WO2025188771A1 - Surface patterning with sequence defined, self sequencing oligourethanes - Google Patents

Surface patterning with sequence defined, self sequencing oligourethanes

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Publication number
WO2025188771A1
WO2025188771A1 PCT/US2025/018365 US2025018365W WO2025188771A1 WO 2025188771 A1 WO2025188771 A1 WO 2025188771A1 US 2025018365 W US2025018365 W US 2025018365W WO 2025188771 A1 WO2025188771 A1 WO 2025188771A1
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WO
WIPO (PCT)
Prior art keywords
group
monomer unit
groups
substituted version
monomer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/018365
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French (fr)
Other versions
WO2025188771A8 (en
Inventor
Hannah MCLAIN
Eric V. Anslyn
Andrew D. Ellington
Hazel FARGHER
Heather GRAHAM
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Texas System
National Aeronautics and Space Administration NASA
University of Texas at Austin
Original Assignee
University of Texas System
National Aeronautics and Space Administration NASA
University of Texas at Austin
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Publication of WO2025188771A1 publication Critical patent/WO2025188771A1/en
Publication of WO2025188771A8 publication Critical patent/WO2025188771A8/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G71/00—Macromolecular compounds obtained by reactions forming a ureide or urethane link, otherwise, than from isocyanate radicals in the main chain of the macromolecule
    • C08G71/04—Polyurethanes
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/14—Libraries containing macromolecular compounds and not covered by groups C40B40/06 - C40B40/12
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/08—Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support
    • C40B50/10—Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support involving encoding steps
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/89—Inverse chromatography

Definitions

  • the present invention relates generally to the field of analytical chemistry. More particularly, it concerns methods and compositions for detecting and/or identifying an analyte.
  • the chemical senses, taste and smell are akin to using supramolecular receptors for binding and eliciting responses, which are typically detected with electrochemistry or spectroscopy, such as with electronic noses and tongues (Rabehi et al., 2024; Li et al., 2023; Ye et al., 2021; Li et al., 2019; Geng et al., 2019; Podrazka et al., 2017; Tahara et al., 2013; Albert et al., 2000).
  • These mammalian senses do not rely on a single receptor for each type of chemical that to be identified (Gravina et al., 2013; Buck et al., 2004).
  • Human noses and tongues contain hundreds of receptors which bind in a cross-reactive manner to odorant and tastant molecules. Over lifetimes, brains are trained to interpret the combination of receptor responses as a specific smell and taste.
  • the sense of touch operates via mechanosensors in our skin which detect sensations such as pressure, vibrations, as well as skin indentation, stretch, and slip (Johansson et al., 2009; Johansson et al., 1983).
  • tactile sensor arrays have been used to detect pressure, strain, and temperature, with applications in electronic skin (Lee et al., 2020) and assessing fruit ripeness (Zhang et al., 2024).
  • the human sense of touch can differentiate surfaces not only by their bulk physical properties, but also by their surface chemistry (Carpenter et al., 2018).
  • the present disclosure provides methods of identifying a surface, such as a surface of a sample, by contacting the surface with a chemical probe comprising at least one oligourethane, preferably a pool or pools of oligourethanes.
  • a chemical probe comprising at least one oligourethane, preferably a pool or pools of oligourethanes.
  • the interaction between the surface and the chemical probe allows the skilled artisan to recognize the surface.
  • the chemical probes of the present are adaptable to incorporate a range of functionality, which aids in accessing distinguishable patterns of interactions between surface and chemical probe.
  • the present oligourethane chemical probes are more easily characterizable than other polymers that could be useful in such methods, such as peptides.
  • the present methods provide a benefit in that the chemical probes can be identified at a picomolar concentration, and as such only small quantities of probes are required.
  • the present disclosure provides methods of determining the identity of a surface comprising:
  • the oligourethane is a polymer comprising at least two monomer units of the formula R O , wherein R is an independently selected chemical group.
  • the oligourethane is a polymer comprising at least two unique monomer units of the formula R O , wherein R is an independently selected chemical group. [0011] In some embodiments, the oligourethane comprises from about three to about 50 monomer units. In further embodiments, the oligourethane comprises from about 5 urethane moieties to about 15 monomer units. In still further embodiments, the oligourethane comprises about 10 monomer units.
  • the chemical group comprises an amino group, an alcohol group, a C1-C12 alkyl group, a C1-C12 cycloalkyl group, a C1-C12 heterocycloalkyl group, a C2-C12 alkenyl group, a C2-C12 alkynyl group, a C1-C12 aryl group, a C1-C12 aralkyl group, a C1-C12 heteroaryl group, a C1-C12 heteroaralkyl group, a C1-C12 alkylamino group, a C2-C24 dialkylamino group, a guanidyl group, a hydroxide group, a C1-C12 alkoxide group, a C1-C12 cycloalkoxide group, a mercaptate group, a sulfate group, a phosphate group, or a C1-C12 carboxylate group, or a substituted
  • At least one of the monomer units has an R group that comprises an aromatic group. In some embodiments, at least one of the monomer units has an R group that is an aromatic group. In some embodiments, at least one of the monomer units has an R group selected from among a C1-C12 aryl group, a C1-C12 heteroaryl group, a Cl- C12 aralkyl group, a C1-C12 heteroaralkyl group, or a substituted version of any of these groups.
  • At least one of the monomer units has an R group that comprises an aliphatic group. In some embodiments, at least one of the monomer units has an R group that is an aliphatic group. In further embodiments, at least one of the monomer units has an R group selected from among a C1-C12 alkyl group, a C1-C12 cycloalkyl group, a Cl- C12 heterocycloalkyl group, or a substituted version of either of these groups.
  • At least one of the monomer units has an R group that comprises an unsaturated group. In some embodiments, at least one of the monomer units has an R group that is an unsaturated group. In some embodiments, at least one of the monomer units has an R group selected from among a C2-C12 alkenyl group, a C2-C12 alkynyl group, or a substituted version of either of these groups.
  • At least one of the monomer units has an R group comprising a positive charge. In some embodiments, at least one of the monomer units has an R group comprising a protonated amino group, a protonated C1-C12 alkylamino group, a protonated C2-C24 dialkylamino group, a protonated guanidyl group, or a substituted version of any of these groups.
  • At least one of the monomer units has an R group comprising a negative charge. In some embodiments, at least one of the monomer units has an R group comprising a hydroxide group, a C1-C12 alkoxide group, a C1-C12 cycloalkoxide group, a mercaptate group, a sulfate group, a phosphate group, or a C1-C12 carboxylate group, a substituted version of any of these groups.
  • At least one of the monomer units has an R group comprising a protecting group.
  • identifying the chemical probe comprises degrading the chemical probe to obtain a degraded solution.
  • the degraded solution comprises at least one oxazolidinone.
  • the method further comprises identifying an oxazolidinone in the degraded solution or the composition of oxazolidinone in the degraded solution.
  • an oxazolidinone in the degraded solution or the composition of oxazolidinone in the degraded solution is identified by spectrometry.
  • the spectrometry is mass spectrometry (MS). In some embodiments, the mass spectrometry is a liquid chromatography mass spectrometry (LC-MS). In some embodiments, the identity of the chemical probe or the retention time of the chemical probe is correlated with the identity of the surface through a microprocessor.
  • the identity of the chemical probe to the identity of the surface comprises correlating the identity of an oxazolidinone in the degraded solution or the composition of oxazolidinone in the degraded solution to the identity of the surface.
  • the identity of the oxazolidinone in the degraded solution or the composition of oxazolidinone in the degraded solution is correlated with the identity of the surface through a microprocessor.
  • the surface is a soil sample.
  • the surface comprises a protein.
  • the surface comprises an enzyme.
  • the surface comprises an antibody.
  • the surface comprises a cell.
  • the surface comprises a virus.
  • the surface comprises a biopolymer. In some embodiments, the surface comprises a polymer. In some embodiments, the surface comprises an organism. In some embodiments, the surface comprises rock. In some embodiments, the surface comprises material. In some embodiments, the surface comprises a mixture of any of the surface alternatives provided above.
  • the present disclosure provides methods of synthesizing a library of oligourethanes for identifying or detecting a surface, wherein the method comprises:
  • R is a chemical group
  • Ri and R2 are each independently hydrogen or a protecting group, or Ri and R2 are taken together and are a protecting group;
  • Rs is an activating group; wherein the unique monomer units in the coupling mixture do not have the same R group;
  • the coupling reaction mixture comprises between 2 and 50 unique monomer units. In some embodiments, the coupling reaction mixture comprises between 5 and 15 unique monomer units. In some embodiments, the coupling reaction mixture comprises 10 unique monomer units. In some embodiments, the library comprises between 5 and 20 pools of oligourethanes. In some embodiments, the library comprises about 16 pools of oligourethanes. In some embodiments, the coupling mixture for each pool of oligourethanes has a different molar ratio of monomer units.
  • At least one of the coupling mixtures comprises at least a first unique monomer unit at a higher concentration than at least a second unique monomer unit. In some embodiments, between 5 and 20 of the coupling mixtures comprise at least a first unique monomer unit at a higher concentration than at least a second unique monomer unit. In some embodiments, 15 of the coupling mixtures comprise at least a first unique monomer unit at a higher concentration than at least a second unique monomer unit. In some embodiments, the first unique monomer unit has a concentration between about 2 and about 30 times higher than concentration of the second unique monomer unit. In some embodiments, the first unique monomer unit has a concentration between about 4 and about 30 times higher than the concentration of the second unique monomer unit. In some embodiments, the first unique monomer unit has a concentration about 4, about 5, about 6, or about 7 times higher than the concentration of the second unique monomer unit.
  • the R group of the first monomer unit is selected from among optionally protonated -Nth, -OH, a C1-C12 alkyl group, a C1-C12 cycloalkyl group, a C1-C12 heterocycloalkyl group, a C1-C12 alkoxy group, a C2-C12 alkenyl group, a C2-C12 alkynyl group, a C1-C12 acyl group, a C1-C12 aryl group, a C1-C12 aralkyl group, a C1-C12 heteroaryl group, a C1-C12 heteroaralkyl group, an optionally protonated C1-C12 alkylamino group, an optionally protonated C2-C24 dialkylamino group, an optionally protonated guanidyl group, a hydroxide group, a Cl -Cl 2 alkoxide group, a Cl -C
  • the R group of the first monomer unit is selected from among a C1-C12 aryl group, a C1-C12 heteroaryl group, a C1-C12 aralkyl group, a C1-C12 heteroaralkyl group, or a substituted version of any of these groups.
  • the R group of the first monomer unit is selected from among a C1-C12 alkyl group, a C1-C12 cycloalkyl group, a C1-C12 heterocycloalkyl group, or a substituted version of either of these groups. In some embodiments, the R group of the first monomer unit is selected from among a C2-C12 alkenyl group, a C2-C12 alkynyl group, or a substituted version of either of these groups. In some embodiments, the R group of the first monomer unit comprises a positive charge.
  • the R group of the first monomer unit comprises an optionally protonated amino group, an optionally protonated Cl- C12 alkylamino group, an optionally protonated C2-C24 dialkylamino group, an optionally protonated guanidyl group, or a substituted version of any of these groups. In some embodiments, the R group of the first monomer unit comprises a negative charge.
  • the R group of the first monomer unit comprises a hydroxide group, a C1-C12 alkoxide group, a C1-C12 cycloalkoxide group, a mercaptate group, a sulfate group, a sulfoxide group, a phosphate group, or a carboxylate group, a substituted version of any of these groups.
  • the R group of a monomer unit comprises a protecting group.
  • At least one of the coupling mixtures comprises at least a first monomer unit and a third monomer unit at a higher concentration than at least a second monomer unit.
  • between 5 and 20 of the coupling mixtures comprise at least a first monomer unit and a third monomer unit at a higher concentration than at least a second monomer unit.
  • 15 of the coupling mixtures of the pools of oligourethanes of the chemical probe comprises at least a first monomer unit and a third monomer unit at a higher concentration than at least a second monomer unit.
  • the R group of the third monomer unit is selected from among optionally protonated -NH2, -OH, a C1-C12 alkyl group, a C1-C12 cycloalkyl group, a C1-C12 heterocycloalkyl group, a C1-C12 alkoxy group, a C2-C12 alkenyl group, a C2-C12 alkynyl group, a C1 -C12 acyl group, a C1-C12 aryl group, a C1-C12 aralkyl group, a C1 -C12 heteroaryl group, a C1-C12 heteroaralkyl group, an optionally protonated C1-C12 alkylamino group, an optionally protonated C2-C24 dialkylamino group, an optionally protonated guanidyl group, a hydroxide group, a Cl -Cl 2 alkoxide group, a Cl -
  • the R group of the third monomer unit is selected from among a C1-C12 aryl group, a C1-C12 heteroaryl group, a C1-C12 aralkyl group, a C1-C12 heteroaralkyl group, or a substituted version of any of these groups.
  • the R group of the third monomer unit is selected from among a C1-C12 alkyl group, a C1-C12 cycloalkyl group, a C1-C12 heterocycloalkyl group, or a substituted version of either of these groups.
  • the R group of the third monomer unit is selected from among a C2-C12 alkenyl group, a C2-C12 alkynyl group, or a substituted version of either of these groups.
  • the R group of the third monomer unit comprises a positive charge.
  • the R group of the third monomer unit comprises a protonated amino group, a protonated C1-C12 alkylamino group, a protonated C2-C24 dialkylamino group, a protonated guanidyl group, or a substituted version of any of these groups.
  • the R group of the third monomer unit comprises a negative charge.
  • the R group of the third monomer unit comprises a hydroxide group, a Cl -Cl 2 alkoxide group, a Cl -Cl 2 cycloalkoxide group, a mercaptate group, a sulfate group, a phosphate group, or a carboxylate group, a substituted version of any of these groups.
  • the R group of a monomer unit comprises a protecting group.
  • the R group of the first monomer unit comprises an aliphatic group; and the R group of the third monomer unit comprises an aromatic group.
  • the R group of the first monomer unit is selected from among a C1-C12 alkyl group, a C1-C12 cycloalkyl group, a C1-C12 heterocycloalkyl group, or a substituted version of either of these groups; and the R group of the third monomer unit is selected from among a C1-C12 aryl group, a C1-C12 heteroaryl group, a C1-C12 aralkyl group, a C1-C12 heteroaralkyl group, or a substituted version of any of these groups.
  • the R group of the first monomer unit comprises an aliphatic group; and the R group of the third monomer unit comprises an unsaturated group.
  • the R group of the first monomer unit is selected from among a Cl -Cl 2 alkyl group, a C1-C12 cycloalkyl group, a C1-C12 heterocycloalkyl group, or a substituted version of either of these groups; and the R group of the third monomer unit is selected from among a C2-C12 alkenyl group, a C2-C12 alkynyl group, or a substituted version of either of these groups.
  • the R group of the first monomer unit comprises an aliphatic group; and the R group of the third monomer unit comprises a positive charge.
  • the R group of the first monomer unit is selected from among a Cl -Cl 2 alkyl group, a C1-C12 cycloalkyl group, a C1-C12 heterocycloalkyl group, or a substituted version of either of these groups; and the R group of the third monomer unit is selected from among a protonated amino group, a protonated C1-C12 alkylamino group, a protonated C2- C24 dialkylamino group, a protonated guanidyl group, or a substituted version of any of these groups.
  • the R group of the first monomer unit comprises an aliphatic group; and the R group of the third monomer unit comprises a negative charge.
  • the R group of the first monomer unit is selected from among a Cl -Cl 2 alkyl group, a C1-C12 cycloalkyl group, a C1-C12 heterocycloalkyl group, or a substituted version of either of these groups; and the R group of the third monomer unit is selected from among hydroxide group, a C1-C12 alkoxide group, a C1-C12 cycloalkoxide group, a mercaptate group, a sulfate group, a phosphate group, or a carboxylate group, a substituted version of any of these groups.
  • the R group of the first monomer unit comprises an aromatic group; and the R group of the third monomer unit comprises an unsaturated group.
  • the R group of the first monomer unit is selected from among a C1 -C12 aryl group, a C1-C12 heteroaryl group, a C1-C12 aralkyl group, a C1-C12 heteroaralkyl group, or a substituted version of any of these groups; and the R group of the third monomer unit is selected from among aC2-C12 alkenyl group, a C2-C12 alkynyl group, or a substituted version of either of these groups.
  • the R group of the first monomer unit comprises an aromatic group; and the R group of the third monomer unit comprises a positive charge.
  • the R group of the first monomer unit is selected from among a Cl -Cl 2 aryl group, a C1-C12 heteroaryl group, a C1-C12 aralkyl group, a C1-C12 heteroaralkyl group, or a substituted version of any of these groups; and the R group of the third monomer unit is selected from among a protonated amino group, a protonated C1-C12 alkylamino group, a protonated C2-C24 dialkylamino group, a protonated guanidyl group, or a substituted version of any of these groups.
  • the R group of the first monomer unit comprises an aromatic group; and the R group of the third monomer unit comprises a negative charge.
  • the R group of the first monomer unit is selected from among a Cl -Cl 2 aryl group, a C1-C12 heteroaryl group, a C1-C12 aralkyl group, a C1-C12 heteroaralkyl group, or a substituted version of any of these groups; and the R group of the third monomer unit is selected from among hydroxide group, a C1-C12 alkoxide group, a C1-C12 cycloalkoxide group, a mercaptate group, a sulfate group, a phosphate group, or a carboxylate group, a substituted version of any of these groups.
  • the R group of the first monomer unit comprises an unsaturated group; and the R group of the third monomer unit comprises a positive charge.
  • the R group of the first monomer unit is selected from among a C2-C12 alkenyl group, a C2-C12 alkynyl group, or a substituted version of either of these groups; and the R group of the third monomer unit is selected from among a protonated amino group, a protonated C1-C12 alkylamino group, a protonated C2-C24 dialkylamino group, a protonated guanidyl group, or a substituted version of any of these groups.
  • the R group of the first monomer unit comprises an unsaturated group; and the R group of the third monomer unit comprises a negative charge.
  • the R group of the first monomer unit is selected from among a C2-C12 alkenyl group, a C2-C12 alkynyl group, or a substituted version of either of these groups; and the R group of the third monomer unit is selected from among hydroxide group, a C1-C12 alkoxide group, a C1-C12 cycloalkoxide group, a mercaptate group, a sulfate group, a phosphate group, or a carboxylate group, a substituted version of any of these groups
  • the R group of the first monomer unit comprises a positive charge; and the R group of the third monomer unit comprises a negative charge.
  • the R group of the first monomer unit is selected from among a protonated amino group, a protonated C1-C12 alkylamino group, a protonated C2-C24 dialkylamino group, a protonated guanidyl group, or a substituted version of any of these groups; and the R group of the third monomer unit is selected from among hydroxide group, a Cl -Cl 2 alkoxide group, a C1-C12 cycloalkoxide group, a mercaptate group, a sulfate group, a phosphate group, or a carboxylate group, a substituted version of any of these groups.
  • the total concentration of between about 10% and about 60% of the unique monomer units in the coupling mixture is about 2, about 3, about 4, about 5, about 6, about 7, or about 8 times higher than the total concentration of the other unique monomer units in the coupling mixture.
  • the sum of the concentrations of about 30% of the unique monomer units in the coupling mixture is about 2, about 3, about 4, about 5, about 6, about 7, or about 8 times higher than the total concentration of the other unique monomer units in the coupling mixture.
  • the present disclosure provides biased libraries of oligourethanes for detecting or identifying a surface, wherein the biased library comprises at least one pool of oligourethanes synthesized according to any one of the embodiments disclosed above.
  • the present disclosure provides methods of determining the identity of a surface comprising:
  • the chemical probe comprises from about three to about 20 unique oligourethanes. In further embodiments, the chemical probe comprises from about five to about 15 unique oligourethanes. In some embodiments, the unique oligourethane comprises a polyurethane, wherein the polyurethane comprises from about three to about 50 repeating units. In further embodiments, the polyurethane comprises from about 5 repeating units to about 15 repeating units.
  • the method further comprises degrading the oligourethane to obtain an oxazolidinone.
  • the oxazolidinone is identified by spectrometry.
  • the spectrometry is mass spectrometry (MS).
  • the mass spectrometry is a liquid chromatography mass spectrometry (LC-MS).
  • the oligourethane comprises a chemical group attached to a urethane moiety.
  • each oligourethane comprises a distinct chemical group.
  • the chemical group is a C1-C12 alkyl, a C1-C12 cycloalkyl group, a C1-C12 alkenyl, a C1-C12 alkynyl, a C1-C12 aryl, a C1-C12 aralkyl, a C1-C12 heteroaryl, a C1-C12 heteroaralkyl, or a substituted version of any of these groups, wherein the substituted version includes a polar functional group, a halide, a hydrogen bond donating group, a hydrogen bond accepting group, an acid group, or a basic group.
  • the oligourethane comprises a single repeating unit in each oligourethane. In some embodiments, the oligourethane comprises two or more repeating units in each oligourethane. In some embodiments, the oligourethane comprises a distinct sequence of different repeating units. In some embodiments, the identity of the chemical probe or the
  • the retention time of the chemical probe is correlated with the identity of the surface through a microprocessor.
  • the surface is a soil sample.
  • the present disclosure provides methods of determining the presence of a chemical analyte in a sample comprising:
  • the present disclosure provides methods of determining the identity of a substrate in a sample or the identity of the sample comprising:
  • the present disclosure provides methods of determining the identity of a substrate in a sample or the identity of the sample comprising:
  • essentially free in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts.
  • the total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%.
  • Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.
  • FIGS. 1A-1E illustrate the design of illustrative oligourethane libraries useful with the methods disclosed herein and the workflow of the experiments described in the Examples section.
  • FIG. 1C Biased monomer coupling mixtures were used in solid-phase OU synthesis to make 16 libraries of OUs that comprised a statistically random sequences of monomers, but which were biased towards specific monomers and/or classes of monomers as outlined in FIG. 1A-1B.
  • FIG. ID After cleavage from the solid support, all libraries were combined to create a pool of OUs with 108 possible different sequences.
  • FIG. IE A schematic of the experimental workflow.
  • oligourethane probes were sent through a column packed with an analyte (or stationary phase) of interest. Eluting OUs were separated into 1 mL fractions and then completely degraded to 5-membered oxazolidinone rings. The identity and relative amount of oxazolidinones in each fraction was determined by LC-MS EIC.
