solvents were used as received. 9-bromoanthracene, potassium formate, nitric acid, Fmoc chloride, pyridine, and acetonitrile were purchased from Sigma-Aldrich (St. Louis, Mo.). Methyl acrylate, triethylamine (Et3N), tri-o-tolylphosphine, palladium(II) acetate, cesium fluoride, and Pd/C were purchased from Acros Organics. Methanol, dichloromethane (DCM), dimethylformamide (DMF) were purchased from Fisher Scientific (Waltham, Mass.). (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxide hexafluorophosphate) (HATU) was purchased from Oakwood Products, Inc. (West Colombia, S.C.), 2-chlorotrityl chloride resin was purchased from Advanced ChemTech (Louisville, Ky.), diisopropylethylamine (DIEA), trifluoroacetic acid (TFA), and 2,2,2-trifluoroethanol (TFE) were purchased from Alfa Aesar (Ward Hill, Mass.), and piperidine was purchased from American Bioanalytical (Natick, Mass.). Chloroform-d, methanol-d4, dimethylsulfoxide-d6 were purchased from Cambridge Isotope Laboratories (Tewksbury, Mass.). Thin-layer chromatography was done using Sorbent Technologies (Norcross, Ga.) silica plates (250 μm thickness). Flash chromatography was performed on a Teledyne Isco (Lincoln, Nebr.) CombiFlash Rf system using RediSep Rf silica columns.
TNR DNA 3WJ (5′-GCGGAGCAGCCCTTGGGCAGCACCTTGGTGCTGCTCCGC-3′) and DNA inhibitor 10 (5′-GCTGCTCCGC-3′) were purchased from Integrated DNA Technologies (IDT). HPLC-purified TNR DNA 3WJ oligo modified with a 5′-FAM and a 3′-IowaBlack was purchased from IDT.
1H and 13C NMR were recorded on a Bruker UNI 500 NMR at 500 and 125 MHz, respectively. High resolution mass spectra were obtained at the University of Pennsylvania Mass Spectrometry Center on a Waters LC-TOF mass spectrometer (model LCT-XE Premier) using electrospray ionization in positive or negative mode, depending on the analyte. High performance liquid chromatography was performed on a JASCO HPLC (Easton, Md.) equipped with a Phenomenx (Torrance, Calif.) column (Analytical: Luna 5μ, C18(2) 100 A; 250×4.60 mm, 5 μm Semi-prep: 5μ C18(2) 100 A; 250×10.00 mm, 5 μm) using aqueous (H2O+0.1% CF3CO2H) and organic (CH3CN) phases. Matrix-assisted laser desorption ionization (MALDI) mass spectra were obtained on a Bruker Ultraflex III MALDI-TOF-TOF mass spectrometer (Billerica, Mass.) using α-cyano-4-hydroxycinnamic acid (CHCA). Fluorescence measurements were collected on a Tecan M1000 plate reader (Mannedorf, Switzerland).
Synthesis:
A solution of 9-bromoanthracene (192 mg, 0.746 mmol), methyl acrylate (642 mg, 7.46 mmol), Et3N (755 mg, 7.46 mmol), tri-o-tolylphosphine (25 mg, 0.082 mmol), and Pd(OAc)2 (8.37 mg, 0.0373 mmol) in DMF (7 mL) was heated at 120° C. in a sealed tube for 5 h. Upon cooling, the mixture was filtered through Celite and washed with ethyl acetate. The filtrate was extracted with ethyl acetate and water several times. Combined organic layers were then dried over Na2SO4. The crude mixture was purified by column chromatography on silica gel (5% EtOAc/hexanes) to give 1 (164 mg, 84%). 1H NMR (500 MHz, CDCl3) δ 8.65 (d, 1H, J=16.3 Hz), 8.45 (s, 1H), 8.25-8.23 (m, 2H), 8.03-8.01 (m, 2H), 7.53-7.48 (m, 4H), 6.45 (d, 1H, J=16.3 Hz), 3.93 (s, 3H);
13C NMR (125 MHz, CDCl3) δ 167.0, 142.4, 131.4, 129.5, 129.4, 129.0, 128.4, 126.9, 126.5, 125.5, 125.3, 52.1; IR (neat) 3051, 2949, 1719, 1635, 1435, 1265, 1170, 988, 886, 733; HRMS m/z calcd for CBH15O2+ [M+H]+ 263.1067, observed 263.1074.