  • FIG. 2 is a bar chart showing the relative composition of monomers in the coupling mixtures for each of the 16 biased libraries.
  • FIGS. 3A-3D show the heat maps of the oxazolidinone LC-MS EIC peak areas vs. fraction volume and demonstrate the effect of each normalizing operation on fraction component evaluation.
  • FIG. 3A Raw oxazolidinone LC-MS EIC peak area values.
  • FIG. 3B LC-MS EIC peak area values which have been divided by the value for VaLoxazolidinone in each fraction.
  • FIG. 3C Internal standard adjusted values are min-max adjusted for each oxazolidnone, for a better comparison across all oxazolidinones.
  • FIG. 3A Raw oxazolidinone LC-MS EIC peak area values.
  • FIG. 3B LC-MS EIC peak area values which have been divided by the value for VaLoxazolidinone in each fraction.
  • FIG. 3C Internal standard adjusted values are min-max adjusted for each oxazolidnone, for a better comparison across all oxazolidinones.
  • FIG. 3A
  • FIG. 4 shows heat maps of standard adjusted oxazolidinone EIC peak areas for all 32 fractions and all five substrates.
  • FIG. 5 shows heat maps of Phe-oxazolidinone and min-max adjusted oxazolidinone EIC peak areas across all fractions, oxazolidinones, and column substrates.
  • FIG. 6 is a PCA plot using the min-max standard adjusted data, which shows poor column substrate differentiation.
  • FIG. 7 shows standard normalized oxazolidinone EIC peak areas for fractions 5, 7, 10, 15, and 20 for all five replicates and all five substrates. See Example 4 section xviii. When used with PCA, these data points gave overlapping clusters with large scatter (FIG. 6).
  • FIG. 8 shows the results of PCA using the min-max Phe-ox adjusted data shows clear clustering of each unique column stationary phase.
  • FIG. 9 shows the top 10 variables for PCI, PC2 and PC3, plotted as vectors from the graph origin. Values for each loading can vary from -1 to 1, with a value near 0 contributing very little to the principal component axis, while a value near -1 or 1 strongly contributing to the principal component axis.
  • FIGS. 10A-10C show bar charts depicting the top 25 variables (i.e. oxazolidinone and fraction number) contributing to FIG. 10A) PCI; FIG. 10B) PC2; FIG. 10C) PC3.
  • variables i.e. oxazolidinone and fraction number
  • FIG. 11 shows the 3D LDA of the Phe-adjusted oxazolidinone EIC peaks, which reveals tight clustering of each of the five column substrates. A jackknife analysis was able to classify substrates with 96% accuracy.
  • FIG. 12 shows the monomers that were used in solid phase oligourethane synthesis. Monomers were chosen to introduce functional group chemical diversity into the oligourethanes.
  • FIG. 13 provides overlayed LC spectra of all degraded OU trimers at 280 nm. After subjecting each trimer to base and heat according to the general degradation procedures outlined in the Materials and Methods example below, all trimers had chain-end degraded to the methyl tyrisinol acetamide.
  • FIG. 14 is a heat map of adjusted R-oxazolidinone EIC values in the degradation mixture of each of the 16 directed libraries. These heat maps act as an estimation of the relative monomer composition of the OU in each directed library.
  • FIG. 15 shows the MSD1 total ion chromatogram and mass spectrum from 6- 14 minutes, taken from the LC-MS analysis of the combined pool of all 16 OU directed libraries.
  • FIG. 16 shows the overlayed FTIR spectra of BSA, acyl chloride functionalized silica, and the isolated reaction product.
  • the isolated reaction product has IR stretches which are present in both starting materials.
  • FIG. 17 provides overlayed MSD1 total ion chromatograms demonstrating that bovine serum albumin does not leach out of the silica gel support following the reaction conditions outlined in Example 4 section xvi.
  • the MSD1 mass spectrum of the peak at 6.9 minutes in the BSA sample shows a mass spectrum pattern indicative of a protein.
  • the MSD1 mass spectrum at this retention time in the acyl chloride functionalized silica filtrate and the reaction product filtrate do not show a similar pattern and there is no significant peak in the MSD1 total ion chromatogram.
  • FIG. 18 shows retention times of all oxazolidinone EIC peaks for all 32 fractions for all five substrates as described in section xvii of Example 4. If an EIC peak was not recorded because the signal-to-noise ratio was too low, it was given a retention time of 0.
  • FIG. 19 shows that the ratio of Tyr(OMe)Ac/Phe LC-MS EIC peak areas for the sensor array column chromatography outlined in section xvii of Example 4 indicates most samples were degraded to a similar extent.
  • a Tyr(OMe)Ac/Phe ratio of 0, for example charcoal fractions 1 and 2 was assigned if a peak could not be assigned for either Phe or Tyr(OMe)Ac due to a high signal-to-noise ratio.
  • FIG. 21 shows that the ratio of Tyr(OMe)Ac/Phe LC-MS EIC peak areas for the samples used in the PCA outlined in section xviii of Example 4 shows that most samples were degraded to a similar extent.
  • a Tyr(OMe)Ac/Phe ratio of 0 e.g. celite 4 fraction 20
  • FIG. 22 shows the Phe-oxazolidinone adjusted oxazolidinone EIC peak areas for fractions 5, 7, 10, 15, and 20 for all five replicates and all five substrates. When used with PCA, these data points differentiated each substrate type (FIG. 8).
  • the present disclosure relates to methods of distinguishing and/or identifying an analyte using a chemical probe comprising an oligourethane or, preferably, a mixture of oligourethanes.
  • the chemical probe is passed over the analyte in a contact area, such as in a chromatography column.
  • the composition of the chemical probe leaving the contact area varies over time in a surface-specific manner due to the interactions between the analyte and the chemical probe.
  • Measurement and deconvolution of the chemical probe composition over time provides an analyte-specific method of characterizing and identifying an analyte. In this way, the inventors presently provide an agnostic approach to allow differentiation of any analyte, such as a surface.
  • the presently disclosed methods represents an improvement in differentiating analytes that do not have obvious binding sites, such as many bulk surfaces.
  • the chemical probes of the present methods harness a chemical diversity available for the monomers of the constituent oligourethanes that is not seen in other comparable methods known in the art.
  • Oligourethanes (OUs) a chemical diversity available for the monomers of the constituent oligourethanes that is not seen in other comparable methods known in the art.
  • the present disclosure relates to the use of chemical probes which comprise a solution of one or more unique oligourethanes.
  • the chemical probe of the present disclosure is useful to, for example, identify an analyte and/or distinguish analytes from each other.
  • the oligourethanes of the presently disclosed chemical probes chain-end degrade in the presence
  • Scheme 1 Synthesis and degradation of oligourethanes (OUs) [0086]
  • the oligourethanes of the presently disclosed chemical probes are degraded into monomer-derived 5-membered carbamate rings called an oxazolidinone.
  • the chemical probe comprises at least one oxazolidinone derived from an oligourethane.
  • Oxazolidinones formed by the degradation of oligourethanes of the presently disclosed chemical probes may be identified through any means known to the skilled artisan, including by mass spectrometry.
  • Oligourethanes of the presently disclosed chemical probes are able to be synthesized according to any method known to the skilled artisan.
  • oligourethanes may be synthesized from a solid-support with Fmoc- protected, p-nitrophenyl carbonate activated amino alcohols (Fmoc-R(PG)-PNOC) with a large variety of R-functional groups.
  • Oligourethanes are favorable as a compound for use in the presently disclosed analytical technique because synthetic routes to these compounds allow control over the length, sequence, and functional group composition of the oligourethane.
  • Solid phase peptide synthesis methods are well known and practiced in the art. In such methods the synthesis of oligourethanes of the present disclosure can be carried out by sequentially
  • the oligourethanes of the present methods comprise at least one urethane monomer of the formula R O , wherein R is hydrogen or a functional group.
  • the side chain or R group of the urethane monomer is protected.
  • the backbone amino of the urethane monomer is protected while that entity reacts at a carboxyl group, such as the activated alcohol of the urethane monomer, followed by the selective removal of the backbone amino protecting group to allow a subsequent reaction to take place at that site.
  • Specific protecting groups have been disclosed and are known in solid phase synthesis methods and solution phase synthesis methods.
  • the backbone amino group and/or the R group of a urethane monomer may be protected by a suitable protecting group, including a urethane-type protecting group, such as benzyloxycarbonyl and substituted benzyloxycarbonyl, such as p-chlorobenzy loxycarbony 1, /?- nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, / ⁇ -biphenyl- isopropoxycarbonyl, 9- fluorenylmethoxycarbonyl (Fmoc) and p-methoxybenzyloxycarbonyl (Moz) and aliphatic urethane-type protecting groups, such as z-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl, isopropoxycarbonyl, and allyloxycarbonyl (Alloc).
  • a suitable protecting group including a urethane-type protecting group, such as benzyl
  • Fmoc is preferred for backbone amino protection.
  • Guanidino groups may be protected by a suitable protecting group, such as nitro, p-toluenesulfonyl (Tos), benzyloxycarbonyl, pentamethylchromanesulfonyl (Pmc), adamantyloxycarbonyl, pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) and Boc.
  • Pbf and Pmc are preferred protecting groups for Arg.
  • Boc is the preferred protecting group for Lys and Trp.
  • a tert-butyl group is the preferred protecting group for Asp and Tyr. Trityl is the preferred protecting group for His.
  • the alcohol group of the urethane monomer is pre-activated to facilitate the coupling reaction with an amino group on the growing oligourethane.
  • Specific activating groups have been disclosed and are known in solid phase synthesis methods and solution phase synthesis methods.
  • the urethane monomers are activated with p-nitrophenyl carbonate (PNOC).
  • Solid phase synthesis is commenced from the C-terminal end by coupling a protected alpha amino acid to a suitable resin.
  • a suitable resin Such starting material is prepared by attaching an alpha amino-protected amino acid by an ester linkage to a p-benzyloxybenzyl alcohol (Wang) resin, a 2-chlorotrityl chloride resin or an oxime resin, by an amide bond between an Fmoc-Linker, such as p-[(R,S)-a-[l-(9H-fhior-en-9-yl)-methoxyformamido]-2,4- dimethyloxybenzyl] -phenoxy acetic acid (Rink linker) to a benzhydrylamine (BHA) resin, or by other means well known in the art.
  • Fmoc-Linker such as p-[(R,S)-a-[l-(9H-fhior-en-9-yl)-methoxyformamid
  • the resins are carried through repetitive cycles as necessary to add urethane monomers sequentially.
  • the alpha amino Fmoc protecting groups are removed under basic conditions, piperidine, piperazine, diethylamine, or morpholine (20- 40% v/v) in A,A-dimethylformamide (DMF) may be used for this purpose.
  • DMF A,A-dimethylformamide
  • the subsequent protected monomers are coupled stepwise in the desired order to obtain an intermediate, protected oligourethane-resin.
  • the activating reagents used for coupling of the monomers in the solid phase synthesis of the oligourethanes are well known in the art.
  • the orthogonally protected side chain protecting groups may be removed using methods well known in the art for further derivatization of the peptide. Typically, orthogonal protecting groups are used as appropriate.
  • Reactive groups in an oligourethane can be selectively modified, either during solid phase synthesis or after removal from the resin.
  • oligourethanes can be modified to obtain N-terminal modifications, such as acetylation, while on resin, or may be removed from the resin by use of a cleaving reagent and then modified.
  • methods for modifying side chains of amino acids are well known to those skilled in the art. The choice of modifications made to reactive groups present on the oligourethane will be determined, in part, by the characteristics that are desired in the oligourethane.
  • the R group of the urethane monomer may have a particular characteristic or property.
  • the R group may be, for example, aromatic, aliphatic, or comprise an unsaturated group such as an alkyne.
  • the R group may have an overall net charge.
  • the R group may have a positive charge.
  • the R group may have a negative charge.
  • the chemical probe of the presently disclosed methods may comprise a pool or mixture of unique oligourethanes distinguished by different R groups.
  • the synthesis of the pool of oligourethanes may comprise a coupling step wherein the reaction mixture, referenced herein as the “coupling mixture”, comprises a mixture of urethane monomers.
  • the coupling mixture is an equimolar mixture of the urethane monomers.
  • certain urethane monomers have a higher molar concentration in the coupling mixture than other urethane monomers.
  • the urethane monomers with relatively higher molar concentrations in the coupling mixture have R groups that share a particular characteristic or property.
  • the urethane monomers with relatively higher molar concentrations have R groups that comprise an aromatic group.
  • the urethane monomers with relatively higher molar concentrations have R groups that comprise an aliphatic group.
  • the urethane monomers with relatively higher molar concentrations have R groups that comprise an alkyne group.
  • the urethane monomers with relatively higher molar concentrations have R groups that have an overall charge.
  • the urethane monomers with relatively higher molar concentrations have R groups that have an overall positive charge. In some embodiments, the urethane monomers with relatively higher molar concentrations have R groups that have an overall negative charge. Pools of urethane monomers with higher molar concentrations of at least one urethane monomer are termed herein as a biased pool. Equivalent terms to “pool” include “mixture” and “library”. Equivalent terms to “biased” include “directed”. In some embodiments, the one or more urethane monomers having a higher molar concentration has a particular characteristic or property.
  • a pool of urethane monomers may be biased towards one, two, or more characteristics or properties by, for example, having a relatively higher molar concentration of at least one urethane monomer with an R group having each characteristic or property in comparison to urethane monomers with an R group that does not have the characteristic or property.
  • a Oligourethanes of the chemical probes of the present methods may be formed by methods involving coupling mixtures that comprise an equimolar pool of urethane monomers, a biased pool of urethane monomers, a single urethane monomer, or any combination thereof. Biased pools of oligourethanes are formed through the use of coupling mixtures comprising biased pools of urethane monomers.
  • the total concentration of the monomer units with higher concentration is about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 11 times, about 12 times, about 13 times, about 14 times about 15 times, about 16 times, about 17 times, about 18 times, about 19 times, or about 20 times higher than the sum total of all other monomer units in the coupling mixture, or any range derivable therein
  • the chemical probe of the present disclosure is a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 biased pools of oligourethanes. In some embodiments, the chemical probe of the present disclosure is a combination of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 biased pools of oligourethanes.
  • a biased pool may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 urethane monomers with relatively higher molar concentration in the coupling mixture.
  • a biased pool may have 1, 2, 3, 4, 5, or 6 urethane monomers with relatively higher molar concentration in the coupling mixture.
  • about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or any range derivable therein of the monomer units of a pool of urethane monomers may have a relatively higher molar concentration in the pool of urethane monomer units than another monomer unit.
  • about 10%, about 20%, about 30%, or about 40% of monomer units, or any range derivable therein may have a higher concentration in the pool of urethane monomer units than another monomer unit.
  • about 30% of monomer units may have a higher concentration in the pool of urethane monomer units than another monomer unit.
  • the coupling mixture comprises at least one urethane monomer with a concentration that is about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 11 times, about 12 times, about 13 times, about 14 times, about 15 times, about 16 times, about 17 times, about 18 times, about 19 times, or about 20 times as high as the concentration of another urethane monomer in the coupling mixture.
  • the coupling mixture comprises at least one urethane monomer with a concentration that is about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 times as high as the concentration of another urethane monomer in the coupling mixture. If more than one urethane monomer has a higher molar concentration in the coupling mixture than another urethane monomer, the urethane monomers with relatively higher molar concentration need not have the same molar concentration.
  • the oligourethane of the chemical probe of the present disclosure comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
  • the oligourethane of the chemical probe of the present disclosure comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 repeating units.
  • the oligourethane of the chemical probe of the present disclosure comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 repeating units.
  • the oligourethane of the chemical probe of the present disclosure comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 repeating units.
  • the repeating unit is of the formula described above for the urethane monomer.
  • the chemical probe of the present disclosure comprises more than one distinct oligourethane.
  • the chemical probe of the present disclosure comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 distinct oligourethanes.
  • the chemical probe of the present disclosure comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 distinct oligourethanes. In some embodiments, the chemical probe of the present disclosure comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 distinct oligourethanes. In some embodiments, the present disclosure comprises five or more distinct oligourethanes. In some embodiments, the present disclosure comprises ten or more distinct oligourethanes.
  • the chemical probe of the present disclosure is biased.
  • a biased chemical probe comprises oligourethanes that are formed from at least one coupling step involving a biased pool of urethane monomers, as discussed in more detail above.
  • the chemical probe is degraded to facilitate identification of the components of the chemical probe.
  • oligourethanes can undergo chain-end degradation to oxazolidinone derivatives of the monomer oligourethanes. Further details on this aspect are provided in the Examples.
  • the identity of the resultant degraded chemical probe can be determined and used to characterize the source oligourethane. In this manner, chemical probes comprising oligourethanes represent an improvement over other possible compounds which could be used in a similar manner, such as peptides, which are more difficult to sequence.
  • the present methods involve analysis of the chemical probe and/or the degraded chemical probe.
  • the identity of the chemical probe, the degraded chemical probe, or any portion, fraction, or mixture thereof can be determined using any analytical technique known to the skilled artisan.
  • the analytical technique is chromatography, such as high-performance liquid chromatography.
  • the analytical technique is spectrometry, such as mass spectrometry.
  • the analytical technique is spectroscopy.
  • the analytical technique is a hybrid technique, such as liquid chromatography-mass spectrometry.
  • the symbol — represents an optional bond, which if present is either single or double.
  • the symbol represents a single bond or a double bond.
  • ' /vv means a single bond where the geometry around a double bond (e.g., either E or Z) is undefined. Both options, as well as combinations thereof are therefore intended. Any undefined valency on an atom of a structure shown in this application implicitly represents a hydrogen atom bonded to that atom. A bold dot on a carbon atom indicates that the hydrogen attached to that carbon is oriented out of the plane of the paper.
  • variable When a variable is depicted as a “floating group” on a ring system, for example, the group “R” in the formula: then the variable may replace any hydrogen atom attached to any of the ring atoms, including a depicted, implied, or expressly defined hydrogen, so long as a stable structure is formed.
  • variable When a variable is depicted as a “floating group” on a fused ring system, as for example the group “R” in the formula: then the variable may replace any hydrogen attached to any of the ring atoms of either of the fused rings unless specified otherwise.
  • Replaceable hydrogens include depicted hydrogens (e.g. , the hydrogen attached to the nitrogen in the formula above), implied hydrogens (e.g., a hydrogen of the formula above that is not shown but understood to be present), expressly defined hydrogens, and optional hydrogens whose presence depends on the identity of a ring atom (e.g., a hydrogen attached to group X, when X equals -CH-), so long as a stable structure is formed.
  • R may reside on either the 5 -membered or the 6-membered ring of the fused ring system.
  • the subscript letter “y” immediately following the R enclosed in parentheses represents a numeric variable. Unless specified otherwise, this variable can be 0, 1, 2, or any integer greater than 2, only limited by the maximum number of replaceable hydrogen atoms of the ring or ring system.
  • the minimum number of carbon atoms in the groups “alkyl ( c ⁇ 8)”, “alkanediyl(c ⁇ 8)”, “heteroaryl(c ⁇ s)”, and “acyl(c ⁇ 8)” is one
  • the minimum number of carbon atoms in the groups “alkenyl(c ⁇ 8)”, “alkynyl(c ⁇ 8)”, and “heterocycloalkyl(c ⁇ 8)” is two
  • the minimum number of carbon atoms in the group “cycloalkyl(c ⁇ 8)” is three
  • the minimum number of carbon atoms in the groups “aryl(c ⁇ 8)” and “arenediyl(c ⁇ 8)” is six.
  • Cn-n' defines both the minimum (n) and maximum number (n') of carbon atoms in the group.
  • alkyl(C2-io) designates those alkyl groups having from 2 to 10 carbon atoms.
  • These carbon number indicators may precede or follow the chemical groups or class it modifies and it may or may not be enclosed in parenthesis, without signifying any change in meaning.
  • the terms “Ci-4-alkyl”, “Cl-4-alkyl”, “alkyl(ci-4)”, and “alkyl(c ⁇ 4)” are all synonymous. Except as noted below, every carbon atom is counted to determine whether the group or compound falls with the specified number of carbon atoms.
  • the group dihexylamino is an example of a dialkylamino ( ci2) group; however, it is not an example of a dialkylaminojce) group.
  • any of the chemical groups or compound classes defined herein is modified by the term “substituted”, any carbon atom in the moiety replacing the hydrogen atom is not counted.
  • methoxyhexyl which has a total of seven carbon atoms, is an example of a substituted alkyl ( ci-6).
  • any chemical group or compound class listed in a claim set without a carbon atom limit has a carbon atom limit of less than or equal to twelve.
  • saturated when used to modify a compound or chemical group means the compound or chemical group has no carbon-carbon double and no carboncarbon triple bonds, except as noted below.
  • the term when used to modify an atom, it means that the atom is not part of any double or triple bond.
  • substituted versions of saturated groups one or more carbon oxygen double bond or a carbon nitrogen double bond may be present. And when such a bond is present, then carbon-carbon double bonds that may occur as part of keto-enol tautomerism or imine/enamine tautomerism are not precluded.
  • saturated when used to modify a solution of a substance, it means that no more of that substance can dissolve in that solution.
  • aliphatic signifies that the compound or chemical group so modified is an acyclic or cyclic, but non-aromatic compound or group.
  • the carbon atoms can be joined together in straight chains, branched chains, or non-aromatic rings (alicyclic).
  • Aliphatic compounds/groups can be saturated, that is joined by single carbon-carbon bonds (alkanes/alkyl), or unsaturated, with one or more carbon-carbon double bonds (alkenes/alkenyl) or with one or more carbon-carbon triple bonds (alky nes/alkynyl) .
  • aromatic signifies that the compound or chemical group so modified has a planar unsaturated ring of atoms with 4/? +2 electrons in a fully conjugated cyclic 7i system.
  • An aromatic compound or chemical group may be depicted as a single resonance structure; however, depiction of one resonance structure is taken to also refer to any other resonance structure. For example:
  • Aromatic compounds may also be depicted using a circle to represent the delocalized nature of the electrons in the fully conjugated cyclic n system, two non-limiting examples of which are shown below:
  • alkyl refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, a linear or branched acyclic structure, and no atoms other than carbon and hydrogen.