1-3 To a solution of 2 (102 mg, 0.389 mmol) in DMF (5 mL) was added potassium formate (654 mg, 7.78 mmol) and Pd(OAc)2 (4.4 mg, 0.02 mmol) and stirred at 60° C. for 4 h. After cooling, the mixture was filtered through Celite and washed with ethyl acetate. The filtrate was extracted with ethyl acetate and water. The combined organic layer was washed with water and brine, then dried over Na2SO4. The crude mixture was purified by column chromatography on silica gel (5% EtOAc/hexanes) to yield 3 (87.3 mg, 85%). 1H NMR (500 MHz, CDCl3) δ 8.38 (s, 1H), 8.28 (dd, 2H, J=8.8, 0.6 Hz), 8.02 (dd, 2H, J=8.4, 0.5), 7.56-7.53 (m, 2H), 7.50-7.46 (m, 2H), 4.00-3.96 (m, 2H), 3.75 (s, 3H), 2.82-2.79 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 173.6, 132.4, 131.7, 129.6, 129.6, 126.5, 126.1, 125.1, 124.0, 52.0, 35.2, 23.4; IR (neat) 3053, 2950, 1734, 1436, 1174, 885, 732; HRMS m/z calcd for C18H17O2+ [M+H]+ 265.1223, observed 265.1226.
4 To a solution of 3 (443 mg, 1.68 mmol) in acetonitrile (2.8 mL) was added CsF (764 mg, 5.03 mmol) and 2-(trimethylsilyl)phenyl trifluoromethanesulfonate (1.0 g, 3.35 mmol) and stirred at 80° C. for 4 h. Upon cooling, saturated NH4Cl solution was added to the mixture and then extracted with dichloromethane. The combined organic layer was washed with brine and dried over Na5SO4. The crude mixture was purified by column chromatography on silica gel (5-10% EtOAc/hexanes) to yield 4 (542 mg, 95%). 1H NMR (500 MHz, CDCl3) δ 7.40-7.30 (m, 6H), 7.04-6.95 (m, 6H), 5.34 (s, 1H), 3.85 (s, 3H), 3.36-3.31 (m, 2H), 3.21-3.15 (m, 2H);
13C NMR (125 MHz, CDCl3) δ 174.7, 147.0, 125.2, 125.1, 123.8, 122.1, 54.6, 53.4, 52.2, 30.7, 22.7; IR (neat) 2952, 1733, 1450, 1176, 628; HRMS m/z calcd for C24H20NaO2+ [M+Na]+ 363.1356, observed 363.1369.
A solution of 4 (424.5 mg, 1.25 mmol) in concentrated HNO3 (15 mL) was stirred at 75° C. overnight. The solution was cooled to room temperature, neutralized, and extracted with EtOAc. The organic layers were combined, washed with brine, and dried over Na2SO4. The crude mixture was then reesterified by stirring in methanol (50 mL) with catalytic H2SO4 under reflux overnight. The solution was concentrated under vacuum. Water was added and then basified by the addition of 1M NaOH. The water was extracted with EtOAc immediately. The organic layer was washed with brine and dried over Na2SO4. The crude mixture was purified by column chromatography on silica gel (30% EtOAc/hexanes) to give 5a (110 mg, 19%), 5b (130 mg, 22%), 5c (37.2 mg, 6.3%), and 5d (88.3 mg, 15%). 