  • the groups -CH3 (Me), -CH2CH3 (Et), -CH2CH2CH3 (n-Pr or propyl), -CH(CH 3 )2 (z-Pr, 'Pr or isopropyl), -CH 2 CH 2 CH 2 CH 3 (n-Bu), -CH(CH3)CH 2 CH3 sec -butyl), -CH 2 CH(CH3) 2 (isobutyl), -C(CH3)3 (tert-butyl, t-butyl, t-Bu or 'Bu), and -CH 2 C(CH3)3 (neo-pentyl) are non-limiting examples of alkyl groups.
  • alkanediyl refers to a divalent saturated aliphatic group, with one or two saturated carbon atom(s) as the point(s) of attachment, a linear or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen.
  • the groups -CH 2 - (methylene), -CH2CH2-, -CH 2 C(CH3) 2 CH2-, and -CH 2 CH 2 CH 2 - are non-limiting examples of alkanediyl groups.
  • An “alkane” refers to the class of compounds having the formula H-R, wherein R is alkyl as this term is defined above.
  • cycloalkyl refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, said carbon atom forming part of one or more non-aromatic ring structures, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. If more than one ring is present, the rings may be fused, bridged, or spirocyclic. Non-limiting examples include: -CH(CH 2 ) 2 (cyclopropyl), cyclobutyl, cyclopentyl, or cyclohexyl (Cy).
  • cycloalkanediyl refers to a divalent saturated aliphatic group with two carbon atoms as points of attachment, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen.
  • the group j s a non _ limiting example of cycloalkanediyl group.
  • a “cycloalkane” refers to the class of compounds having the formula H-R, wherein R is cycloalkyl as this term is defined above.
  • alkenyl refers to a monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a linear or branched, acyclic structure, at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen.
  • alkenediyl refers to a divalent unsaturated aliphatic group, with two carbon atoms as points of attachment, a linear or branched acyclic structure, at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen.
  • alkene and olefin are synonymous and refer to the class of compounds having the formula H-R, wherein R is alkenyl as this term is defined above.
  • terminal alkene and a-olefin are synonymous and refer to an alkene having just one carbon-carbon double bond, wherein that bond is part of a vinyl group at an end of the molecule.
  • alkynyl refers to a monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a linear or branched acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. As used herein, the term alkynyl does not preclude the presence of one or more non-aromatic carbon-carbon double bonds.
  • An “alkyne” refers to the class of compounds having the formula H-R, wherein R is alkynyl.
  • aryl refers to a monovalent unsaturated aromatic group with an aromatic carbon atom as the point of attachment, said carbon atom forming part of a one or more aromatic ring structures, each with six ring atoms that are all carbon, and wherein the group consists of no atoms other than carbon and hydrogen. If more than one ring is present, the rings may be fused or unfused. Unfused rings are connected with a covalent bond. As used herein, the term aryl does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present.
  • Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl, (dimethyl)phenyl, -C6H4CH2CH3 (ethylphenyl), naphthyl, and a monovalent group derived from biphenyl (e.g. , 4-phenylphenyl).
  • aromaticiyl refers to a divalent aromatic group with two aromatic carbon atoms as points of attachment, said carbon atoms forming part of one or more six-membered aromatic ring structures, each with six ring atoms that are all carbon, and wherein the divalent group consists of no atoms other than carbon and hydrogen.
  • arenediyl does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. If more than one ring is present, the rings may be fused or unfused. Unfused rings are connected with a covalent bond.
  • alkyl groups carbon number limitation permitting
  • An “arene” refers to the class of compounds having the formula H-R, wherein R is aryl as that term is defined above. Benzene and toluene are non-limiting examples of arenes.
  • aralkyl refers to the monovalent group -alkanediyl-aryl, in which the terms alkanediyl and aryl are each used in a manner consistent with the definitions provided above.
  • Non-limiting examples are: phenylmethyl (benzyl, Bn) and 2-phenyl-ethyl.
  • heteroaryl refers to a monovalent aromatic group with an aromatic carbon atom or nitrogen atom as the point of attachment, said carbon atom or nitrogen atom forming part of one or more aromatic ring structures, each with three to eight ring atoms, wherein at least one of the ring atoms of the aromatic ring structure(s) is nitrogen, oxygen or sulfur, and wherein the heteroaryl group consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. If more than one ring is present, the rings are fused; however, the term heteroaryl does not preclude the presence of one or more alkyl or aryl groups (carbon number limitation permitting) attached to one or more ring atoms.
  • heteroaryl groups include benzoxazolyl, benzimidazolyl, furanyl, imidazolyl (Im), indolyl, indazolyl, isoxazolyl, methylpyridinyl, oxazolyl, oxadiazolyl, phenylpyridinyl, pyridinyl (pyridyl), pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl.
  • W-heteroaryl refers to a heteroaryl group with a nitrogen atom as the point of attachment.
  • a “heteroarene” refers to the class of compounds having the formula H-R, wherein R is heteroaryl. Pyridine and quinoline are non-limiting examples of heteroarenes.
  • heterocycloalkyl refers to a monovalent non-aromatic group with a carbon atom or nitrogen atom as the point of attachment, said carbon atom or nitrogen atom forming part of one or more non-aromatic ring structures, each with three to eight ring atoms, wherein at least one of the ring atoms of the non-aromatic ring structure(s) is nitrogen, oxygen or sulfur, and wherein the heterocycloalkyl group consists of no atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur. If more than one ring is present, the rings may be fused, bridged, or spirocyclic.
  • the term does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to one or more ring atoms. Also, the term does not preclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic.
  • heterocycloalkyl groups include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydro thiofuranyl, tetrahydropyranyl, tetrahydropyridinyl, pyranyl, oxiranyl, and oxetanyl.
  • W-heterocycloalkyl refers to a heterocycloalkyl group with a nitrogen atom as the point of attachment.
  • A-pyrrolidinyl is an example of such a group.
  • acyl refers to the group -C(O)R, in which R is a hydrogen, alkyl, cycloalkyl, or aryl as those terms are defined above.
  • the groups, -CHO, -C(O)CH3 (acetyl, Ac), -C(O)CH 2 CH 3 , -C(O)CH(CH 3 ) 2 , -C(O)CH(CH 2 ) 2 , -C(O)C 6 H 5 , and -C(O)CeH4CH 3 are non-limiting examples of acyl groups.
  • a “thioacyl” is defined in an analogous manner, except that the oxygen atom of the group -C(O)R has been replaced with a sulfur atom, -C(S)R.
  • aldehyde corresponds to an alkyl group, as defined above, attached to a -CHO group.
  • carboxylate means an alkyl group, as defined above, attached to a -C(O)O group.
  • alkoxy refers to the group -OR, in which R is an alkyl, as that term is defined above.
  • Non-limiting examples include: -OCH 3 (methoxy), -OCH 2 CH 3 (ethoxy), -OCH2CH2CH3, -OCH(CH3)2 (isopropoxy), or -OC(CH3)3 (tert-butoxy).
  • cycloalkoxy refers to groups, defined as -OR, in which R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and acyl, respectively.
  • alkylthio and “acylthio” refers to the group -SR, in which R is an alkyl and acyl, respectively.
  • alcohol corresponds to an alkane or cycloalkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with a hydroxy group.
  • alkoxide and cycloalkoxide refer to a negatively charged group of the form OR wherein R is alkyl or cycloalkyl, respectively.
  • ether corresponds to an alkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with an alkoxy group.
  • alkylamino refers to the group -NHR, in which R is an alkyl, as that term is defined above. Non-limiting examples include: -NHCH3 and -NHCH2CH3.
  • dialkylamino refers to the group -NRR', in which R and R' can be the same or different alkyl groups. Non-limiting examples of dialkylamino groups include: -N(CH3)2 and -N(CH3)(CH2CH3).
  • acylamino when used without the “substituted” modifier, refers to the group -NHR, in which R is acyl, as that term is defined above.
  • a nonlimiting example of an amido group is -NHC(O)CH3.
  • Chemical groups refers to any functional group or substituent covalently bound to a core structure.
  • Chemical groups may include but are not limited to hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -OH, -F, -Cl, -Br, -I, -NH 2 , -NO 2 , -CO2H, -CO2CH3, -CO2CH2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH 3 , -NHCH3, -NHCH2CH3, -N(CH 3 )2, -C(O)NH 2 , -C(O)NHCH 3 , -C(O)N(CH 3 )2, -OC(O)CH 3 , -NHC(O)CH 3 , -NHC(O)CH 3 ,
  • substituted alkyl groups are non-limiting examples of substituted alkyl groups: -CH2OH, -CH2CI, -CF3, -CH2CN, -CH 2 C(O)OH, -CH 2 C(O)OCH 3 , -CH 2 C(O)NH 2 , -CH 2 C(O)CH 3 , -CH2OCH3, -CH 2 OC(O)CH 3 , -CH2NH2, -CH 2 N(CH 3 )2, and -CH2CH2CI.
  • haloalkyl is a subset of substituted alkyl, in which the hydrogen atom replacement is limited to halo (i.e.
  • -F, -Cl, -Br, or -I such that no other atoms aside from carbon, hydrogen and halogen are present.
  • the group, -CH 2 C1 is a non- limiting example of a haloalkyl.
  • fluoroalkyl is a subset of substituted alkyl, in which the hydrogen atom replacement is limited to fluoro such that no other atoms aside from carbon, hydrogen and fluorine are present.
  • the groups -CH2F, -CF 3 , and -CH 2 CF 3 are non-limiting examples of fluoroalkyl groups.
  • Non-limiting examples of substituted aralkyls are: (3-chlorophenyl)-methyl, and 2-chloro-2-phenyl-eth-l-yl.
  • the groups, -C(O)CH 2 CF 3 , -CO 2 H (carboxyl), -CO 2 CH 3 (methylcarboxyl), -CO 2 CH2CH 3 , -C(O)NH 2 (carbamoyl), and -CON(CH 3 )2, are non-limiting examples of substituted acyl groups.
  • the groups -NHC(O)OCH 3 and -NHC(O)NHCH 3 are non-limiting examples of substituted amido groups.
  • An “isomer” of a first compound is a separate compound in which each molecule contains the same constituent atoms as the first compound, but where the configuration of those atoms in three dimensions differs.
  • “Pharmaceutically acceptable salts” means salts of compounds disclosed herein which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as 1 ,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3 -phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene- 1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene- 1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic
  • Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases.
  • Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide.
  • Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, A-methylglucamine and the like. It should be recognized that the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).
  • a “stereoisomer” or “optical isomer” is an isomer of a given compound in which the same atoms are bonded to the same other atoms, but where the configuration of those atoms in three dimensions differs.
  • “Enantiomers” are stereoisomers of a given compound that are mirror images of each other, like left and right hands.
  • “Diastereomers” are stereoisomers of a given compound that are not enantiomers.
  • Chiral molecules contain a chiral center, also referred to as a stereocenter or stereogenic center, which is any point, though not necessarily an atom, in a molecule bearing groups such that an interchanging of any two groups leads to a stereoisomer.
  • the chiral center is typically a carbon, phosphorus or sulfur atom, though it is also possible for other atoms to be stereocenters in organic and inorganic compounds.
  • a molecule can have multiple stereocenters, giving it many stereoisomers.
  • compounds whose stereoisomerism is due to tetrahedral stereogenic centers e.g. , tetrahedral carbon
  • the total number of hypothetically possible stereoisomers will not exceed 2 n , where n is the number of tetrahedral stereocenters.
  • Molecules with symmetry frequently have fewer than the maximum possible number of stereoisomers.
  • a 50:50 mixture of enantiomers is referred to as a racemic mixture.
  • a mixture of enantiomers can be enantiomerically enriched so that one enantiomer is present in an amount greater than 50%.
  • enantiomers and/or diastereomers can be resolved or separated using techniques known in the art. It is contemplated that for any stereocenter or axis of chirality for which stereochemistry has not been defined, that stereocenter or axis of chirality can be present in its R form, S form, or as a mixture of the R and .S' forms, including racemic and non-racemic mixtures.
  • the phrase “substantially free from other stereoisomers” means that the composition contains ⁇ 15%, more preferably ⁇ 10%, even more preferably ⁇ 5%, or most preferably ⁇ 1% of another stereoisomer(s).
  • An “amine protecting group” or “amino protecting group” is well understood in the art.
  • An amine protecting group is a group which modulates the reactivity of the amine group during a reaction which modifies some other portion of the molecule.
  • Amine protecting groups can be found at least in Greene and Wuts, 1999, which is incorporated herein by reference.
  • amino protecting groups include formyl, acetyl, propionyl, pivaloyl, /-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, o-nitrophenoxyacetyl, a-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4- bromobenzoyl, 4-nitrobenzoyl, and the like; sulfonyl groups such as benzenesulfonyl, p- toluenesulfonyl and the like; alkoxy- or aryloxycarbonyl groups (which form urethanes with the protected amine) such as benzyloxycarbonyl (Cbz), -chlorobenzyloxycarbonyl, p- methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitro
  • the “amine protecting group” can be a divalent protecting group such that both hydrogen atoms on a primary amine are replaced with a single protecting group.
  • the amine protecting group can be phthalimide (phth) or a substituted derivative thereof wherein the term “substituted” is as defined above.
  • the halogenated phthalimide derivative may be tetrachlorophthalimide (TCphth).
  • a “protected amino group” is a group of the formula PGMANH- or PGDAN- wherein PGMA is a monovalent amine protecting group, which may also be described as a “monovalently protected amino group” and PGDA is a divalent amine protecting group as described above, which may also be described as a “divalently protected amino group”.
  • a surface references the portion of a substrate, sample, analyte, or the like which is capable of interacting with other compounds or compositions.
  • a surface is an atom, molecule, or macromolecule capable of participating in intermolecular interactions with a compound or composition, such as an oligourethane or a mixture of oligourethanes.
  • the samples may be either organic material, inorganic material, or a mixture thereof.
  • the samples may be manipulated or processed to improve the suitability of the surface for use in the methods disclosed herein.
  • Nonlimiting examples of samples which have surfaces that may be analyzed according to the methods disclosed herein include proteins, enzymes, antibodies, cells, viruses, biopolymers, polymers, organisms, soil, rocks, material, and combinations thereof.
  • the present disclosure describes the development of an agnostic approach to surface differentiation, which facilitates analysis of any type of surface.
  • This approach represents an improvement techniques known in the art, such as a lock-and-key type detection approach which requires advanced knowledge of the surfaces to be differentiated and careful design of probes with specific molecular recognition motifs.
  • the agnostic approach described herein is also beneficial for analyzing surfaces, such as bulk surfaces, that do not have obvious or particularly reactive binding sites.
  • the inventors describe in further detail below the creation of a library of oligomers with various monomer compositions.
  • the oligomers self-sort during elution based on their individual compositions, the overall monomer composition of the oligomer library, and any cooperativity between oligomer monomers.
  • Use of oligomers instead of discrete small molecules facilitated creation of a pool of molecules with similar structures and sizes, so that differences in molecular retention times would, without being bound by theory, primarily arise from the different supramolecular interactions of their functional groups with the substrate surface, without prior knowledge of the surfaces to be differentiated.
  • Oligourethane (OU) structures (Dahlhauser et al., 2020) recently developed by the inventors were selected for use in creating the probe library (Scheme 1).
  • OUs may be synthesized from a solid-support with Fmoc-protected, p-nitrophenyl carbonate activated amino alcohols (Fmoc-R(PG)-PNOC) with a large variety of R-functional groups with control over the length, sequence, and functional group composition of the OUs (Scheme 1). These OUs have been shown to chain-end degrade in the presence of base and heat through a 5-exo-trig intramolecular cyclization from the terminal alcohol (Scheme 1).
  • OUs are degraded into monomerderived 5-membered carbamate rings called an oxazolidinone.
  • This degradation mechanism has been used to decode the monomer sequence of unknown OUs with discrete sequences (Dahlhauser et al., 2021; Dahlhauser et al., 2022; Zhang et al., 2024; Soete et al., 2022).
  • An OU comprising an entirely random sequence of monomers and an equal distribution of said monomers would, without being bound by theory, disfavor bias or selectivity towards any one surface and accordingly would carry variance primarily along one PCA axis which, as mentioned above, would be disfavored for use in classification of analyte surfaces. Instead, surface differentiation would, still without being bound by theory, be facilitated by mixtures of O s that comprise a bias towards a monomer or a class of monomer (FIG. ID).
  • sixteen libraries of OUs were synthesized from 16 coupling mixtures using different mixtures of monomers (FIG. IB, FIG. 1C).
  • one library was biased towards aliphatic monomers, with the aliphatic monomers Fmoc-Leu-PNOC and Fmoc-Cha-PNOC at a combined concentration of 75 mM in the monomer coupling mixture and the remaining eight monomers at a combined concentration of 25 mM.
  • Another library for example, was biased towards both aromatic monomers and polar protic monomers that bear a negative charge in water.
  • the monomer coupling mixture contained aromatic monomers Fmoc-Trp(Boc)-PNOC, Fmoc-Tyr(tBu)-PNOC, and Fmoc-BnTrz-PNOC and polar protic monomer Fmoc-Asp(tBu)-PNOC at a combined concentration of 81 mM and the remaining six monomers had a combined concentration of 19 mM.
  • FIG. 2 and Table 1 show the relative monomer composition used to synthesize all biased libraries.
  • the combined pool of all OU libraries had 108 possible different sequences, with the diversity of sequences artificially engineered by using the biased monomer mixtures.
  • Reversed phase C18-silica gel made by capping surface silanols with an 18- carbon alkyl chain, is nonpolar.
  • Celite is a highly porous and amorphous silicon oxide but does not bind strongly to most organic molecules because it does not have many surface silanols (Ren et al., 2022).
  • Activated carbon is made primarily of aromatic carbon allotropes and is commonly used in purification due to its high porosity and absorbency. While celite is nearly non-adsorbent, activated carbon is the opposite. Activated carbon was diluted to 0.1 w% with celite to facilitate elution of the OUs to elute from the column under the experimental conditions disclosed in other sections.
  • Silica gel functionalized with bovine serum albumin (BS A), a protein isolated from cows with a molecular weight of 66.5 kDa, has highly complex surface chemistry and has previously been studied to separate enantiomers by high performance liquid chromatography (HPLC) (Allenmark et al., 1984; Giplin et al., 1991; Haginaka et al., 1997; Zhang et al., 2000).
  • BSA-functionalized silica gel was synthesized using a commercially available propionyl chloride-functionalized silica gel starting material (see Example 4 for synthesis and characterization).
  • EIC peak areas were scaled from 0 to 1 (that is, min-max adjusted) based on the highest peak area for each R-group across all samples (FIG. 3C) to facilitate comparison of R-oxazolidinone responses across R-groups.
  • FIG. 3 demonstrates these adjustments using data collected from a column run with silica as the substrate.
  • FIG. 5 shows a heatmap of the Phe-oxazolidinone adjusted EIC peaks for all oxazolidinones over all 32 fractions and all five column substrates.
  • PCA Principal component analysis
  • This technique is an unsupervised model because no input is provided on the substrate identity of each sample.
  • a clustering pattern on PCA plots of replicates from the same substrates that is clear and distinct from that of PCA plots of replicates from another substrate would indicate that the sensor array is useful for differentiating between the substrates.
  • this substrate is the least biased towards any one of the linear combinations of oxazolidinones and fractions used to make the principal components, perhaps because, without being bound by theory, it is the most chemically complex surface.
  • the high dimensionality of the PCA space as well as the tight clustering of the replicates using this unsupervised chemometric method validates the probe and library design.
  • a loading plot analysis was performed for PCI, PC2 and PC3 (FIG. 9).
  • a loading plot reveals the contribution of each variable towards the principal component on each axis. None of the variables (OU side chains at specific retention times) contributed very strongly to any principal component, while many variables contributed a similar amount. Furthermore, there was no overlap between the top ten variables contributing to PCI, PC2 and PC3. This suggests, without being bound by theory, that nearly all the oligourethane side chain interactions were used to differentiate each sample instead of there being a single important side chain or combination of side chains.
  • top 10 variables influencing PCI, PC2, and PC3 were analyzed.
  • the top 10 variables for the PCI axis move data points exclusively in a positive direction along the axis, and thus are most responsible for differentiating Cl 8- silica from the other four substrates.
  • Eight of the ten top PCI components are aromatic oxazolidinones, suggesting that, without being bound by theory, aromatic biased OUs were most important for differentiating Cl 8-silica in this study.
  • PC2 is responsible for differentiating the remaining four substrates.
  • PC3 had the variables with the largest contribution to the principal component axis.
  • the top four variables are the aspartic acid derived oxazolidinone, and seven of the top 10 components are derived from charged amino acids. This suggests that the OUs with a charged monomer bias contributed the most to differentiating celite in PC3.
  • LDA Linear discriminatory analysis
  • Monomers for oligourethane synthesis were selected to introduce functional group diversity into the OU probes (FIG. 12).
  • Leucine and cyclohexyl-alanine derived monomers (Fmoc-Leu-PNOC and Fmoc-Cha-PNOC, respectively) were used as aliphatic monomers.
  • Tryptophan, tyrosine, and l-benzyl-lH-l,2,3-triazole-alanine derived monomers Fmoc-Trp(Boc)-PNOC, Fmoc-Tyr(tBu)-PNOC, Fmoc-BnTrz-PNOC, respectively) were introduced as aromatic monomers.
  • Aspartic acid, lysine, arginine, and histidine derived monomers (Fmoc-Asp(tBu)-PNOC, Fmoc-Lys(Boc)-PNOC, Fmoc- Arg(Pbf)-PNOC, and Fmoc-His(Trt)-PNOC, respectively) were used because they bear a charge in water and are polar protic in organic media.
  • a propargyl-glycine derived monomer (Fmoc-PGly-PNOC) served as a monomer comprising an alkynyl functional group.
  • Fmoc-Lys(Boc)-PNOC (9H-fluoren-9-yl)methyl tert-butyl (6-(((4- nitrophenoxy)carbonyI)oxy)hexane-l,5-diyl)(S)-dicarbamate:
  • a coupling solution of HOBt (0.2 M, 6 equiv.), diisopropyl ethylamine (0.05 M, 1.6 equiv.), and Fmoc-R(PG)-PNOC monomer or monomer mixture (0.1 M, 3 equiv.) in NMP was added to the resin (1 equiv. active sites), and the suspension was heated to 35 °C and periodically shaken for 8 hours. After coupling, the resin was filtered and washed 3 times with 2 mL DMF. Following washing, the Fmoc protecting group on the previously attached monomer was deprotected by suspending the resin in 100 pL of a solution of 20% piperidine in DMF for 10 minutes and filtering.
  • OUs were cleaved from the solid support by suspending the resin in 500 pL of a solution of 95% TFA/2.5% TIPS/2.5% DCM for 4 hours. The resin was then filtered and washed 5 times with 500 pL DCM. The filtrate was collected and dried first under flowing nitrogen and then under vacuum in a Genevac, producing a brown residue.
  • Sequence-defined oligourethanes of the presently disclosed methods depolymerize through chain-end degradation.
  • the terminal alcohol undergoes a 5-exo-trig cyclization and attacks its neighboring carbamate bond.
  • Subsequent cleavage of a five-membered oxazolidinone ring leaves a shortened oligourethane with a terminal alcohol.
  • This chain-end degradation continues until the entire oligourethane is depolymerized into monomer-derived oxazolidinones and the final monomer, which in the present Example is methyl tyrosinol acetamide (Scheme 1).
  • trimers were dissolved in 300 pL MeCN and 100 pL 20 mM K3PO4 and shaken at room temperature for 4 hours. Samples were evaporated to dryness overnight with a Genevac, redissolved in 300 pL MeCN, and filtered. Filtrates were submitted to LC-MS and HRMS for analysis as described in sections above. Characterization data for the trimers is provided in Table 2 below.
  • Table 2 Characterization data for oligourethane trimer standards.
  • Each monomer-derived oxazolidinone was observed in the degradation mixture of their respective trimers as the [M+H] + m/z intensity in the MSD1 total ion chromatogram (TIC) using Agilent ChemStation.
  • the oxazolidinone extracted ion chromatogram (EIC) peaks of a given monomer were not observed at a high intensity in the degradation mixtures of trimers that were not the trimer of said monomer.
  • the degradation mixture of all trimers contained the Val-oxazolidinone internal standard, Phe-oxazolidinone, and Tyr(OMe)Ac. High resolution mass spectrometry on each of the degradation mixtures corroborated the presence of the respective oxazolidinones. Characterization data for the Vai standard and monomer oxazolidinones is provided in Table 3 below.
  • Each directed library represents a pool of oligourethanes that could contain any number of 10 8 different possible sequences, making it untenable to identify individual OUs on the LC-MS.
  • a portion of each directed library was fully chain-end degraded and the resulting R-oxazolidinone composition profile was used to estimate the average monomer composition of the OU in each directed library, according to the following procedure.
  • oligourethane libraries were dissolved in 400 mL 25% PLO/MeCN and 6.25 mL of each directed library was removed and diluted with the degradation mixture described above. These aliquots of the directed libraries were fully degraded according to the method outlined above and submitted for LC-MS analysis, using the LC-MS instrumentation and method described above.
  • Oxazolidinones were identified by searching for their [M+H] + m/z intensities in the MSD1 total ion chromatogram (TIC) using Agilent ChemStation. The software automatically identified significant peaks in the resulting extracted ion chromatogram (EIC) and calculated the area under each curve. Oxazolidinone EIC peak signals were recorded if they could be identified above the noise by human eye and if they had a retention time that was satisfactorily close to that of the oxazolidinone standards run in the study above (summarized in Table 3).
  • oxazolidinone EIC peak areas had to be adjusted as discussed in further detail in Examples above (see also FIGS. 3A-3D). More particularly, oxazolidinone EIC peak areas were normalized by dividing by the peak area of the Val-oxazolidinone internal standard in its sample, which was added during OU degradation. Because every oligourethane in the pool begins with phenylalaninol (already present on the solid-phase support used for synthesis) Phe- oxazolidinone can be used as a proxy for the number of OU in a sample.
  • oxazolidinone EIC peak areas were also normalized by dividing by the area of the Phe-oxazolidinone EIC peak in the same library sample. Finally, some oxazolidinones were more easily ionized through the positive mode of ESI than others.
  • Phe-oxazolidinone adjusted EIC peak areas were scaled from 0 to 1 (or min-max adjusted) based on the largest adjusted value for each R- group across all libraries. This facilitated improved comparison of relative monomer composition across all oxazolidinones, regardless of their signal intensities.
  • oxazolidinone values were plotted as a heatmap, comparing all directed libraries and the final combined probe library (FIG. 14).
  • Oxazolidinones derived from monomers that were present at a greater concentration during a library synthesis have the highest adjusted values in that directed library.
  • Leu- oxazolidinone and Cha-oxazolidinone read the highest adjusted values in the aliphatic-biased library.
  • the adjusted values for Trp-oxazolidinone, Tyr-oxazolidinone, and BnTrz- oxazolidinone are the highest in the aromatic -biased library.
  • the total ion chromatogram of the pool of oligourethanes in the positive mode shows a broad peak over several minutes instead of unique peaks (FIG. 15).
  • the 10-mer oligourethanes forming the mixture are calculated to have a range of exact masses between 1400 and 2465 amu, and a multitude of signals in this m/z range is seen in the positive mode mass spectrum between 6 and 14 minutes of the LC-MS analysis.
  • a broad shoulder between 700 and 1230 m/z typically corresponds, without being bound by theory, to the signals for oligourethanes which have gained two positive charges during electrospray ionization.
  • BSA bovine serum albumin
  • R-oxazolidinones were measured by identifying for their [M+H] + m/z intensities in the MSD1 total ion chromatogram (TIC) using Agilent ChemStation.
  • the software automatically identified significant peaks in the resulting extracted ion chromatogram (E1C) and calculated the area under each curve.
  • Oxazolidinone EIC peak signals were recorded if they could be identified above the noise by human eye and if they had a retention time that was satisfactorily close to that of the oxazolidinone standards as shown in Table 3 above.
  • Boeijen et al. Solid- Phase Synthesis of Oligourea Peptidomimetics Employing the Fmoc Protection Strategy, Journal of Organic Chemistry, 66(25): 8454-8462, 2001.
  • van Zutphen et al. Combinatorial discovery of new asymmetric cis platinum anticancer complexes is made possible with solid-phase synthetic methods, Journal of Inorganic Biochemistry, 99(10):2032-2038, 2005.
  • Triazolo-Peptidomimetics Novel Radiolabeled Minigastrin Analogs for Improved Tumor Targeting, J Med Chem, 63(9): 4484-4495, 2020.

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Abstract

Provided herein are methods for identifying or differentiating an analyte, such as a bulk surface or a soil sample. The presently disclosed methods involve contact between an oligourethane solution and an analyte, wherein interactions between the oligourethane chemical probe solution and the analyte result in unique data patterns that can be interpreted to identify or differentiate the analyte.

Description

DESCRIPTION
SURFACE PATTERNING WITH SEQUENCE DEFINED, SELF
SEQUENCING OLIGOURETHANES
REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the priority benefit of United States provisional application number 63/561,277, filed March 4, 2024, the entire contents of which is incorporated herein by reference.
STATEMENT OF FEDERALLY SPONSORED RESEARCH
[0002] The invention described herein was made in the performance of work under a NASA contract and by an employee of the United States Government and is subject to the provisions of Public Law 96-517 (35 U.S.C. §202) and may be manufactured and used by or for the Government for governmental purposes without the payment of any royalties thereon or therefore.
BACKGROUND
1. Field
[0003] The present invention relates generally to the field of analytical chemistry. More particularly, it concerns methods and compositions for detecting and/or identifying an analyte.
2. Description of Related Art
[0004] The senses used by animals are typically classified into five categories: vision, smell, taste, hearing, and touch. Recent reports on optical (Chen et al., 2022; Liao et al., 2022; Huang et al., 2023) and auditory (Lenk, 2024) sensors are working towards emulating their biological counterparts. The chemical senses, taste and smell, are akin to using supramolecular receptors for binding and eliciting responses, which are typically detected with electrochemistry or spectroscopy, such as with electronic noses and tongues (Rabehi et al., 2024; Li et al., 2023; Ye et al., 2021; Li et al., 2019; Geng et al., 2019; Podrazka et al., 2017; Tahara et al., 2013; Albert et al., 2000). These mammalian senses do not rely on a single receptor for each type of chemical that to be identified (Gravina et al., 2013; Buck et al., 2004). Human noses and tongues contain hundreds of receptors which bind in a cross-reactive manner to odorant and tastant molecules. Over lifetimes, brains are trained to interpret the combination of receptor responses as a specific smell and taste.
[0005] Differential sensing is an analytical technique inspired by these two chemicalbased senses (Jo et al., 2024; Fargher et al., 2023; Wong et al., 2019; Lavigne et al., 2001). This approach to sensing has become a highly successful technique for individual analyte detection (Sergeant et al., 2023; Zeng et al., 2022; Harrison et al., 2021; Li et al., 2020; Liu et al., 2010) and mixture differentiation (Hoon Lee et al., 2024; Jiang et al., 2022; Crook et al., 2021; Suslick et al., 2010; Palacios et al., 2007). Instead of relying on a single, highly selective supramolecular receptor, differential sensing employs an array of sensors which have different responses to an analyte and mixtures thereof. Instead of detection using a single probe, the pattern of responses from a sensor array are interpreted by various chemometric protocols.
[0006] The sense of touch operates via mechanosensors in our skin which detect sensations such as pressure, vibrations, as well as skin indentation, stretch, and slip (Johansson et al., 2009; Johansson et al., 1983). Recently, tactile sensor arrays have been used to detect pressure, strain, and temperature, with applications in electronic skin (Lee et al., 2020) and assessing fruit ripeness (Zhang et al., 2024). However, the human sense of touch can differentiate surfaces not only by their bulk physical properties, but also by their surface chemistry (Carpenter et al., 2018). The differentiation of surfaces arises from adhesion forces produced from a tapping motion, or discerned through slip-stick friction forces made from sliding a finger over the surface. Methods which mimic the human sense of touch could represent a useful route to differentiating surfaces. Furthermore, there remains an unmet need for analytical techniques which expand the dimensionality of the analysis, thereby facilitating improved classification of samples and/or analytes.
SUMMARY
[0007] The present disclosure provides methods of identifying a surface, such as a surface of a sample, by contacting the surface with a chemical probe comprising at least one oligourethane, preferably a pool or pools of oligourethanes. The interaction between the surface and the chemical probe allows the skilled artisan to recognize the surface. The chemical probes of the present are adaptable to incorporate a range of functionality, which aids in accessing distinguishable patterns of interactions between surface and chemical probe. The present oligourethane chemical probes are more easily characterizable than other polymers that could be useful in such methods, such as peptides. In addition, the present methods provide a benefit in that the chemical probes can be identified at a picomolar concentration, and as such only small quantities of probes are required.
[0008] In one aspect, the present disclosure provides methods of determining the identity of a surface comprising:
(A) loading the surface into a chamber; and
(B) exposing a length of the surface in the chamber to a chemical probe, wherein the chemical probe comprises a solution of one or more oligourethanes in the presence of a mobile phase;
(C) identifying the chemical probe and/or measuring a retention time of the chemical probe; and
(D) correlating the identity of the chemical probe or the retention time of the chemical probe to the identity of the surface.
[0009] In some embodiments, the oligourethane is a polymer comprising at least two monomer units of the formula R O , wherein R is an independently selected chemical group.
[0010] In some embodiments, the oligourethane is a polymer comprising at least two unique monomer units of the formula R O , wherein R is an independently selected chemical group. [0011] In some embodiments, the oligourethane comprises from about three to about 50 monomer units. In further embodiments, the oligourethane comprises from about 5 urethane moieties to about 15 monomer units. In still further embodiments, the oligourethane comprises about 10 monomer units.
[0012] In some embodiments, the chemical group comprises an amino group, an alcohol group, a C1-C12 alkyl group, a C1-C12 cycloalkyl group, a C1-C12 heterocycloalkyl group, a C2-C12 alkenyl group, a C2-C12 alkynyl group, a C1-C12 aryl group, a C1-C12 aralkyl group, a C1-C12 heteroaryl group, a C1-C12 heteroaralkyl group, a C1-C12 alkylamino group, a C2-C24 dialkylamino group, a guanidyl group, a hydroxide group, a C1-C12 alkoxide group, a C1-C12 cycloalkoxide group, a mercaptate group, a sulfate group, a phosphate group, or a C1-C12 carboxylate group, or a substituted version of any of these groups, wherein the substituted version includes a polar functional group, a halide, a hydrogen bond donating group, a hydrogen bond accepting group, an acidic group, or a basic group.
[0013] In some embodiments, at least one of the monomer units has an R group that comprises an aromatic group. In some embodiments, at least one of the monomer units has an R group that is an aromatic group. In some embodiments, at least one of the monomer units has an R group selected from among a C1-C12 aryl group, a C1-C12 heteroaryl group, a Cl- C12 aralkyl group, a C1-C12 heteroaralkyl group, or a substituted version of any of these groups.
[0014] In some embodiments, at least one of the monomer units has an R group that comprises an aliphatic group. In some embodiments, at least one of the monomer units has an R group that is an aliphatic group. In further embodiments, at least one of the monomer units has an R group selected from among a C1-C12 alkyl group, a C1-C12 cycloalkyl group, a Cl- C12 heterocycloalkyl group, or a substituted version of either of these groups.
[0015] In some embodiments, at least one of the monomer units has an R group that comprises an unsaturated group. In some embodiments, at least one of the monomer units has an R group that is an unsaturated group. In some embodiments, at least one of the monomer units has an R group selected from among a C2-C12 alkenyl group, a C2-C12 alkynyl group, or a substituted version of either of these groups.
[0016] In some embodiments, at least one of the monomer units has an R group comprising a positive charge. In some embodiments, at least one of the monomer units has an R group comprising a protonated amino group, a protonated C1-C12 alkylamino group, a protonated C2-C24 dialkylamino group, a protonated guanidyl group, or a substituted version of any of these groups.
[0017] In some embodiments, at least one of the monomer units has an R group comprising a negative charge. In some embodiments, at least one of the monomer units has an R group comprising a hydroxide group, a C1-C12 alkoxide group, a C1-C12 cycloalkoxide group, a mercaptate group, a sulfate group, a phosphate group, or a C1-C12 carboxylate group, a substituted version of any of these groups.
[0018] In some embodiments, at least one of the monomer units has an R group comprising a protecting group.
[0019] In some embodiments, identifying the chemical probe comprises degrading the chemical probe to obtain a degraded solution. In some embodiments, the degraded solution comprises at least one oxazolidinone. In some embodiments, the method further comprises identifying an oxazolidinone in the degraded solution or the composition of oxazolidinone in the degraded solution. In some embodiments, an oxazolidinone in the degraded solution or the composition of oxazolidinone in the degraded solution is identified by spectrometry.
[0020] In some embodiments, the spectrometry is mass spectrometry (MS). In some embodiments, the mass spectrometry is a liquid chromatography mass spectrometry (LC-MS). In some embodiments, the identity of the chemical probe or the retention time of the chemical probe is correlated with the identity of the surface through a microprocessor.
[0021] In some embodiments, the identity of the chemical probe to the identity of the surface comprises correlating the identity of an oxazolidinone in the degraded solution or the composition of oxazolidinone in the degraded solution to the identity of the surface. In some embodiments, the identity of the oxazolidinone in the degraded solution or the composition of oxazolidinone in the degraded solution is correlated with the identity of the surface through a microprocessor. In some embodiments, the surface is a soil sample. In some embodiments, the surface comprises a protein. In some embodiments, the surface comprises an enzyme. In some embodiments, the surface comprises an antibody. In some embodiments, the surface comprises a cell. In some embodiments, the surface comprises a virus. In some embodiments, the surface comprises a biopolymer. In some embodiments, the surface comprises a polymer. In some embodiments, the surface comprises an organism. In some embodiments, the surface comprises rock. In some embodiments, the surface comprises material. In some embodiments, the surface comprises a mixture of any of the surface alternatives provided above.
[0022] In another aspect, the present disclosure provides methods of synthesizing a library of oligourethanes for identifying or detecting a surface, wherein the method comprises:
A) coupling at least two unique monomer units of the formula: in a coupling reaction mixture to provide a pool of oligourethanes, wherein: the coupling reaction mixture comprises the unique monomer units and a solvent;
R is a chemical group;
Ri and R2 are each independently hydrogen or a protecting group, or Ri and R2 are taken together and are a protecting group; and
Rs is an activating group; wherein the unique monomer units in the coupling mixture do not have the same R group; and
B) combining at least one pool of oligourethanes to provide the library of oligourethanes.
[0023] In some embodiments, the coupling reaction mixture comprises between 2 and 50 unique monomer units. In some embodiments, the coupling reaction mixture comprises between 5 and 15 unique monomer units. In some embodiments, the coupling reaction mixture comprises 10 unique monomer units. In some embodiments, the library comprises between 5 and 20 pools of oligourethanes. In some embodiments, the library comprises about 16 pools of oligourethanes. In some embodiments, the coupling mixture for each pool of oligourethanes has a different molar ratio of monomer units.
[0024] In some embodiments, at least one of the coupling mixtures comprises at least a first unique monomer unit at a higher concentration than at least a second unique monomer unit. In some embodiments, between 5 and 20 of the coupling mixtures comprise at least a first unique monomer unit at a higher concentration than at least a second unique monomer unit. In some embodiments, 15 of the coupling mixtures comprise at least a first unique monomer unit at a higher concentration than at least a second unique monomer unit. In some embodiments, the first unique monomer unit has a concentration between about 2 and about 30 times higher than concentration of the second unique monomer unit. In some embodiments, the first unique monomer unit has a concentration between about 4 and about 30 times higher than the concentration of the second unique monomer unit. In some embodiments, the first unique monomer unit has a concentration about 4, about 5, about 6, or about 7 times higher than the concentration of the second unique monomer unit.
[0025] In some embodiments, the R group of the first monomer unit is selected from among optionally protonated -Nth, -OH, a C1-C12 alkyl group, a C1-C12 cycloalkyl group, a C1-C12 heterocycloalkyl group, a C1-C12 alkoxy group, a C2-C12 alkenyl group, a C2-C12 alkynyl group, a C1-C12 acyl group, a C1-C12 aryl group, a C1-C12 aralkyl group, a C1-C12 heteroaryl group, a C1-C12 heteroaralkyl group, an optionally protonated C1-C12 alkylamino group, an optionally protonated C2-C24 dialkylamino group, an optionally protonated guanidyl group, a hydroxide group, a Cl -Cl 2 alkoxide group, a Cl -Cl 2 cycloalkoxide group, a mercaptate group, a sulfate group, a phosphate group, a C1-C12 carboxylate group or a substituted version of any of these groups, wherein the substituted version includes a polar functional group, a halide, a hydrogen bond donating group, a hydrogen bond accepting group, an acid group, or a basic group.
[0026] In some embodiments, the R group of the first monomer unit is selected from among a C1-C12 aryl group, a C1-C12 heteroaryl group, a C1-C12 aralkyl group, a C1-C12 heteroaralkyl group, or a substituted version of any of these groups.
[0027] In some embodiments, the R group of the first monomer unit is selected from among a C1-C12 alkyl group, a C1-C12 cycloalkyl group, a C1-C12 heterocycloalkyl group, or a substituted version of either of these groups. In some embodiments, the R group of the first monomer unit is selected from among a C2-C12 alkenyl group, a C2-C12 alkynyl group, or a substituted version of either of these groups. In some embodiments, the R group of the first monomer unit comprises a positive charge. In some embodiments, the R group of the first monomer unit comprises an optionally protonated amino group, an optionally protonated Cl- C12 alkylamino group, an optionally protonated C2-C24 dialkylamino group, an optionally protonated guanidyl group, or a substituted version of any of these groups. In some embodiments, the R group of the first monomer unit comprises a negative charge. In some embodiments, the R group of the first monomer unit comprises a hydroxide group, a C1-C12 alkoxide group, a C1-C12 cycloalkoxide group, a mercaptate group, a sulfate group, a sulfoxide group, a phosphate group, or a carboxylate group, a substituted version of any of these groups. In some embodiments, the R group of a monomer unit comprises a protecting group.
[0028] In some embodiments, wherein at least one of the coupling mixtures comprises at least a first monomer unit and a third monomer unit at a higher concentration than at least a second monomer unit. In some embodiments, between 5 and 20 of the coupling mixtures comprise at least a first monomer unit and a third monomer unit at a higher concentration than at least a second monomer unit. In some embodiments, 15 of the coupling mixtures of the pools of oligourethanes of the chemical probe comprises at least a first monomer unit and a third monomer unit at a higher concentration than at least a second monomer unit.
[0029] In some embodiments, the R group of the third monomer unit is selected from among optionally protonated -NH2, -OH, a C1-C12 alkyl group, a C1-C12 cycloalkyl group, a C1-C12 heterocycloalkyl group, a C1-C12 alkoxy group, a C2-C12 alkenyl group, a C2-C12 alkynyl group, a C1 -C12 acyl group, a C1-C12 aryl group, a C1-C12 aralkyl group, a C1 -C12 heteroaryl group, a C1-C12 heteroaralkyl group, an optionally protonated C1-C12 alkylamino group, an optionally protonated C2-C24 dialkylamino group, an optionally protonated guanidyl group, a hydroxide group, a Cl -Cl 2 alkoxide group, a Cl -Cl 2 cycloalkoxide group, a mercaptate group, a sulfate group, a phosphate group, a C1-C12 carboxylate group or a substituted version of any of these groups, wherein the substituted version includes a polar functional group, a halide, a hydrogen bond donating group, a hydrogen bond accepting group, an acid group, or a basic group.
[0030] In some embodiments, the R group of the third monomer unit is selected from among a C1-C12 aryl group, a C1-C12 heteroaryl group, a C1-C12 aralkyl group, a C1-C12 heteroaralkyl group, or a substituted version of any of these groups.
[0031] In some embodiments, the R group of the third monomer unit is selected from among a C1-C12 alkyl group, a C1-C12 cycloalkyl group, a C1-C12 heterocycloalkyl group, or a substituted version of either of these groups.
[0032] In some embodiments, the R group of the third monomer unit is selected from among a C2-C12 alkenyl group, a C2-C12 alkynyl group, or a substituted version of either of these groups. [0033] In some embodiments, the R group of the third monomer unit comprises a positive charge. In some embodiments, the R group of the third monomer unit comprises a protonated amino group, a protonated C1-C12 alkylamino group, a protonated C2-C24 dialkylamino group, a protonated guanidyl group, or a substituted version of any of these groups.