5a 1H NMR (500 MHz, CDCl3) δ 8.32 (d, 2H, J=1.3 Hz), 8.28 (s, 1H), 8.01 (dd, 3H, J=8.3, 2.2 Hz), 7.69 (d, 1H, J=8.1 Hz), 7.62 (d, 2H, J=8.4 Hz), 5.87 (s, 1H), 3.91 (s, 3H), 3.52 (t, 2H, J=7.4 Hz), 3.17 (t, 2H, J=7.4 Hz); 13C NMR (125 MHz, CDCl3) δ 173.4, 151.3, 150.2, 146.2, 146.1, 145.8, 145.0, 125.2, 123.6, 122.5, 122.1, 119.4, 118.3, 54.8, 53.3, 52.7, 30.2, 22.0; IR (neat) 2953, 1734, 1523, 1344, 1201, 738; HRMS m/z calcd for C24H18N3NaO8+ [M+Na]+ 498.0908, observed 498.0919; mp 143-146° C.; 5b 1H NMR (500 MHz, CDCl3) δ 8.32-8.27 (m, 3H), 8.04-7.97 (m, 3H), 7.69 (d, 2H, J=8.1 Hz), 7.64 (d, 1H, J=8.4 Hz), 5.88 (s, 1H), 3.92 (s, 3H), 3.56 (t, 2H, J=7.3 Hz), 3.20 (t, 2H, J=7.3 Hz); 13C NMR (125 MHz, CDCl3) δ 173.4, 150.9, 150.5, 146.2, 145.8, 145.7, 145.3 125.2, 123.6, 122.4, 122.1, 119.4, 118.1, 54.6, 53.4, 52.7, 30.2, 21.8; IR (neat) 3091, 2953, 2848, 1733, 1520, 1342, 1201, 736; HRMS m/z calcd for C24H18N3NaO8+ [M+Na]+ 498.0908, observed 498.0919; mp 139-142° C.; 5c 1H NMR (500 MHz, CDCl3) δ 8.32 (d, 3H, J=2.2 Hz), 8.03 (dd, 3H, J=8.4, 2.2 Hz), 7.60 (d, 3H, J=8.4 Hz), 5.79 (s, 1H), 3.92 (s, 3H), 3.48 (t, 2H, J=7.6 Hz), 3.13 (t, 2H, J=7.6 Hz); 13C NMR (125 MHz, CDCl3) δ 173.4, 149.9, 146.7, 146.4, 123.7, 122.2, 119.5, 55.2, 53.6, 52.7, 30.5, 22.6; HRMS m/z calcd for C24H16N3O8 474.0943, observed 474.0931; 5d 1H NMR (500 MHz, CDCl3) δ 8.31 (d, 3H, J=1.7 Hz), 8.04 (dd, 3H, J=8.1, 1.8 Hz), 7.63 (d, 3H, J=8.1 Hz), 5.78 (s, 1H), 3.96 (s, 3H), 3.57 (t, 2H, J=7.3 Hz), 3.20 (t, 2H, J=7.3 Hz); 13C NMR (125 MHz, CDCl3) δ 173.4, 150.5, 146.4, 125.2, 122.5, 118.3, 54.6, 53.8, 52.9, 30.3, 21.8; IR (neat) 3093, 2954, 2851, 1736, 1525, 1453, 1343, 1202, 1076, 903, 823; HRMS m/z calcd for C24H18N3NaO8+ [M+Na]+ 498.0908, observed [M+Na]+ 498.0910; mp 147-150° C.
To a solution of 5d (259 mg, 0.544 mmol) in methanol was added Pd/C (25 mg). The solution was purged with a H2 gas balloon and kept under H2 gas for 1 h. The mixture was filtered through Celite and washed with methanol. The filtrate was concentrated and purified by column chromatography on silica gel (5% MeOH/DCM) to give 6 (204 mg, 97%). 1H NMR (500 MHz, MeOD) δ 7.01 (d, 3H, J=7.7 Hz), 6.79 (d, 3H, J=1.7 Hz), 6.33 (dd, 3H, J=7.7, 1.5 Hz), 4.98 (s, 1H), 3.85 (s, 3H), 3.18-3.13 (m, 4H); 13C NMR (125 MHz, MeOD) δ 176.5, 144.6, 140.7, 124.1, 112.6, 112.2, 54.2, 53.3, 52.5, 31.4, 23.8; IR (neat) 3354, 2951, 1724, 1605, 1473, 1326, 1181, 582 HRMS m/z calcd for C24H24N3O2+ [M+H]+ 386.1863, observed 386.1848.