[0034] In some embodiments, the R group of the third monomer unit comprises a negative charge.In some embodiments, the R group of the third monomer unit comprises a hydroxide group, a Cl -Cl 2 alkoxide group, a Cl -Cl 2 cycloalkoxide group, a mercaptate group, a sulfate group, a phosphate group, or a carboxylate group, a substituted version of any of these groups.
[0035] In some embodiments, the R group of a monomer unit comprises a protecting group.
[0036] In some embodiments, the R group of the first monomer unit comprises an aliphatic group; and the R group of the third monomer unit comprises an aromatic group. In some embodiments, the R group of the first monomer unit is selected from among a C1-C12 alkyl group, a C1-C12 cycloalkyl group, a C1-C12 heterocycloalkyl group, or a substituted version of either of these groups; and the R group of the third monomer unit is selected from among a C1-C12 aryl group, a C1-C12 heteroaryl group, a C1-C12 aralkyl group, a C1-C12 heteroaralkyl group, or a substituted version of any of these groups.
[0037] In some embodiments, the R group of the first monomer unit comprises an aliphatic group; and the R group of the third monomer unit comprises an unsaturated group. In some embodiments, the R group of the first monomer unit is selected from among a Cl -Cl 2 alkyl group, a C1-C12 cycloalkyl group, a C1-C12 heterocycloalkyl group, or a substituted version of either of these groups; and the R group of the third monomer unit is selected from among a C2-C12 alkenyl group, a C2-C12 alkynyl group, or a substituted version of either of these groups.
[0038] In some embodiments, the R group of the first monomer unit comprises an aliphatic group; and the R group of the third monomer unit comprises a positive charge. In some embodiments, the R group of the first monomer unit is selected from among a Cl -Cl 2 alkyl group, a C1-C12 cycloalkyl group, a C1-C12 heterocycloalkyl group, or a substituted version of either of these groups; and the R group of the third monomer unit is selected from among a protonated amino group, a protonated C1-C12 alkylamino group, a protonated C2- C24 dialkylamino group, a protonated guanidyl group, or a substituted version of any of these groups.
[0039] In some embodiments, the R group of the first monomer unit comprises an aliphatic group; and the R group of the third monomer unit comprises a negative charge. In some embodiments, the R group of the first monomer unit is selected from among a Cl -Cl 2 alkyl group, a C1-C12 cycloalkyl group, a C1-C12 heterocycloalkyl group, or a substituted version of either of these groups; and the R group of the third monomer unit is selected from among hydroxide group, a C1-C12 alkoxide group, a C1-C12 cycloalkoxide group, a mercaptate group, a sulfate group, a phosphate group, or a carboxylate group, a substituted version of any of these groups.
[0040] In some embodiments, the R group of the first monomer unit comprises an aromatic group; and the R group of the third monomer unit comprises an unsaturated group. In some embodiments, the R group of the first monomer unit is selected from among a C1 -C12 aryl group, a C1-C12 heteroaryl group, a C1-C12 aralkyl group, a C1-C12 heteroaralkyl group, or a substituted version of any of these groups; and the R group of the third monomer unit is selected from among aC2-C12 alkenyl group, a C2-C12 alkynyl group, or a substituted version of either of these groups.
[0041] In some embodiments, the R group of the first monomer unit comprises an aromatic group; and the R group of the third monomer unit comprises a positive charge. In some embodiments, the R group of the first monomer unit is selected from among a Cl -Cl 2 aryl group, a C1-C12 heteroaryl group, a C1-C12 aralkyl group, a C1-C12 heteroaralkyl group, or a substituted version of any of these groups; and the R group of the third monomer unit is selected from among a protonated amino group, a protonated C1-C12 alkylamino group, a protonated C2-C24 dialkylamino group, a protonated guanidyl group, or a substituted version of any of these groups.
[0042] In some embodiments, the R group of the first monomer unit comprises an aromatic group; and the R group of the third monomer unit comprises a negative charge. In some embodiments, the R group of the first monomer unit is selected from among a Cl -Cl 2 aryl group, a C1-C12 heteroaryl group, a C1-C12 aralkyl group, a C1-C12 heteroaralkyl group, or a substituted version of any of these groups; and the R group of the third monomer unit is selected from among hydroxide group, a C1-C12 alkoxide group, a C1-C12 cycloalkoxide group, a mercaptate group, a sulfate group, a phosphate group, or a carboxylate group, a substituted version of any of these groups.
[0043] In some embodiments, the R group of the first monomer unit comprises an unsaturated group; and the R group of the third monomer unit comprises a positive charge. In some embodiments, the R group of the first monomer unit is selected from among a C2-C12 alkenyl group, a C2-C12 alkynyl group, or a substituted version of either of these groups; and the R group of the third monomer unit is selected from among a protonated amino group, a protonated C1-C12 alkylamino group, a protonated C2-C24 dialkylamino group, a protonated guanidyl group, or a substituted version of any of these groups.
[0044] In some embodiments, the R group of the first monomer unit comprises an unsaturated group; and the R group of the third monomer unit comprises a negative charge. In some embodiments, the R group of the first monomer unit is selected from among a C2-C12 alkenyl group, a C2-C12 alkynyl group, or a substituted version of either of these groups; and the R group of the third monomer unit is selected from among hydroxide group, a C1-C12 alkoxide group, a C1-C12 cycloalkoxide group, a mercaptate group, a sulfate group, a phosphate group, or a carboxylate group, a substituted version of any of these groups
[0045] In some embodiments, the R group of the first monomer unit comprises a positive charge; and the R group of the third monomer unit comprises a negative charge. In some embodiments, the R group of the first monomer unit is selected from among a protonated amino group, a protonated C1-C12 alkylamino group, a protonated C2-C24 dialkylamino group, a protonated guanidyl group, or a substituted version of any of these groups; and the R group of the third monomer unit is selected from among hydroxide group, a Cl -Cl 2 alkoxide group, a C1-C12 cycloalkoxide group, a mercaptate group, a sulfate group, a phosphate group, or a carboxylate group, a substituted version of any of these groups.
[0046] In some embodiments, the total concentration of between about 10% and about 60% of the unique monomer units in the coupling mixture is about 2, about 3, about 4, about 5, about 6, about 7, or about 8 times higher than the total concentration of the other unique monomer units in the coupling mixture. In some embodiments, the sum of the concentrations of about 30% of the unique monomer units in the coupling mixture is about 2, about 3, about 4, about 5, about 6, about 7, or about 8 times higher than the total concentration of the other unique monomer units in the coupling mixture.
[0047] In another aspect, the present disclosure provides biased libraries of oligourethanes for detecting or identifying a surface, wherein the biased library comprises at least one pool of oligourethanes synthesized according to any one of the embodiments disclosed above.
[0048] In another aspect, the present disclosure provides methods of determining the identity of a surface comprising:
(A) loading the surface into a chamber; and
(B) exposing a length of the surface in the chamber to a chemical probe, wherein the chemical probe comprises a solution of one or more unique oligourethanes, in the presence of a mobile phase;
(C) identifying the chemical probe and/or measuring a retention time of the chemical probe; and
(D) correlating the identity of the chemical probe or the retention time of the chemical probe to the identity of the surface.
[0049] In some embodiments, the chemical probe comprises from about three to about 20 unique oligourethanes. In further embodiments, the chemical probe comprises from about five to about 15 unique oligourethanes. In some embodiments, the unique oligourethane comprises a polyurethane, wherein the polyurethane comprises from about three to about 50 repeating units. In further embodiments, the polyurethane comprises from about 5 repeating units to about 15 repeating units.
[0050] In some embodiments, the method further comprises degrading the oligourethane to obtain an oxazolidinone. In some embodiments, the oxazolidinone is identified by spectrometry. In some embodiments, the spectrometry is mass spectrometry (MS). In further embodiments, the mass spectrometry is a liquid chromatography mass spectrometry (LC-MS).
[0051] In some embodiments, the oligourethane comprises a chemical group attached to a urethane moiety. In some embodiments, each oligourethane comprises a distinct chemical group. In some embodiments, the chemical group is a C1-C12 alkyl, a C1-C12 cycloalkyl group, a C1-C12 alkenyl, a C1-C12 alkynyl, a C1-C12 aryl, a C1-C12 aralkyl, a C1-C12 heteroaryl, a C1-C12 heteroaralkyl, or a substituted version of any of these groups, wherein the substituted version includes a polar functional group, a halide, a hydrogen bond donating group, a hydrogen bond accepting group, an acid group, or a basic group. In some embodiments, the oligourethane comprises a single repeating unit in each oligourethane. In some embodiments, the oligourethane comprises two or more repeating units in each oligourethane. In some embodiments, the oligourethane comprises a distinct sequence of different repeating units. In some embodiments, the identity of the chemical probe or the
retention time of the chemical probe is correlated with the identity of the surface through a microprocessor. In some embodiments, the surface is a soil sample.
[0052] In another aspect, the present disclosure provides methods of determining the presence of a chemical analyte in a sample comprising:
(A) loading the sample into a chamber; and
(B) exposing a length of the sample in the chamber to a chemical probe, wherein the chemical probe comprises a solution of one or more unique oligourethanes, in the presence of a mobile phase;
(C) identifying the chemical probe and/or measuring a retention time of the chemical probe; and
(D) correlating the identity of the chemical probe or the retention time of the chemical probe to the identity of the substrate in the sample or to the identity of the sample.
[0053] In another aspect, the present disclosure provides methods of determining the identity of a substrate in a sample or the identity of the sample comprising:
(A) loading the sample into a chamber; and
(B) exposing a length of the sample in the chamber to a chemical probe, wherein the chemical probe comprises a solution of one or more unique oligourethanes, in the presence of a mobile phase;
(C) identifying the chemical probe and/or measuring a retention time of the chemical probe; and
(D) correlating the identity of the chemical probe or the retention time of the chemical probe to the identity of the sample.
[0054] In another aspect, the present disclosure provides methods of determining the identity of a substrate in a sample or the identity of the sample comprising:
(A) loading the sample into a chamber; and (B) exposing a length of the sample in the chamber to a chemical probe, wherein the chemical probe comprises a solution of one or more unique oligourethanes, in the presence of a mobile phase, wherein the oligourethane comprises one or more distinct sequences;
(C) identifying the chemical probe and/or measuring a retention time of the chemical probe; and
(D) correlating the identity of the chemical probe or the retention time of the chemical probe to the identity of the substrate in the sample or to the identity of the sample.
[0055] As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.
[0056] As used herein, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.
[0057] The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” As used herein “another” may mean at least a second or more.
[0058] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the inherent variation in the method being employed to determine the value, the variation that exists among the study subjects, or a value that is within 10% of a stated value.
[0059] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF DRAWINGS
[0061] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
[0062] FIGS. 1A-1E illustrate the design of illustrative oligourethane libraries useful with the methods disclosed herein and the workflow of the experiments described in the Examples section. FIG. 1A) Ten OU monomers were synthesized to promote functional group diversity. Monomers were chosen to have aliphatic (R = Leu, Cha), aromatic (R = BnTrz, Tyr(tBu), Trp(Boc)), alkynyl (R = PGly), or polar protic (R = Arg(Pbf), Lys(Boc), His(Trt), Asp(tBu)) side chains represented. FIG. IB) Monomers were mixed in different ratios to create coupling mixtures which were biased towards aliphatic, aromatic, alkynyl, and/or polar protic monomers. FIG. 1C) Biased monomer coupling mixtures were used in solid-phase OU synthesis to make 16 libraries of OUs that comprised a statistically random sequences of monomers, but which were biased towards specific monomers and/or classes of monomers as outlined in FIG. 1A-1B. FIG. ID) After cleavage from the solid support, all libraries were combined to create a pool of OUs with 108 possible different sequences. FIG. IE) A schematic of the experimental workflow. A portion of the pool of oligourethane probes were sent through a column packed with an analyte (or stationary phase) of interest. Eluting OUs were separated into 1 mL fractions and then completely degraded to 5-membered oxazolidinone rings. The identity and relative amount of oxazolidinones in each fraction was determined by LC-MS EIC.
[0063] FIG. 2 is a bar chart showing the relative composition of monomers in the coupling mixtures for each of the 16 biased libraries.
[0064] FIGS. 3A-3D show the heat maps of the oxazolidinone LC-MS EIC peak areas vs. fraction volume and demonstrate the effect of each normalizing operation on fraction component evaluation. FIG. 3A) Raw oxazolidinone LC-MS EIC peak area values. FIG. 3B) LC-MS EIC peak area values which have been divided by the value for VaLoxazolidinone in each fraction. FIG. 3C) Internal standard adjusted values are min-max adjusted for each oxazolidnone, for a better comparison across all oxazolidinones. FIG. 3D) Oxazolidinone LC- MS EIC peak area values were divided by that of Phe-oxazolidinone and the min-max adjusted for each oxazolidinone. The heat map shown in FIG. 3D demonstrates that this results in an improved evaluation of monomer OU composition of the eluent.
[0065] FIG. 4 shows heat maps of standard adjusted oxazolidinone EIC peak areas for all 32 fractions and all five substrates.
[0066] FIG. 5 shows heat maps of Phe-oxazolidinone and min-max adjusted oxazolidinone EIC peak areas across all fractions, oxazolidinones, and column substrates.
[0067] FIG. 6 is a PCA plot using the min-max standard adjusted data, which shows poor column substrate differentiation.
[0068] FIG. 7 shows standard normalized oxazolidinone EIC peak areas for fractions 5, 7, 10, 15, and 20 for all five replicates and all five substrates. See Example 4 section xviii. When used with PCA, these data points gave overlapping clusters with large scatter (FIG. 6).
[0069] FIG. 8 shows the results of PCA using the min-max Phe-ox adjusted data shows clear clustering of each unique column stationary phase.
[0070] FIG. 9 shows the top 10 variables for PCI, PC2 and PC3, plotted as vectors from the graph origin. Values for each loading can vary from -1 to 1, with a value near 0 contributing very little to the principal component axis, while a value near -1 or 1 strongly contributing to the principal component axis.
[0071] FIGS. 10A-10C show bar charts depicting the top 25 variables (i.e. oxazolidinone and fraction number) contributing to FIG. 10A) PCI; FIG. 10B) PC2; FIG. 10C) PC3.
[0072] FIG. 11 shows the 3D LDA of the Phe-adjusted oxazolidinone EIC peaks, which reveals tight clustering of each of the five column substrates. A jackknife analysis was able to classify substrates with 96% accuracy.
[0073] FIG. 12 shows the monomers that were used in solid phase oligourethane synthesis. Monomers were chosen to introduce functional group chemical diversity into the oligourethanes.
[0074] FIG. 13 provides overlayed LC spectra of all degraded OU trimers at 280 nm. After subjecting each trimer to base and heat according to the general degradation procedures outlined in the Materials and Methods example below, all trimers had chain-end degraded to the methyl tyrisinol acetamide.
[0075] FIG. 14 is a heat map of adjusted R-oxazolidinone EIC values in the degradation mixture of each of the 16 directed libraries. These heat maps act as an estimation of the relative monomer composition of the OU in each directed library.
[0076] FIG. 15 shows the MSD1 total ion chromatogram and mass spectrum from 6- 14 minutes, taken from the LC-MS analysis of the combined pool of all 16 OU directed libraries.
[0077] FIG. 16 shows the overlayed FTIR spectra of BSA, acyl chloride functionalized silica, and the isolated reaction product. The isolated reaction product has IR stretches which are present in both starting materials.
[0078] FIG. 17 provides overlayed MSD1 total ion chromatograms demonstrating that bovine serum albumin does not leach out of the silica gel support following the reaction conditions outlined in Example 4 section xvi. The MSD1 mass spectrum of the peak at 6.9 minutes in the BSA sample shows a mass spectrum pattern indicative of a protein. The MSD1 mass spectrum at this retention time in the acyl chloride functionalized silica filtrate and the reaction product filtrate do not show a similar pattern and there is no significant peak in the MSD1 total ion chromatogram.
[0079] FIG. 18 shows retention times of all oxazolidinone EIC peaks for all 32 fractions for all five substrates as described in section xvii of Example 4. If an EIC peak was not recorded because the signal-to-noise ratio was too low, it was given a retention time of 0.
[0080] FIG. 19 shows that the ratio of Tyr(OMe)Ac/Phe LC-MS EIC peak areas for the sensor array column chromatography outlined in section xvii of Example 4 indicates most samples were degraded to a similar extent. Low Tyr(OMe)Ac/Phe ratios, for example charcoal fractions 4-6, suggest incomplete degradation of OUs. A Tyr(OMe)Ac/Phe ratio of 0, for example charcoal fractions 1 and 2, was assigned if a peak could not be assigned for either Phe or Tyr(OMe)Ac due to a high signal-to-noise ratio.
[0081] FIG. 20 shows plots of retention times of the 13 LC-MS EIC peaks recorded in each of the five samples for the five substrates used in PCA as described in Example 4 section xviii. If an EIC peak was not recorded because the signal-to-noise ratio was too low, it was given a retention time of 0.
[0082] FIG. 21 shows that the ratio of Tyr(OMe)Ac/Phe LC-MS EIC peak areas for the samples used in the PCA outlined in section xviii of Example 4 shows that most samples were degraded to a similar extent. Low Tyr(OMe)Ac/Phe ratios, for example charcoal 1-5 fraction 5, suggest incomplete degradation of OUs. A Tyr(OMe)Ac/Phe ratio of 0 (e.g. celite 4 fraction 20) was assigned if a peak could not be assigned for either Phe or Tyr(OMe)Ac due to a high signal-to-noise ratio.
[0083] FIG. 22 shows the Phe-oxazolidinone adjusted oxazolidinone EIC peak areas for fractions 5, 7, 10, 15, and 20 for all five replicates and all five substrates. When used with PCA, these data points differentiated each substrate type (FIG. 8).
DETAILED DESCRIPTION
[0084] The present disclosure relates to methods of distinguishing and/or identifying an analyte using a chemical probe comprising an oligourethane or, preferably, a mixture of oligourethanes. The chemical probe is passed over the analyte in a contact area, such as in a chromatography column. The composition of the chemical probe leaving the contact area varies over time in a surface-specific manner due to the interactions between the analyte and the chemical probe. Measurement and deconvolution of the chemical probe composition over time provides an analyte-specific method of characterizing and identifying an analyte. In this way, the inventors presently provide an agnostic approach to allow differentiation of any analyte, such as a surface. The presently disclosed methods represents an improvement in differentiating analytes that do not have obvious binding sites, such as many bulk surfaces. The chemical probes of the present methods harness a chemical diversity available for the monomers of the constituent oligourethanes that is not seen in other comparable methods known in the art. I. Oligourethanes (OUs)
[0085] The present disclosure relates to the use of chemical probes which comprise a solution of one or more unique oligourethanes. The chemical probe of the present disclosure is useful to, for example, identify an analyte and/or distinguish analytes from each other. The oligourethanes of the presently disclosed chemical probes chain-end degrade in the presence
of base and heat through a 5-exo-trig intramolecular cyclization from the terminal alcohol as shown in Scheme 1.
Scheme 1: Synthesis and degradation of oligourethanes (OUs) [0086] Through this mechanism, the oligourethanes of the presently disclosed chemical probes are degraded into monomer-derived 5-membered carbamate rings called an oxazolidinone. In some embodiments, the chemical probe comprises at least one oxazolidinone derived from an oligourethane. Oxazolidinones formed by the degradation of oligourethanes of the presently disclosed chemical probes may be identified through any means known to the skilled artisan, including by mass spectrometry. Oligourethanes of the presently disclosed chemical probes are able to be synthesized according to any method known to the skilled artisan. For example, oligourethanes may be synthesized from a solid-support with Fmoc- protected, p-nitrophenyl carbonate activated amino alcohols (Fmoc-R(PG)-PNOC) with a large variety of R-functional groups. Oligourethanes are favorable as a compound for use in the presently disclosed analytical technique because synthetic routes to these compounds allow control over the length, sequence, and functional group composition of the oligourethane. Solid phase peptide synthesis methods are well known and practiced in the art. In such methods the synthesis of oligourethanes of the present disclosure can be carried out by sequentially
incorporating the desired monomers one at a time according to the general principles of solid phase methods.
In some embodiments, the oligourethanes of the present methods comprise at least one urethane monomer of the formula R O , wherein R is hydrogen or a functional group.
[0087] Principles of peptide synthesis, which involve stepwise coupling reactions between amino groups and carbonyl groups, may be relevant to the synthesis of oligourethanes. In chemical syntheses of peptides, reactive side chain groups of the various amino acid residues are protected with suitable protecting groups, which prevent a chemical reaction from occurring at that site until the protecting group is removed. To form oligourethanes of presently disclosed R chemical probes, urethane monomers of the form , or mixtures thereof, are sequentially coupled to form the oligourethane. In some embodiments, the amino group of the formula, also referenced below as the backbone amino group, is protected with a protecting group. In some embodiments, the R group comprises a reactive functional group. In some embodiments, a reactive functional group of the R group is protected with a protecting group. In preferred embodiments, the alcohol group of the urethane monomer is activated with an activating group, such as p-nitrophenyl carbonate (PNOC).
[0088] As mentioned above, in some embodiments, the side chain or R group of the urethane monomer is protected. In some embodiments, the backbone amino of the urethane monomer is protected while that entity reacts at a carboxyl group, such as the activated alcohol of the urethane monomer, followed by the selective removal of the backbone amino protecting group to allow a subsequent reaction to take place at that site. Specific protecting groups have been disclosed and are known in solid phase synthesis methods and solution phase synthesis methods. The backbone amino group and/or the R group of a urethane monomer may be protected by a suitable protecting group, including a urethane-type protecting group, such as benzyloxycarbonyl and substituted benzyloxycarbonyl, such as p-chlorobenzy loxycarbony 1, /?- nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, /^-biphenyl- isopropoxycarbonyl, 9- fluorenylmethoxycarbonyl (Fmoc) and p-methoxybenzyloxycarbonyl (Moz) and aliphatic urethane-type protecting groups, such as z-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl, isopropoxycarbonyl, and allyloxycarbonyl (Alloc). Fmoc is preferred for backbone amino protection. Guanidino groups may be protected by a suitable protecting group, such as nitro, p-toluenesulfonyl (Tos), benzyloxycarbonyl, pentamethylchromanesulfonyl (Pmc), adamantyloxycarbonyl, pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) and Boc. Pbf and Pmc are preferred protecting groups for Arg. Boc is the preferred protecting group for Lys and Trp. A tert-butyl group is the preferred protecting group for Asp and Tyr. Trityl is the preferred protecting group for His.
[0089] Also as mentioned above, in preferred embodiments, the alcohol group of the urethane monomer is pre-activated to facilitate the coupling reaction with an amino group on the growing oligourethane. Specific activating groups have been disclosed and are known in solid phase synthesis methods and solution phase synthesis methods. In some embodiments, the urethane monomers are activated with p-nitrophenyl carbonate (PNOC).
[0090] Solid phase synthesis is commenced from the C-terminal end by coupling a protected alpha amino acid to a suitable resin. Such starting material is prepared by attaching an alpha amino-protected amino acid by an ester linkage to a p-benzyloxybenzyl alcohol (Wang) resin, a 2-chlorotrityl chloride resin or an oxime resin, by an amide bond between an Fmoc-Linker, such as p-[(R,S)-a-[l-(9H-fhior-en-9-yl)-methoxyformamido]-2,4- dimethyloxybenzyl] -phenoxy acetic acid (Rink linker) to a benzhydrylamine (BHA) resin, or by other means well known in the art. The resins are carried through repetitive cycles as necessary to add urethane monomers sequentially. The alpha amino Fmoc protecting groups are removed under basic conditions, piperidine, piperazine, diethylamine, or morpholine (20- 40% v/v) in A,A-dimethylformamide (DMF) may be used for this purpose.
[0091] Following removal of the alpha amino protecting group, the subsequent protected monomers are coupled stepwise in the desired order to obtain an intermediate, protected oligourethane-resin. The activating reagents used for coupling of the monomers in the solid phase synthesis of the oligourethanes are well known in the art. After the oligourethane is synthesized, if desired, the orthogonally protected side chain protecting groups may be removed using methods well known in the art for further derivatization of the peptide. Typically, orthogonal protecting groups are used as appropriate.
[0092] Reactive groups in an oligourethane can be selectively modified, either during solid phase synthesis or after removal from the resin. For example, oligourethanes can be modified to obtain N-terminal modifications, such as acetylation, while on resin, or may be removed from the resin by use of a cleaving reagent and then modified. Similarly, methods for modifying side chains of amino acids are well known to those skilled in the art. The choice of modifications made to reactive groups present on the oligourethane will be determined, in part, by the characteristics that are desired in the oligourethane.
[0093] In some embodiments, the R group of the urethane monomer may have a particular characteristic or property. In some embodiments, the R group may be, for example, aromatic, aliphatic, or comprise an unsaturated group such as an alkyne. In some embodiments, the R group may have an overall net charge. In some embodiments, the R group may have a positive charge. In some embodiments, the R group may have a negative charge.
[0094] In some embodiments, the chemical probe of the presently disclosed methods may comprise a pool or mixture of unique oligourethanes distinguished by different R groups. The synthesis of the pool of oligourethanes may comprise a coupling step wherein the reaction mixture, referenced herein as the “coupling mixture”, comprises a mixture of urethane monomers. In some embodiments, the coupling mixture is an equimolar mixture of the urethane monomers. In some embodiments, certain urethane monomers have a higher molar concentration in the coupling mixture than other urethane monomers. In some embodiments, the urethane monomers with relatively higher molar concentrations in the coupling mixture (that is, higher molar concentration in comparison to the molar concentration of other urethane monomers in the coupling mixture) have R groups that share a particular characteristic or property. In some embodiments, the urethane monomers with relatively higher molar concentrations have R groups that comprise an aromatic group. In some embodiments, the urethane monomers with relatively higher molar concentrations have R groups that comprise an aliphatic group. In some embodiments, the urethane monomers with relatively higher molar concentrations have R groups that comprise an alkyne group. In some embodiments, the urethane monomers with relatively higher molar concentrations have R groups that have an overall charge. In some embodiments, the urethane monomers with relatively higher molar concentrations have R groups that have an overall positive charge. In some embodiments, the urethane monomers with relatively higher molar concentrations have R groups that have an overall negative charge. Pools of urethane monomers with higher molar concentrations of at least one urethane monomer are termed herein as a biased pool. Equivalent terms to “pool” include “mixture” and “library”. Equivalent terms to “biased” include “directed”. In some embodiments, the one or more urethane monomers having a higher molar concentration has a particular characteristic or property. A pool of urethane monomers may be biased towards one, two, or more characteristics or properties by, for example, having a relatively higher molar concentration of at least one urethane monomer with an R group having each characteristic or property in comparison to urethane monomers with an R group that does not have the characteristic or property. A Oligourethanes of the chemical probes of the present methods may be formed by methods involving coupling mixtures that comprise an equimolar pool of urethane monomers, a biased pool of urethane monomers, a single urethane monomer, or any combination thereof. Biased pools of oligourethanes are formed through the use of coupling mixtures comprising biased pools of urethane monomers. In some embodiments, the total concentration of the monomer units with higher concentration is about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 11 times, about 12 times, about 13 times, about 14 times about 15 times, about 16 times, about 17 times, about 18 times, about 19 times, or about 20 times higher than the sum total of all other monomer units in the coupling mixture, or any range derivable therein
[0095] In some embodiments, the chemical probe of the present disclosure is a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 biased pools of oligourethanes. In some embodiments, the chemical probe of the present disclosure is a combination of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 biased pools of oligourethanes.
[0096] In some embodiments, a biased pool may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 urethane monomers with relatively higher molar concentration in the coupling mixture. In some embodiments, a biased pool may have 1, 2, 3, 4, 5, or 6 urethane monomers with relatively higher molar concentration in the coupling mixture. In some embodiments, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or any range derivable therein of the monomer units of a pool of urethane monomers may have a relatively higher molar concentration in the pool of urethane monomer units than another monomer unit. In some embodiments, about 10%, about 20%, about 30%, or about 40% of monomer units, or any range derivable therein, may have a higher concentration in the pool of urethane monomer units than another monomer unit. In some embodiments, about 30% of monomer units may have a higher concentration in the pool of urethane monomer units than another monomer unit. In some embodiments, the coupling mixture comprises at least one urethane monomer with a concentration that is about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 11 times, about 12 times, about 13 times, about 14 times, about 15 times, about 16 times, about 17 times, about 18 times, about 19 times, or about 20 times as high as the concentration of another urethane monomer in the coupling mixture. In some embodiments, the coupling mixture comprises at least one urethane monomer with a concentration that is about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 times as high as the concentration of another urethane monomer in the coupling mixture. If more than one urethane monomer has a higher molar concentration in the coupling mixture than another urethane monomer, the urethane monomers with relatively higher molar concentration need not have the same molar concentration.
[0097] In some embodiments, the oligourethane of the chemical probe of the present disclosure comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,
73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100 repeating units. In some embodiments, the oligourethane of the chemical probe of the present disclosure comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 repeating units. In some embodiments, the oligourethane of the chemical probe of the present disclosure comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 repeating units. In some embodiments, the oligourethane of the chemical probe of the present disclosure comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 repeating units. In some embodiments, the repeating unit is of the formula described above for the urethane monomer. In some embodiments, the chemical probe of the present disclosure comprises more than one distinct oligourethane. In some embodiments, the chemical probe of the present disclosure comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 distinct oligourethanes. In some embodiments, the chemical probe of the present disclosure comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 distinct oligourethanes. In some embodiments, the chemical probe of the present disclosure comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 distinct oligourethanes. In some embodiments, the present disclosure comprises five or more distinct oligourethanes. In some embodiments, the present disclosure comprises ten or more distinct oligourethanes.
[0098] In some embodiments, the chemical probe of the present disclosure is biased. A biased chemical probe comprises oligourethanes that are formed from at least one coupling step involving a biased pool of urethane monomers, as discussed in more detail above.
[0099] In some embodiments, the chemical probe is degraded to facilitate identification of the components of the chemical probe. For example, oligourethanes can undergo chain-end degradation to oxazolidinone derivatives of the monomer oligourethanes. Further details on this aspect are provided in the Examples. The identity of the resultant degraded chemical probe can be determined and used to characterize the source oligourethane. In this manner, chemical probes comprising oligourethanes represent an improvement over other possible compounds which could be used in a similar manner, such as peptides, which are more difficult to sequence.
[00100] In some embodiments, the present methods involve analysis of the chemical probe and/or the degraded chemical probe. The identity of the chemical probe, the degraded chemical probe, or any portion, fraction, or mixture thereof can be determined using any analytical technique known to the skilled artisan. In some embodiments, the analytical technique is chromatography, such as high-performance liquid chromatography. In some embodiments, the analytical technique is spectrometry, such as mass spectrometry. In some embodiments, the analytical technique is spectroscopy. In some embodiments, the analytical technique is a hybrid technique, such as liquid chromatography-mass spectrometry.
IL Chemical Definitions
Common Definitions
[00101] When used in the context of a chemical group: “hydrogen” means -H; “hydroxy” means -OH; “oxo” means =0; “carbonyl” means -C(=O)-; “carboxy” means -C(=O)OH (also written as -COOH or -CC H); “halo” means independently -F, -Cl, -Br or -I; “amino” means -NH2; “hydroxyamino” means -NHOH; “nitro” means -NO2; imino means =NH; “cyano” means -CN; “isocyanyl” means -N=C=O; “aQdo” means -N3; in a monovalent context “phosphate” means -OP(O)(OH)2 or a deprotonated form thereof; in a divalent context “phosphate” means -OP(O)(OH)O- or a deprotonated form thereof; “sulfate” means -OS(O2)(OH) or a deprotonated form thereof; “mercapto” means -SH; “mercaptate” means a deprotonated mercapto; “thio” means =S; “thiocarbonyl” means -C(=S)~: “sulfonyl means -S(0)2-; and “sulfinyl” means -S(0)-.
[00102] In the context of chemical formulas, the symbol means a single bond, means a double bond, and “=” means triple bond. The symbol — ” represents an optional bond, which if present is either single or double. The symbol represents a single bond or a double bond. Thus, the formula covers, for example,
[00103] It is understood that no one such ring atom forms part of more than one double bond. Furthermore, it is noted that the covalent bond symbol when connecting one or two stereogenic atoms, does not indicate any preferred stereochemistry. Instead, it covers all stereoisomers as well as mixtures thereof. The symbol “ v'wx. ”, when drawn for methyl) indicates a point of attachment of the group. It is noted that the point of attachment is typically only identified in this manner for larger groups in order to assist the reader in unambiguously identifying a point of attachment. The symbol “ ” means a single bond where the group attached to the thick end of the wedge is “out of the page.” The symbol “""HI ” means a single bond where the group attached to the thick end of the wedge is “into the page”. The symbol “ '/vv ” means a single bond where the geometry around a double bond (e.g., either E or Z) is undefined. Both options, as well as combinations thereof are therefore intended. Any undefined valency on an atom of a structure shown in this application implicitly represents a hydrogen atom bonded to that atom. A bold dot on a carbon atom indicates that the hydrogen attached to that carbon is oriented out of the plane of the paper.
[00104] When a variable is depicted as a “floating group” on a ring system, for example, the group “R” in the formula: then the variable may replace any hydrogen atom attached to any of the ring atoms, including a depicted, implied, or expressly defined hydrogen, so long as a stable structure is formed.
[00105] When a variable is depicted as a “floating group” on a fused ring system, as for example the group “R” in the formula: then the variable may replace any hydrogen attached to any of the ring atoms of either of the fused rings unless specified otherwise. Replaceable hydrogens include depicted hydrogens (e.g. , the hydrogen attached to the nitrogen in the formula above), implied hydrogens (e.g., a hydrogen of the formula above that is not shown but understood to be present), expressly defined hydrogens, and optional hydrogens whose presence depends on the identity of a ring atom (e.g., a hydrogen attached to group X, when X equals -CH-), so long as a stable structure is formed. In the example depicted, R may reside on either the 5 -membered or the 6-membered ring of the fused ring system. In the formula above, the subscript letter “y” immediately following the R enclosed in parentheses, represents a numeric variable. Unless specified otherwise, this variable can be 0, 1, 2, or any integer greater than 2, only limited by the maximum number of replaceable hydrogen atoms of the ring or ring system.
[00106] For the chemical groups and compound classes, the number of carbon atoms in the group or class is as indicated as follows: “Cn” or “C=n” defines the exact number (n) of carbon atoms in the group/class. “C<n” defines the maximum number (n) of carbon atoms that can be in the group/class, with the minimum number as small as possible for the group/class in question. For example, it is understood that the minimum number of carbon atoms in the groups “alkyl(c<8)”, “alkanediyl(c<8)”, “heteroaryl(c<s)”, and “acyl(c<8)” is one, the minimum number of carbon atoms in the groups “alkenyl(c<8)”, “alkynyl(c<8)”, and “heterocycloalkyl(c<8)” is two, the minimum number of carbon atoms in the group “cycloalkyl(c<8)” is three, and the minimum number of carbon atoms in the groups “aryl(c<8)” and “arenediyl(c<8)” is six. “Cn-n'” defines both the minimum (n) and maximum number (n') of carbon atoms in the group. Thus, “alkyl(C2-io)” designates those alkyl groups having from 2 to 10 carbon atoms. [00107] These carbon number indicators may precede or follow the chemical groups or class it modifies and it may or may not be enclosed in parenthesis, without signifying any change in meaning. Thus, the terms “Ci-4-alkyl”, “Cl-4-alkyl”, “alkyl(ci-4)”, and “alkyl(c<4)” are all synonymous. Except as noted below, every carbon atom is counted to determine whether the group or compound falls with the specified number of carbon atoms. For example, the group dihexylamino is an example of a dialkylamino(ci2) group; however, it is not an example of a dialkylaminojce) group. Likewise, phenylethyl is an example of an aralkylic=8) group. When any of the chemical groups or compound classes defined herein is modified by the term “substituted”, any carbon atom in the moiety replacing the hydrogen atom is not counted. Thus methoxyhexyl, which has a total of seven carbon atoms, is an example of a substituted alkyl(ci-6). Unless specified otherwise, any chemical group or compound class listed in a claim set without a carbon atom limit has a carbon atom limit of less than or equal to twelve.
[00108] The term “saturated” when used to modify a compound or chemical group means the compound or chemical group has no carbon-carbon double and no carboncarbon triple bonds, except as noted below. When the term is used to modify an atom, it means that the atom is not part of any double or triple bond. In the case of substituted versions of saturated groups, one or more carbon oxygen double bond or a carbon nitrogen double bond may be present. And when such a bond is present, then carbon-carbon double bonds that may occur as part of keto-enol tautomerism or imine/enamine tautomerism are not precluded. When the term “saturated” is used to modify a solution of a substance, it means that no more of that substance can dissolve in that solution.
[00109] The term “aliphatic” signifies that the compound or chemical group so modified is an acyclic or cyclic, but non-aromatic compound or group. In aliphatic compounds/groups, the carbon atoms can be joined together in straight chains, branched chains, or non-aromatic rings (alicyclic). Aliphatic compounds/groups can be saturated, that is joined by single carbon-carbon bonds (alkanes/alkyl), or unsaturated, with one or more carbon-carbon double bonds (alkenes/alkenyl) or with one or more carbon-carbon triple bonds (alky nes/alkynyl) .
[00110] The term “aromatic” signifies that the compound or chemical group so modified has a planar unsaturated ring of atoms with 4/? +2 electrons in a fully conjugated cyclic 7i system. An aromatic compound or chemical group may be depicted as a single resonance structure; however, depiction of one resonance structure is taken to also refer to any other resonance structure. For example:
[00111] Aromatic compounds may also be depicted using a circle to represent the delocalized nature of the electrons in the fully conjugated cyclic n system, two non-limiting examples of which are shown below:
[00112] The term “alkyl” refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, a linear or branched acyclic structure, and no atoms other than carbon and hydrogen. The groups -CH3 (Me), -CH2CH3 (Et), -CH2CH2CH3 (n-Pr or propyl), -CH(CH3)2 (z-Pr, 'Pr or isopropyl), -CH2CH2CH2CH3 (n-Bu), -CH(CH3)CH2CH3 sec -butyl), -CH2CH(CH3)2 (isobutyl), -C(CH3)3 (tert-butyl, t-butyl, t-Bu or 'Bu), and -CH2C(CH3)3 (neo-pentyl) are non-limiting examples of alkyl groups. The term “alkanediyl” refers to a divalent saturated aliphatic group, with one or two saturated carbon atom(s) as the point(s) of attachment, a linear or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups -CH2- (methylene), -CH2CH2-, -CH2C(CH3)2CH2-, and -CH2CH2CH2- are non-limiting examples of alkanediyl groups. The term “alkylidene” refers to the divalent group =CRR' in which R and R' are independently hydrogen or alkyl. Non-limiting examples of alkylidene groups include: =CH2, =CH(CH2CH3), and =C(CH3)2. An “alkane” refers to the class of compounds having the formula H-R, wherein R is alkyl as this term is defined above.
[00113] The term “cycloalkyl” refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, said carbon atom forming part of one or more non-aromatic ring structures, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. If more than one ring is present, the rings may be fused, bridged, or spirocyclic. Non-limiting examples include: -CH(CH2)2 (cyclopropyl), cyclobutyl, cyclopentyl, or cyclohexyl (Cy). As used herein, the term does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to a carbon atom of the non-aromatic ring structure. The term “cycloalkanediyl” refers to a divalent saturated aliphatic group with two carbon atoms as points of attachment, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The group js a non_ limiting example of cycloalkanediyl group. A “cycloalkane” refers to the class of compounds having the formula H-R, wherein R is cycloalkyl as this term is defined above.
[00114] The term “alkenyl” refers to a monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a linear or branched, acyclic structure, at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. Non-limiting examples include: -CH=CH2 (vinyl), -CH=CHCH3, -CH=CHCH2CH3, -CH2CH=CH2 (allyl), -CH2CH=CHCH3, and — CH=CHCH=CH2. The term “alkenediyl” refers to a divalent unsaturated aliphatic group, with two carbon atoms as points of attachment, a linear or branched acyclic structure, at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. The groups -CH=CH-, -CH=C(CH3)CH2-, -CH=CHCH2-, and -CH2CH=CHCH2- are non-limiting examples of alkenediyl groups. It is noted that while the alkenediyl group is aliphatic, once connected at both ends, this group is not precluded from forming part of an aromatic structure. The terms “alkene” and “olefin” are synonymous and refer to the class of compounds having the formula H-R, wherein R is alkenyl as this term is defined above. Similarly, the terms “terminal alkene” and “a-olefin” are synonymous and refer to an alkene having just one carbon-carbon double bond, wherein that bond is part of a vinyl group at an end of the molecule.
[00115] The term “alkynyl” refers to a monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a linear or branched acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. As used herein, the term alkynyl does not preclude the presence of one or more non-aromatic carbon-carbon double bonds. The groups -C=CH, -C=CCH3, and -CH2C=CCH3 are non-limiting examples of alkynyl groups. An “alkyne” refers to the class of compounds having the formula H-R, wherein R is alkynyl.
[00116] The term “aryl” refers to a monovalent unsaturated aromatic group with an aromatic carbon atom as the point of attachment, said carbon atom forming part of a one or more aromatic ring structures, each with six ring atoms that are all carbon, and wherein the group consists of no atoms other than carbon and hydrogen. If more than one ring is present, the rings may be fused or unfused. Unfused rings are connected with a covalent bond. As used herein, the term aryl does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl, (dimethyl)phenyl, -C6H4CH2CH3 (ethylphenyl), naphthyl, and a monovalent group derived from biphenyl (e.g. , 4-phenylphenyl). The term “arenediyl” refers to a divalent aromatic group with two aromatic carbon atoms as points of attachment, said carbon atoms forming part of one or more six-membered aromatic ring structures, each with six ring atoms that are all carbon, and wherein the divalent group consists of no atoms other than carbon and hydrogen. As used herein, the term arenediyl does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. If more than one ring is present, the rings may be fused or unfused. Unfused rings are connected with a covalent bond. Non-limiting examples of arenediyl groups include:
[00117] An “arene” refers to the class of compounds having the formula H-R, wherein R is aryl as that term is defined above. Benzene and toluene are non-limiting examples of arenes.
[00118] The term “aralkyl” refers to the monovalent group -alkanediyl-aryl, in which the terms alkanediyl and aryl are each used in a manner consistent with the definitions provided above. Non-limiting examples are: phenylmethyl (benzyl, Bn) and 2-phenyl-ethyl.
[00119] The term “heteroaryl” refers to a monovalent aromatic group with an aromatic carbon atom or nitrogen atom as the point of attachment, said carbon atom or nitrogen atom forming part of one or more aromatic ring structures, each with three to eight ring atoms, wherein at least one of the ring atoms of the aromatic ring structure(s) is nitrogen, oxygen or sulfur, and wherein the heteroaryl group consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. If more than one ring is present, the rings are fused; however, the term heteroaryl does not preclude the presence of one or more alkyl or aryl groups (carbon number limitation permitting) attached to one or more ring atoms. Non-limiting examples of heteroaryl groups include benzoxazolyl, benzimidazolyl, furanyl, imidazolyl (Im), indolyl, indazolyl, isoxazolyl, methylpyridinyl, oxazolyl, oxadiazolyl, phenylpyridinyl, pyridinyl (pyridyl), pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl. The term ‘W-heteroaryl” refers to a heteroaryl group with a nitrogen atom as the point of attachment. A “heteroarene” refers to the class of compounds having the formula H-R, wherein R is heteroaryl. Pyridine and quinoline are non-limiting examples of heteroarenes.
[00120] The term “heterocycloalkyl” refers to a monovalent non-aromatic group with a carbon atom or nitrogen atom as the point of attachment, said carbon atom or nitrogen atom forming part of one or more non-aromatic ring structures, each with three to eight ring atoms, wherein at least one of the ring atoms of the non-aromatic ring structure(s) is nitrogen, oxygen or sulfur, and wherein the heterocycloalkyl group consists of no atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur. If more than one ring is present, the rings may be fused, bridged, or spirocyclic. As used herein, the term does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to one or more ring atoms. Also, the term does not preclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkyl groups include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydro thiofuranyl, tetrahydropyranyl, tetrahydropyridinyl, pyranyl, oxiranyl, and oxetanyl. The term ‘W-heterocycloalkyl” refers to a heterocycloalkyl group with a nitrogen atom as the point of attachment. A-pyrrolidinyl is an example of such a group.