To a solution of 6 (116 mg, 0.301 mmol) in DCM (2.5 mL) was added excess pyridine. The solution was cooled to 0° C., then added Fmoc chloride in DCM (2.5 mL) slowly. The solution was allowed to warm to room temperature over time and stirred overnight. The mixture was extracted with DCM and acidic water. The organic layer was washed with brine and dried over MgSO4. The crude mixture was purified by column chromatography on silica gel (30% EtOAc/hexanes). A solution of the ester (209 mg, 0.199 mmol) in dioxane (5 mL), H2O (5 mL), and catalytic H2SO4 was stirred at 80° C. overnight. The reaction mixture was neutralized and concentrated under vacuum. Water was added to the mixture and was extracted with DCM. The organic layer was washed with brine and dried over Na2SO4. The crude mixture was purified by column chromatography on silica gel (50% EtOAc/hexanes) to yield 7 (165 mg, 80%). 1H NMR (500 MHz, DMSO-d6) δ 12.44 (bs, 1H), 9.58 (s, 3H), 7.89 (d, 6H, J=7.6 Hz), 7.72 (d, 6H, J=7.0 Hz), 7.50 (s, 3H), 7.40 (t, 6H, J=7.4 Hz), 7.36-7.22 (m, 9H), 7.14 (bs, 3H), 5.37 (s, 1H), 4.44 (d, 6H, J=6.7 Hz), 4.28 (t, 3H, J=6.7 Hz), 3.13-2.92 (m, 4H); 13C NMR (125 MHz, DMSO) δ 174.2, 153.4, 143.7, 141.3, 140.7, 135.7, 127.6, 127.1, 125.1, 123.2, 120.1, 114.4, 113.5, 65.5, 52.2, 50.9, 46.6, 29.2, 22.2 IR (neat) 3375, 3325, 3075, 2950, 1709, 1605, 1528, 1450, 1212, 738; HRMS m/z calcd for C69H52N3O8+ [M−H]+ 1038.3749, observed 1038.3737; mp 169-171° C.
Solid Phase Synthesis:
All triptycenes were synthesized on 2-chlorotrityl chloride resin (100-200 mesh, 1.5 mmol substitution/g). The resin was added to a dry glass reaction vessel and swollen by stirring in dichloromethane (DCM) for 30 min. After swelling, the DCM was removed by vacuum and Fmoc-Trip-OH (8d) was coupled to the resin. Fmoc-Trip-OH (1.5 equiv) in 1:5 DMF:DCM and DIEA (5 equiv) were added and stirred for 5 min. DIEA (1.5 equiv) was added and the resin was stirred overnight. The solution was then drained by vacuum and the resin was washed thoroughly with DMF, then DCM, then DMF. The beads were deprotected by treatment with 20% piperidine in DMF for 1 h with stirring. The deprotection solution was removed by vacuum and the resin was washed thoroughly with DMF, DCM, then DMF. The first Fmoc-protected amino acid was then activated with HATU (9 equiv) in the presence of DIEA (18 equiv) prior to addition to the reaction vessel and allowed to couple overnight. Subsequent deprotections and amino acid couplings were run as described above. Before cleavage from the resin, the terminal Fmoc was removed. The beads were thoroughly washed with DMF then DCM. Peptides were cleaved by addition of trifluoroacetic acid (TFA), 2,2,2-trifluoroethanol (TFE), and DCM (9:1:1). The cleavage solution was collected by vacuum and concentrated using a rotary evaporator. The crude residue was diluted in 1:1 (0.1% TFA/H2O:MeCN), purified by reverse-phase HPLC, and analyzed by MALDI-MS.
Fluorescence Quenching Assay: All experiments were conducted in 50 mM sodium phosphate buffer, pH 7.2. Fluorescence measurements were recorded with excitation at 495 nm and emission at 520 nm using 5 nm bandwidths on a Tecan M1000 plate reader. Inhibitor strand displacement by triptycene curves were obtained by incubating 120 nM TNR DNA with 10 μM inhibitor 10 for 2h, followed by addition of increasing concentrations of triptycenes. Samples were incubated for 2h and measured in triplicate in a 384-well plate.
Gel Shift Assay: Gel shift experiments were conducted in 50 mM sodium phosphate buffer, pH 7.2. Triptycene titration gels were prepared by incubating TNR 3WJ (0.5 μM) with inhibitor strand 10 (1.5 μM) for 2 h followed by titration of triptycenes and incubation at room temperature for 2h. Samples were loaded on a 20% non-denaturing polyacrylamide gel (19:1 monomer:bis) at 50V in 1×TBR buffer at 4° C. for 10 h. Gels were imaged by staining with SYBR Gold for 15 min then visualized using a BioRad GelDoc XR+ imager.
The present application claims benefit of priority to US Provisional Patent Application No. 62/232,324, filed Sep. 24, 2015, which is incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2016/053801 | 9/26/2016 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62232324 | Sep 2015 | US |