[00121] The term “acyl” refers to the group -C(O)R, in which R is a hydrogen, alkyl, cycloalkyl, or aryl as those terms are defined above. The groups, -CHO, -C(O)CH3 (acetyl, Ac), -C(O)CH2CH3, -C(O)CH(CH3)2, -C(O)CH(CH2)2, -C(O)C6H5, and -C(O)CeH4CH3 are non-limiting examples of acyl groups. A “thioacyl” is defined in an analogous manner, except that the oxygen atom of the group -C(O)R has been replaced with a sulfur atom, -C(S)R. The term “aldehyde” corresponds to an alkyl group, as defined above, attached to a -CHO group. The term “carboxylate” means an alkyl group, as defined above, attached to a -C(O)O group.
[00122] The term “alkoxy” refers to the group -OR, in which R is an alkyl, as that term is defined above. Non-limiting examples include: -OCH3 (methoxy), -OCH2CH3 (ethoxy), -OCH2CH2CH3, -OCH(CH3)2 (isopropoxy), or -OC(CH3)3 (tert-butoxy). The terms “cycloalkoxy”, “alkenyloxy”, “alkynyloxy”, “aryloxy”, “aralkoxy”, “heteroaryloxy”, “heterocycloalkoxy”, and “acyloxy”, when used without the “substituted” modifier, refers to groups, defined as -OR, in which R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and acyl, respectively. The term “alkylthio” and “acylthio” refers to the group -SR, in which R is an alkyl and acyl, respectively. The term “alcohol” corresponds to an alkane or cycloalkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with a hydroxy group. The terms “alkoxide” and “cycloalkoxide” refer to a negatively charged group of the form OR wherein R is alkyl or cycloalkyl, respectively. The term “ether” corresponds to an alkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with an alkoxy group.
[00123] The term “alkylamino” refers to the group -NHR, in which R is an alkyl, as that term is defined above. Non-limiting examples include: -NHCH3 and -NHCH2CH3. The term “dialkylamino” refers to the group -NRR', in which R and R' can be the same or different alkyl groups. Non-limiting examples of dialkylamino groups include: -N(CH3)2 and -N(CH3)(CH2CH3). The term “amido” (acylamino), when used without the “substituted” modifier, refers to the group -NHR, in which R is acyl, as that term is defined above. A nonlimiting example of an amido group is -NHC(O)CH3.
[00124] As used herein, the term “chemical group” refers to any functional group or substituent covalently bound to a core structure. Chemical groups may include but are not limited to hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CO2CH2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, -S(O)2NH2 or a combination or substituted version of any of these groups.
[00125] When a chemical group is used with the “substituted” modifier, one or more hydrogen atom has been replaced, independently at each instance, by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CO2CH2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. For example, the following groups are non-limiting examples of substituted alkyl groups: -CH2OH, -CH2CI, -CF3, -CH2CN, -CH2C(O)OH, -CH2C(O)OCH3, -CH2C(O)NH2, -CH2C(O)CH3, -CH2OCH3, -CH2OC(O)CH3, -CH2NH2, -CH2N(CH3)2, and -CH2CH2CI. The term “haloalkyl” is a subset of substituted alkyl, in which the hydrogen atom replacement is limited to halo (i.e. -F, -Cl, -Br, or -I) such that no other atoms aside from carbon, hydrogen and halogen are present. The group, -CH2C1 is a non- limiting example of a haloalkyl. The term “fluoroalkyl” is a subset of substituted alkyl, in which the hydrogen atom replacement is limited to fluoro such that no other atoms aside from carbon, hydrogen and fluorine are present. The groups -CH2F, -CF3, and -CH2CF3 are non-limiting examples of fluoroalkyl groups. Non-limiting examples of substituted aralkyls are: (3-chlorophenyl)-methyl, and 2-chloro-2-phenyl-eth-l-yl. The groups, -C(O)CH2CF3, -CO2H (carboxyl), -CO2CH3 (methylcarboxyl), -CO2CH2CH3, -C(O)NH2 (carbamoyl), and -CON(CH3)2, are non-limiting examples of substituted acyl groups. The groups -NHC(O)OCH3 and -NHC(O)NHCH3 are non-limiting examples of substituted amido groups.
[00126] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps.
[00127] The term “effective,” as that term is used in the specification and/or claims, means adequate to accomplish a desired, expected, or intended result.
[00128] An “isomer” of a first compound is a separate compound in which each molecule contains the same constituent atoms as the first compound, but where the configuration of those atoms in three dimensions differs.
[00129] “Pharmaceutically acceptable salts” means salts of compounds disclosed herein which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as 1 ,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3 -phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene- 1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene- 1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzcnesiil Ionic acid, phenyl-substituted alkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiarybutylacetic acid, trimethylacetic acid, and the like. Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, A-methylglucamine and the like. It should be recognized that the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).
[00130] A “stereoisomer” or “optical isomer” is an isomer of a given compound in which the same atoms are bonded to the same other atoms, but where the configuration of those atoms in three dimensions differs. “Enantiomers” are stereoisomers of a given compound that are mirror images of each other, like left and right hands. “Diastereomers” are stereoisomers of a given compound that are not enantiomers. Chiral molecules contain a chiral center, also referred to as a stereocenter or stereogenic center, which is any point, though not necessarily an atom, in a molecule bearing groups such that an interchanging of any two groups leads to a stereoisomer. In organic compounds, the chiral center is typically a carbon, phosphorus or sulfur atom, though it is also possible for other atoms to be stereocenters in organic and inorganic compounds. A molecule can have multiple stereocenters, giving it many stereoisomers. In compounds whose stereoisomerism is due to tetrahedral stereogenic centers (e.g. , tetrahedral carbon), the total number of hypothetically possible stereoisomers will not exceed 2n, where n is the number of tetrahedral stereocenters. Molecules with symmetry frequently have fewer than the maximum possible number of stereoisomers. A 50:50 mixture of enantiomers is referred to as a racemic mixture. Alternatively, a mixture of enantiomers can be enantiomerically enriched so that one enantiomer is present in an amount greater than 50%. Typically, enantiomers and/or diastereomers can be resolved or separated using techniques known in the art. It is contemplated that for any stereocenter or axis of chirality for which stereochemistry has not been defined, that stereocenter or axis of chirality can be present in its R form, S form, or as a mixture of the R and .S' forms, including racemic and non-racemic mixtures. As used herein, the phrase “substantially free from other stereoisomers” means that the composition contains < 15%, more preferably < 10%, even more preferably < 5%, or most preferably < 1% of another stereoisomer(s).
[00131] An “amine protecting group” or “amino protecting group” is well understood in the art. An amine protecting group is a group which modulates the reactivity of the amine group during a reaction which modifies some other portion of the molecule. Amine protecting groups can be found at least in Greene and Wuts, 1999, which is incorporated herein by reference. Some non-limiting examples of amino protecting groups include formyl, acetyl, propionyl, pivaloyl, /-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, o-nitrophenoxyacetyl, a-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4- bromobenzoyl, 4-nitrobenzoyl, and the like; sulfonyl groups such as benzenesulfonyl, p- toluenesulfonyl and the like; alkoxy- or aryloxycarbonyl groups (which form urethanes with the protected amine) such as benzyloxycarbonyl (Cbz), -chlorobenzyloxycarbonyl, p- methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p- bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5- dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4- methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3 ,4,5-trimethoxy- benzyloxycarbonyl, l-(p-biphenylyl)- 1 -methylethoxycarbonyl, a,a-dimethyl-3,5- dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl, 2-trimethylsilylethyloxycarbonyl (Teoc), phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl (Fmoc), cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl and the like; alkylaminocarbonyl groups (which form ureas with the protect amine) such as ethylaminocarbonyl and the like; aralkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl and the like; and silyl groups such as trimethylsilyl and the like. Additionally, the “amine protecting group” can be a divalent protecting group such that both hydrogen atoms on a primary amine are replaced with a single protecting group. In such a situation the amine protecting group can be phthalimide (phth) or a substituted derivative thereof wherein the term “substituted” is as defined above. In some embodiments, the halogenated phthalimide derivative may be tetrachlorophthalimide (TCphth). When used herein, a “protected amino group”, is a group of the formula PGMANH- or PGDAN- wherein PGMA is a monovalent amine protecting group, which may also be described as a “monovalently protected amino group” and PGDA is a divalent amine protecting group as described above, which may also be described as a “divalently protected amino group”.
[00132] As used herein, the term “surface” references the portion of a substrate, sample, analyte, or the like which is capable of interacting with other compounds or compositions. In some embodiments, a surface is an atom, molecule, or macromolecule capable of participating in intermolecular interactions with a compound or composition, such as an oligourethane or a mixture of oligourethanes. The samples may be either organic material, inorganic material, or a mixture thereof. In some cases, the samples may be manipulated or processed to improve the suitability of the surface for use in the methods disclosed herein. Nonlimiting examples of samples which have surfaces that may be analyzed according to the methods disclosed herein include proteins, enzymes, antibodies, cells, viruses, biopolymers, polymers, organisms, soil, rocks, material, and combinations thereof.
[00133] The above definitions supersede any conflicting definition in any reference that is incorporated by reference herein. The fact that certain terms are defined, however, should not be considered as indicative that any term that is undefined is indefinite. Rather, all terms used are believed to describe the invention in terms such that one of ordinary skill can appreciate the scope and practice the present invention.
III. Examples
[00134] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Example 1: Probe and Sensor Array Design
[00135] The present disclosure describes the development of an agnostic approach to surface differentiation, which facilitates analysis of any type of surface. This approach represents an improvement techniques known in the art, such as a lock-and-key type detection approach which requires advanced knowledge of the surfaces to be differentiated and careful design of probes with specific molecular recognition motifs. The agnostic approach described herein is also beneficial for analyzing surfaces, such as bulk surfaces, that do not have obvious or particularly reactive binding sites.
[00136] The inventors describe in further detail below the creation of a library of oligomers with various monomer compositions. The oligomers self-sort during elution based on their individual compositions, the overall monomer composition of the oligomer library, and any cooperativity between oligomer monomers. Use of oligomers instead of discrete small molecules facilitated creation of a pool of molecules with similar structures and sizes, so that differences in molecular retention times would, without being bound by theory, primarily arise from the different supramolecular interactions of their functional groups with the substrate surface, without prior knowledge of the surfaces to be differentiated. Oligourethane (OU) structures (Dahlhauser et al., 2020) recently developed by the inventors were selected for use in creating the probe library (Scheme 1). OUs may be synthesized from a solid-support with Fmoc-protected, p-nitrophenyl carbonate activated amino alcohols (Fmoc-R(PG)-PNOC) with a large variety of R-functional groups with control over the length, sequence, and functional group composition of the OUs (Scheme 1). These OUs have been shown to chain-end degrade in the presence of base and heat through a 5-exo-trig intramolecular cyclization from the terminal alcohol (Scheme 1). Through this mechanism, OUs are degraded into monomerderived 5-membered carbamate rings called an oxazolidinone. This degradation mechanism has been used to decode the monomer sequence of unknown OUs with discrete sequences (Dahlhauser et al., 2021; Dahlhauser et al., 2022; Zhang et al., 2024; Soete et al., 2022).
[00137] Use of OUs in this manner is beneficial at least because a vast pool of probes with statistically random monomer sequences may be readily synthesized using mixtures of 10 different monomers in eight coupling steps (FIGS. 1A-1D, see monomer and OU synthesis details in Example 4). Classification of analyte surfaces via various chemometric routines, such as PCA score plots, are best with high degrees of cross-reactivity that imparts significant extents of variance across several principal components. High degrees of variance along a single axis reveals little cross-reactivity among sensor elements and would be disfavored (Stewart et al., 2013). An OU comprising an entirely random sequence of monomers and an equal distribution of said monomers would, without being bound by theory, disfavor bias or selectivity towards any one surface and accordingly would carry variance primarily along one PCA axis which, as mentioned above, would be disfavored for use in classification of analyte surfaces. Instead, surface differentiation would, still without being bound by theory, be facilitated by mixtures of O s that comprise a bias towards a monomer or a class of monomer (FIG. ID).
[00138] To that end, sixteen libraries of OUs were synthesized from 16 coupling mixtures using different mixtures of monomers (FIG. IB, FIG. 1C). For example, one library was biased towards aliphatic monomers, with the aliphatic monomers Fmoc-Leu-PNOC and Fmoc-Cha-PNOC at a combined concentration of 75 mM in the monomer coupling mixture and the remaining eight monomers at a combined concentration of 25 mM. Another library, for example, was biased towards both aromatic monomers and polar protic monomers that bear a negative charge in water. For this library, the monomer coupling mixture contained aromatic monomers Fmoc-Trp(Boc)-PNOC, Fmoc-Tyr(tBu)-PNOC, and Fmoc-BnTrz-PNOC and polar protic monomer Fmoc-Asp(tBu)-PNOC at a combined concentration of 81 mM and the remaining six monomers had a combined concentration of 19 mM. FIG. 2 and Table 1 show the relative monomer composition used to synthesize all biased libraries. The combined pool of all OU libraries had 108 possible different sequences, with the diversity of sequences artificially engineered by using the biased monomer mixtures.
Table 1: Fmoc-R(PG)-PNOC concentrations in each directed library coupling mixtures
[00139] Further probe design ensured that every OU sequence began with phenylalaninol (pre-loaded on the solid support used in synthesis), and every OU was terminated at the 10th coupling step with methyl tyrisinol acetamide. These monomers were kept constant so that the presence of Phe-oxazolidinone after OU degradation could be used to approximate the number of OU before degradation. Methyl tyrisinol acetamide terminated every sequence so that the ratio of Phe-oxazolidinone to methyl tyrisinol after degradation could be used to assess completeness of OU degradation. See the Example 4 for OU library and pool characterization and monomer compositional analysis.
[00140] With the pool of combined OUs representing the sensors, the analysis was performed by eluting the OU probes through a column packed with different analytes (stationary phases) and 32 1 mL fractions were collected (FIG. IE). The monomer compositional makeup of the probe mixture at various retention times was determined by completely degrading the OU in each fraction and recording the LC-MS extracted ion chromatogram (EIC) for each oxazolidinone (See Example 4 for degradation and LC-MS sample preparation). Each oxazolidinone LC-MS EIC retention time was compared to that of oxazolidinone standards prepared by synthesizing and degrading a trimer OU bearing the same monomer functional group (see Example 4 for more detail). In this manner, OU monomer composition (measured by the oxazolidinones) at various retention times formed a fingerprint for each unique surface.
Example 2: Sensor Array Analytes as Column Chromatography Stationary Phases
[00141] Five stationary phases were chosen for differentiating via column chromatography by the mixture of biased OU libraries. Supramolecular contacts that were dependent upon monomer composition of OUs, as well as cooperativity between monomers along the OU strands, could, without being bound by theory, be used as a basis for differentiating the surfaces where elution time would be indicative of these phenomena. Stationary phases with distinct surface chemistry were chosen to demonstrate the ability of biased OU libraries to differentiate between the samples. Silica gel, reversed phase C18-silica gel, celite, activated carbon, and silica gel functionalized with bovine serum albumin were chosen as illustrative surfaces. Silica gel made from silicon oxide is highly polar due to surface silanol groups. Reversed phase C18-silica gel, made by capping surface silanols with an 18- carbon alkyl chain, is nonpolar. Celite is a highly porous and amorphous silicon oxide but does not bind strongly to most organic molecules because it does not have many surface silanols (Ren et al., 2022). Activated carbon is made primarily of aromatic carbon allotropes and is commonly used in purification due to its high porosity and absorbency. While celite is nearly non-adsorbent, activated carbon is the opposite. Activated carbon was diluted to 0.1 w% with celite to facilitate elution of the OUs to elute from the column under the experimental conditions disclosed in other sections. Silica gel functionalized with bovine serum albumin (BS A), a protein isolated from cows with a molecular weight of 66.5 kDa, has highly complex surface chemistry and has previously been studied to separate enantiomers by high performance liquid chromatography (HPLC) (Allenmark et al., 1984; Giplin et al., 1991; Haginaka et al., 1997; Zhang et al., 2000). BSA-functionalized silica gel was synthesized using a commercially available propionyl chloride-functionalized silica gel starting material (see Example 4 for synthesis and characterization).
Example 3: Results and Discussion
[00142] Thirty-two fractions collected from columns run with all five substrates were analyzed by degrading the OUs in each fraction down to their oxazolidinone products and then submitting for LC-MS EIC analysis (FIG. 3A). Every LC-MS sample comprised as an internal standard a commercially available (S)-(-)-4-isopropyl-2-oxazolidinone (named here as Val-oxazolidinone), introduced when the OU fractions were degraded. To decrease variation across LC-MS runs, every oxazolidinone EIC peak area was divided by the peak area of the Val-oxazolidinone standard in its fraction (FIG. 3B). As some oxazolidinones more easily pick up a charge from electrospray ionization than others, EIC peak areas were scaled from 0 to 1 (that is, min-max adjusted) based on the highest peak area for each R-group across all samples (FIG. 3C) to facilitate comparison of R-oxazolidinone responses across R-groups.
[00143] As every OU began with a Phe monomer, the total number of eluting OUs could be measured by tracking the peak associated with the Phe monomer, (see FIG. 4). Division of every oxazolidinone EIC peak area of a fraction by the peak area of the Phe- oxazolidinone in that fraction revealed the average monomer composition of each OU, regardless of the number of eluting OUs or of the elution time of any given OU. (FIG. 3D). If the Phe-oxazolidinone EIC peak was too small to identify above the background noise, then Phe-oxazolidinone and all the Phe-oxazolidinone adjusted R-oxazolidinone values were assigned a value of 0. FIG. 3 demonstrates these adjustments using data collected from a column run with silica as the substrate. FIG. 5 shows a heatmap of the Phe-oxazolidinone adjusted EIC peaks for all oxazolidinones over all 32 fractions and all five column substrates. These Phe-oxazolidinone adjusted heat maps reveal that the array response (i.e. OU monomer composition at various retention times) is unique for each of the five substrates.
A. Statistical differentiation of column stationary phases
[00144] To demonstrate that the sensor array described above is able to differentiate each unique surface, five replicate columns were run with each surface and five fractions (fractions 5, 7 , 10, 15, and 20) were analyzed from each replicate. As 10 different oxazolidinones were tracked across each of the five fractions, there was a total of 50 (10 x 5) data points for each surface replicate. Principal component analysis (PCA) was performed on each of the surface replicates. PCA is a statistical technique that linearly transforms highdimensional data (e.g. the 50 data points for each surface replicate) into a 2- or 3-dimensional data set. Principal components are linear combinations of data variables (in this case oxazolidinone EIC peak areas in a specific fraction) that capture the largest variation in the data. This technique is an unsupervised model because no input is provided on the substrate identity of each sample. A clustering pattern on PCA plots of replicates from the same substrates that is clear and distinct from that of PCA plots of replicates from another substrate would indicate that the sensor array is useful for differentiating between the substrates.
[00145] PCA with the min-max standard adjusted data (e.g. data that has undergone the same adjustments as FIG. 3C above) from fractions 5, 7, 15, and 20 from five replicates of the five tested column substrates did not return tight clustering, with many overlapping replicates from BSA-functionalized silica, silica, celite, and 0.1% activated carbon in celite (FIG. 6). The highest min-max standard adjusted values for each oxazolidinone were found in fraction 5 (FIG. 7) for most substrate replicates, biasing the PCA results. However, when PCA was performed with min-max Phe-oxazolidinone adjusted data (e.g. data that has undergone the same adjustments as FIG. 3D) from fractions 5, 7, 15, and 20 from five replicates of the five tested column substrates, clear clusters in 2- and 3-dimensions were observed (FIG. 8). This result reveals that, without being bound by theory, the average monomer composition of later eluting OUs is more useful in differentiating surfaces than the quantity of OUs eluting in each fraction. The silica substrate, the only substrate with an aliphatic surface chemistry, was the only analyte that was able to be differentiated by the first principal component. The BSA-functionalized silica gel, the most chemically complex surface, lies closest to the origin of the graph. This suggests that this substrate is the least biased towards any one of the linear combinations of oxazolidinones and fractions used to make the principal components, perhaps because, without being bound by theory, it is the most chemically complex surface. Finally, the high dimensionality of the PCA space as well as the tight clustering of the replicates using this unsupervised chemometric method validates the probe and library design.
[00146] A loading plot analysis was performed for PCI, PC2 and PC3 (FIG. 9). A loading plot reveals the contribution of each variable towards the principal component on each axis. None of the variables (OU side chains at specific retention times) contributed very strongly to any principal component, while many variables contributed a similar amount. Furthermore, there was no overlap between the top ten variables contributing to PCI, PC2 and PC3. This suggests, without being bound by theory, that nearly all the oligourethane side chain interactions were used to differentiate each sample instead of there being a single important side chain or combination of side chains.
[00147] Because nearly all oxazolidinone responses were used to differentiate samples, many more unique patterns in the sensor array are possible. Therefore, the methods disclosed herein utilizing such a sensor array may be useful in differentiating many more stationary phases, surfaces, samples, or analytes.
[00148] Despite a lack of a single important oxazolidinone (indicative of the OU side chain) or fraction thereof, the top 10 variables influencing PCI, PC2, and PC3 (FIGS. 10A-10C) were analyzed. The top 10 variables for the PCI axis move data points exclusively in a positive direction along the axis, and thus are most responsible for differentiating Cl 8- silica from the other four substrates. Eight of the ten top PCI components are aromatic oxazolidinones, suggesting that, without being bound by theory, aromatic biased OUs were most important for differentiating Cl 8-silica in this study. PC2 is responsible for differentiating the remaining four substrates. The top three components moving data points in a positive direction along the PC2 axis are aliphatic monomers, suggesting that, again without being bound by theory, activated carbon is best differentiated from the remaining three substrates with OU biased towards aliphatic monomers. PC3 had the variables with the largest contribution to the principal component axis. The top four variables are the aspartic acid derived oxazolidinone, and seven of the top 10 components are derived from charged amino acids. This suggests that the OUs with a charged monomer bias contributed the most to differentiating celite in PC3.
[00149] Linear discriminatory analysis (LDA) on was also performed on the data. LDA is statistical technique that uses linear combinations of variables to maximize variance between samples of different classes while simultaneously decreasing variance between samples of the same class. This is a supervised technique and substrate identity must be known and provided to the model. LDA differentiated all five substrates with three principal components using the min-max Phe-oxazolidinone adjusted data (FIG. 11) and could be classified with a 96% accuracy using a jackknife analysis.
Example 4: Materials and Methods
( i ) General Procedures
[00150] All materials used in the synthesis of each compound and related tests were purchased from commercially available sources, including Chem Impex International, Sigma-Aldrich Chemical Co., Acros Organics, Tokyo Chemical Industry, and Fischer Scientific, and were used as received. ]H and 13C NMR spectra were recorded on a Bruker Avance 111 500 MHz equipped with a Prodigy BBO cryoprobe and a 3M 500. *H and 13C chemical shifts (5) are reported in ppm relative to residual CHCh (]H: 7.26 ppm, 13C: 77.16 ppm) shifts
( ii ) LC-MS instrument and method details
[00151] All samples were analyzed on an Agilent Quadrupole LC-MS coupled with an ESI source and interfaced with an Agilent 1200 series liquid chromatography system. The column is an Agilent ZORBAX Eclipse Plus C18 narrow bore column, with a 2.1 mm internal diameter and 50 mm length. The method used to run all samples is a 12-minute program that runs a gradient from 95% H2O/MeCN to 0% H2O/MeCN. The aqueous mobile phase contains 50 mM NH4OAc. 2 uLs of the samples were injected into the column.
( Hi ) Monomer selection
[00152] Monomers for oligourethane synthesis were selected to introduce functional group diversity into the OU probes (FIG. 12). Leucine and cyclohexyl-alanine derived monomers (Fmoc-Leu-PNOC and Fmoc-Cha-PNOC, respectively) were used as aliphatic monomers. Tryptophan, tyrosine, and l-benzyl-lH-l,2,3-triazole-alanine derived monomers (Fmoc-Trp(Boc)-PNOC, Fmoc-Tyr(tBu)-PNOC, Fmoc-BnTrz-PNOC, respectively) were introduced as aromatic monomers. Aspartic acid, lysine, arginine, and histidine derived monomers (Fmoc-Asp(tBu)-PNOC, Fmoc-Lys(Boc)-PNOC, Fmoc- Arg(Pbf)-PNOC, and Fmoc-His(Trt)-PNOC, respectively) were used because they bear a charge in water and are polar protic in organic media. Finally, a propargyl-glycine derived monomer (Fmoc-PGly-PNOC) served as a monomer comprising an alkynyl functional group.
(iv) Monomer synthesis and characterization
[00153] Monomers Fmoc-Leu-PNOC, Fmoc-Cha-PNOC, Fmoc-Tyr(tBu)- PNOC, Fmoc-Trp(Boc)-PNOC, and Fmoc-Tyr(OMe)-PNOC were synthesized as previously reported and matched literature characterization (Cho et al., 1998; Dahlhauser et al., 2020; Dahlhauser et al., 2021; Dahlhauser et al., 2022). The remaining monomers were synthesized from commercially available Fmoc-protected amino acids over two steps (Scheme 2).
Scheme 2: Two-step synthesis for oligourethane monomers ( v) General procedure for synthesis of Fmoc-amino alcohol (Fmoc-R(PG)-ol)
[00154] The procedure for the reduction of Fmoc-amino acids to Fmoc-amino alcohols is adapted from previous reports (Dahlhauser et al., 2020; Dahlhauser et al., 2021; Dahlhauser et al., 2022); . N,N-carbonyldiimidazole (1.33 equiv.) was added to a solution Fmoc-L-amino acid (1 equiv., 0.20 M) in THF and stirred at room temperature for at least 30 minutes. The mixture was cooled to 0 °C and a solution of NaBFU (1.67 equiv., 0.6 M) dissolved in water was added dropwise. The solution was stirred for 3 hours. The reaction was quenched by the addition of 1 M HC1 and extracted with EtOAc (x3). The combined organics were washed with brine (x2), dried over Na2SC>4, and concentrated under vacuum. The product was purified by silica gel chromatography.
Fmoc-PGly-ol (9H-fluoren-9-yl)methyl (S)-(l-hydroxypent-4-yn-2-yl)carbamate
[00155] According to the procedure for the reduction of Fmoc-amino acids: 2- (9H-fluoren-9-ylmethoxycarbonylamino)pent-4-ynoic acid (Fmoc-PGly-OH, 5.09 g, 15.18 mmol) gave a crude product that was purified by silica gel chromatography (DCM and then 25% EtOAc/DCM) and isolated as a white solid (3.84 g, 81%). 1 H NMR (500 MHz, CDCh) 5 7.77 (d, J = 7.6 Hz, 2H), 7.59 (d, J = 7.5 Hz, 2H), 7.42 - 7.39 (m, 2H), 7.33 - 7.30 (m, 2H), 5.23 (d, J = 7.5 Hz, 1H), 4.43 (p, J = 10.2 Hz, 2H), 4.23 (t, J = 6.8 Hz, 1H), 3.96 - 3.49 (m, 3H), 2.53 (s, 2H), 2.05 (t, J = 2.7 Hz, 1H). 13C NMR (101 MHz, CDC13) 5 156.40, 143.95, 143.92, 141.46, 127.86, 127.20, 125.15, 120.14, 80.14, 71.25, 67.00, 63.71, 51.17, 47.35, 21.28. HRMS ESI+: calculated (C20H17NO4) 335.12, found [M+Na]+ 344.1264.
Fmoc-His(Trt)-ol (9H-fluoren-9-yl)methyl (S)-(l-hydroxy-3-(l-trityl-lH-imidazol-4- yl)propan-2-yl)carbamate:
[00156] According to the procedure for the reduction of Fmoc-amino acids: Na- (((9H-fluoren-9-yl)methoxy)carbonyl)-NT-trityl-L-histidine (Fmoc-His(Trt)-OH, 0.51 g, 0.81 mmol) gave a crude product that was purified by silica gel column chromatography (DCM then EtOAc) and isolated as a white solid (0.12 g, 0.20 mmol, 24% yield). 1H 13C HMRS ESI+: calculated (C40H35N3O3) 605.27 found [M+H]+ 606.2757. Fmoc-Arg(Pbf)-ol (9H-fluoren-9-yl)methyl (S)-(l-hydroxy-5-(3-((2,2,4,6,7-pentamethyl- 2,3-dihydrobenzofuran-5-yl)sulfonyl)guanidino)pentan-2-yl)carbamate:
[00157] According to the procedure for the reduction of Fmoc-amino acids: N2- (((9H-fluoren-9-yl)methoxy)carbonyl)-Nro-((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5- yl)sulfonyl)-L-arginine (Fmoc-Arg(Pbf)-OH, 10.04 g, 15.47 mmol) gave a crude product which was purified by silica gel column chromatography (DCM then EtOAc) and isolated as a white powder (9.23 g, 94%), with matching 1 H NMR, 13C NMR, and HRMS as reported in the literature (Sorg et al., 2004; Li et al., 2015; Cussol et al., 2020).
Fmoc-Lys(Boc)-ol (9H-fluoren-9-yl)methyl tert-butyl (6-hydroxyhexane-l,5-diyl)(S)- dicarbamate:
[00158] According to the procedure for the reduction of Fmoc-amino acids: N2- (((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-lysine (Fmoc-Lys(Boc)- OH, 5 g, 10.7 mmol) gave a crude product which was purified by silica gel column chromatography (1: 1 hexanes/EtOAc, then 1:4 hexanes/EtOAc, then EtOAc) to yield a white solid (4.3 g, 9.4 mmol, 88% yield) with matching 1 H NMR, 13C NMR, and HMRS as reported in the literature (Boeijen et al., 2001; Sorg, 2004 et al:, van Zutphen et al., 2005; Yang et al., 2008; Manne et al., 2018; Jadhav et al., 2011).
Fmoc-Asp(‘Bu)-ol tert-butyl (S)-3-((((9H-fhioren-9-yI)methoxy)carbonyl)amino)-4- hydroxybutanoate:
[00159] According to the procedure for the reduction of Fmoc-amino acids: (S)- 2-((((9H-fhioren-9-yl)methoxy)carbonyl)amino)-4-(tert-butoxy)-4-oxobutanoic acid (Fmoc- Asp(’Bu)-OH, 10.06 g, 24.30 mmol) gave a crude product which was purified by silica gel column chromatography (25% EtOAc in DCM, then gradient to EtOAc) and isolated as a white powder (7.31 g, 76% yield), with matching *H NMR, 13C NMR, and HRMS as reported in the literature (Grimm, 2004 et al , Diness, 2004 et al:, Sorg et al., 2004; Wendt et al., 2006; Yang et al., 2008; Jadhav et al., 2011; Grob et al., 2020).
(vi) General procedure for synthesis of Fmoc-amino alcohol (Fmoc-R(PG)-ol)
[00160] The general procedure for the synthesis of activated carbonates was adapted from previous reports. Fmoc-R(PG)-ol (1 equiv, 80 mM) was dissolved in anhydrous DCM and pyridine (0.73 equiv.) was added dropwise. In a separate flask, 4-nitrophenyl chloroformate (1.33 equiv, 110 mM) was dissolved in anhydrous DCM and then added dropwise to the solution of Fmoc-R(PG)-ol. The reaction was left to stir overnight at room temperature. The reaction was then washed with 1 M NaHSO4 (x2), sat. sodium bicarbonate (x4), and then brine (x2). The organic layer was then dried over sodium sulfate and concentrated under vacuum. The product was purified by silica gel column chromatography. If the p-nitrophenol impurity remained after column chromatography, the product was not purified further.
Fmoc-PGly-PNOC (9H-fluoren-9-yI)methyl (S)-(l-(((4-nitrophenoxy)carbonyl)oxy)pent-
4-yn-2-yl)carbamate:
[00161] According to the procedure for the synthesis of activated carbonates: Fmoc-PGly-ol (3.94 g, 12.26 mmol) gave a crude product which was purified by silica gel column chromatography (50% hexanes/DCM, then DCM, then 25% EtOAc/DCM) and isolated as a yellow solid (4.79 g, 80% yield). ’H NMR (500 MHz, CDC13) 5 8.29 - 8.23 (m, 2H), 7.77 (d, J = 7.4 Hz, 2H), 7.62 - 7.56 (m, 2H), 7.44 - 7.34 (m, 4H), 7.31 (td, J = 7.5, 1.1 Hz, 2H), 5.18 (d, J = 8.8 Hz, 1H), 4.63 - 4.31 (m, 4H), 4.23 (t, J = 7.0 Hz, 2H), 2.60 (d, J = 4.3 Hz, 2H), 2.13 (d, J = 2.6 Hz, 1H). 13C NMR (101 MHz, CDCh) 5 155.88, 155.46, 152.42, 145.64, 143.80, 141.47, 127.93, 127.21, 125.45, 125.06, 121.82, 120.19, 78.70, 72.18, 68.96, 67.23, 48.30, 47.32, 21.54. HRMS ESI+: calculated (C27H22N2O7) 486.14, found [M+Na]+ 509.1320.
Fmoc-His(Trt)-PNOC (9H-fluoren-9-yl)methyl (S)-(l-(((4-nitrophenoxy)carbonyl)oxy)- 3-(l-trityI-lH-imidazol-4-yl)propan-2-yl)carbamate:
[00162] According to the procedure for the synthesis of activated carbonates: FMOC-His(Trt)-ol
Fmoc-Arg(Pbf)-PNOC (9H-fluoren-9-yl)methyl (S)-(l-(((4-nitrophenoxy)carbonyl)oxy)-
5-(3-((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)guanidino)pentan-2- yl)carbamate:
[00163] According to the procedure for the synthesis of activated carbonates: Fmoc-Arg(Pbf)-ol (1.025 g, 1.61 mmol) gave a crude product which was purified by silica gel column chromatography (DCM then EtOAc) and isolated as a yellow solid (0.730 g, 0.91 mmol, 56% yield). ] H NMR, 13C NMR, and HRMS matched literature values (Cho et al., 1998). Fmoc-Lys(Boc)-PNOC (9H-fluoren-9-yl)methyl tert-butyl (6-(((4- nitrophenoxy)carbonyI)oxy)hexane-l,5-diyl)(S)-dicarbamate:
[00164] According to the procedure for the synthesis of activated carbonates and like other literature procedures (Lee et al., 2005): Fmoc-Lys(Boc)-ol (1.602 g, 3.52 mmol) gave a crude product which was purified by silica gel column chromatography (DCM then 5% EtOAc/DCM then 50% EtOAc/DCM) to yield a white solid (1.87 g, 3.02 mmol, 86% yield). 'H NMR (500 MHz, CDCh) 8 8.24 (d, J = 9.0 Hz, 2H), 7.76 (d, J = 7.6 Hz, 2H), 7.59 (dd, J = 7.8, 4.1 Hz, 2H), 7.43 - 7.27 (m, 6H), 4.96 (d, J = 9.8 Hz, 1H), 4.58 (s, 1H), 4.47 - 4.42 (m, 2H), 4.31 (dd, J = 11.1, 4.4 Hz, 1H), 4.26 - 4.18 (m, 2H), 4.02 - 3.96 (m, 1H), 3.18 - 3.09 (m, 2H), 1.70 - 1.27 (m, 15H). 13C NMR (126 MHz, CDCh) 8 156.33, 156.27, 155.54, 152.59, 145.58, 143.95, 143.91, 141.47, 127.87, 127.20, 125.42, 125.09, 121.86, 120.14, 79.51, 70.83, 66.91, 50.12, 47.40, 40.21, 30.91, 29.91, 28.55, 22.95. HMRS ESI+: calculated (C33H37N3O9) 619.25, found [M+Na]+ 642.2419
Fmoc-Asp(*Bu)-PNOC tert-butyl (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4- (((4-nitrophenoxy)carbonyl)oxy)butanoate:
[00165] According to the procedure for the synthesis of activated carbonates and like other literature procedures (Cho et al., 1998): Fmoc-Asp(‘Bu)-ol (5.00 g, 12.48 mmol) yielded a yellow solid (5.59 g, 9.94 mmol, 80% yield) which was used without further purification after work-up. 1 H NMR, 13C NMR, and HRMS matched reported literature for the R enantiomer (Cho et al., 1998).
(vii) Synthesis of Fmoc-BnTrz-PNOC: (9H-fluoren-9-yl)methyl (S)-( 1-(1 -benzyl- 1H- 1,2,3- triazol-4-yl )-3-((( 4-nitrophenoxy)carbonyl )oxy )propan-2-yl )carbamate
Scheme 3: Synthesis of Fmoc-BnTrz-PNOC from Fmoc-PGly-PNOC [00166] Fmoc-PGly-PNOC (0.4987 g, 1.03 mmol, 1 equiv.) was dissolved in 12 mL 5: 1 THF/H2O and sparged with N2 for 15 minutes. Benzyl azide (130 uL, 1.04 mmol, 1 equiv.) was added to the solution dropwise. Sodium ascorbate (34 mg, 0.17 mmol, 0.15 equiv.) and CUSO4- 5H2O (14 mg, 0.06 mmol, 0.05 equiv.) were added to the reaction flask and the solution was stirred overnight under N2 at room temperature. TLC showed complete consumption of Fmoc-PGly-PNOC starting material. The reaction was diluted with 100 mL EtOAc and washed with 100 mL H2O (x3) and 100 mL brine (x2). The organic layer was dried over sodium sulfate and concentrated under vacuum. The product was purified by silica gel column chromatography (DCM then gradient to EtOAc) to yield a white solid (0.37 g, 0.60 mmol, 59% yield). ’H NMR (500 MHz, CDCI3) 5 8.25 (d, J = 8.9 Hz, 2H), 7.76 (d, J = 7.6 Hz, 2H), 7.59 - 7.52 (m, 2H), 7.44 - 7.17 (m, 12H), 5.66 (s, 1H), 5.51 (s, 2H), 4.61 - 3.97 (m, 5H), 4.18 (t, J = 7.0 Hz, 1H), 3.07 - 3.04 (m, 2H). 13C NMR (126 MHz, CDCI3) 8 162.57, 156.14, 155.51, 152.46, 145.59, 143.89, 141.42, 134.43, 129.30, 129.03, 128.19, 127.90, 127.84, 127.24, 125.45, 125.23, 121.92, 120.15, 69.63, 67.16, 54.59, 49.63, 47.25, 27.30. HMRS ESI+: calculated (C34H29N5O7) 619.21, found [M+Na]+ 642.1952.
(viii) General procedure for oligourethane synthesis on a solid support
Scheme 4: Synthetic procedure for solid phase oligo urethane synthesis and cleavage
[00167] All oligourethanes were synthesized according to previously published procedures, using a CEM Multipep 2 automated parallel peptide synthesizer (Shuluk et al., 2025). L-phenylalaninol loaded 2-chlorotrityl polystyrene resin (0.21 mmol/g, 200-400 mesh, Chemlmpex) was added to a fritted 96- well plate. The resin was suspended and left to swell in N-methyl-2-pyrrolidine (NMP) for 10 minutes. The suspension was filtered. For each coupling step, a coupling solution of HOBt (0.2 M, 6 equiv.), diisopropyl ethylamine (0.05 M, 1.6 equiv.), and Fmoc-R(PG)-PNOC monomer or monomer mixture (0.1 M, 3 equiv.) in NMP was added to the resin (1 equiv. active sites), and the suspension was heated to 35 °C and periodically shaken for 8 hours. After coupling, the resin was filtered and washed 3 times with 2 mL DMF. Following washing, the Fmoc protecting group on the previously attached monomer was deprotected by suspending the resin in 100 pL of a solution of 20% piperidine in DMF for 10 minutes and filtering. This was performed three times to ensure complete Fmoc deprotection. Following deprotecting, the resin was washed seven times with 2 mL DMF. Coupling and deprotection steps were repeated as needed. The final coupling step for all oligourethanes was with Fmoc-Tyr(OMe)-PNOC. After the final Fmoc-Tyr(OMe)-PNOC coupling and deprotection step, the terminal amine was capped by suspending the resin in 100 pL of 5% acetic anhydride in DMF for 5 minutes at room temperature and filtering. This was performed twice to ensure all terminal amines were capped. Following capping, the resin was washed three times with 2 mL DMF and 5 times with 300 pL DCM.
[00168] After synthesis, OUs were cleaved from the solid support by suspending the resin in 500 pL of a solution of 95% TFA/2.5% TIPS/2.5% DCM for 4 hours. The resin was then filtered and washed 5 times with 500 pL DCM. The filtrate was collected and dried first under flowing nitrogen and then under vacuum in a Genevac, producing a brown residue.
(ix)General procedure for oligourethane chain-end degradation
[00169] Sequence-defined oligourethanes of the presently disclosed methods depolymerize through chain-end degradation. In the presence of base and heat, the terminal alcohol undergoes a 5-exo-trig cyclization and attacks its neighboring carbamate bond. Subsequent cleavage of a five-membered oxazolidinone ring leaves a shortened oligourethane with a terminal alcohol. This chain-end degradation continues until the entire oligourethane is depolymerized into monomer-derived oxazolidinones and the final monomer, which in the present Example is methyl tyrosinol acetamide (Scheme 1).
[00170] All OUs were depolymerized with the following method. Solutions containing oligourethanes were evaporated to dryness under vacuum with a Genevac. Oligourethane residues were then dissolved 500 pL of a solution of 20-40 mM K3PO4 and 300 uM (S)-(-)-4-isopropyl-2-oxazolidinone in 40% HzO/MeCN and heated at 70 °C for 16 hours. To remove K3PO4 before LC-MS analysis, samples were evaporated to dryness under vacuum with a Genevac, redissolved in 300 pL MeCN, and filtered.
(x)Oligourethane trimer standard synthesis
[00171] To determine the instrumentation and method to observe oxazolidinones produced from OU degradation, a series of 10 trimer oligourethanes were synthesized which differed only by the middle monomer. By degrading these OU in the same conditions as the OU in the sensor array, the retention time of each oxazolidinone monomer could be identified in the context of chain-end degradation.
[00172] All 10 oligourethane trimers were synthesized in parallel according to the general coupling procedure described in section (viii) above. Trimers were synthesized on a 2.5 pmol scale using 12 ± 0.2 mg of L-phenylalaninol 2-chlorotrityl polystyrene resin with a 0.21 mmol/g loading. Each coupling step used 76 pL of the coupling mixture described previously. For the first coupling step, which installed the second monomer, the coupling step was performed twice before deprotection, to improve coupling efficiency. After synthesis, OU trimers were cleaved from the solid support according to the steps described above. To remove any trifluoroacetylation at the primary alcohol of the cleaved OU, trimers were dissolved in 300 pL MeCN and 100 pL 20 mM K3PO4 and shaken at room temperature for 4 hours. Samples were evaporated to dryness overnight with a Genevac, redissolved in 300 pL MeCN, and filtered. Filtrates were submitted to LC-MS and HRMS for analysis as described in sections above. Characterization data for the trimers is provided in Table 2 below.
Table 2: Characterization data for oligourethane trimer standards.
(xi) Oligourethane tri me r standard degradation
[00173] After characterization, trimers were fully degraded using the degradation method outlined above, including the Val-oxazolidinone internal standard. Samples were submitted to LC-MS to ensure complete degradation. The overlay ed spectra of all the LC traces of the degraded trimers at 280 nm (FIG. 13) show that all the trimers had completely degraded down to the final methyl tyrisinol acetamide monomer (retention time = 2.70 min). The traces for Tyr and Trp monomers also have peaks for their corresponding oxazolidinones (Tyr-oxazolidinone retention time = 1.25 min, Trp-oxazolidinone retention time = 3.80 min). The determination of oxazolidinone and methyl tyrisinol acetamide retention times is described in the section that follows.
(xii) Determination of LC-MS retention times of R-oxazolidinones from extracted ion chromatograms (EICs) of the degraded OU trimers
[00174] Each monomer-derived oxazolidinone was observed in the degradation mixture of their respective trimers as the [M+H]+ m/z intensity in the MSD1 total ion chromatogram (TIC) using Agilent ChemStation. The oxazolidinone extracted ion chromatogram (EIC) peaks of a given monomer were not observed at a high intensity in the degradation mixtures of trimers that were not the trimer of said monomer. The degradation mixture of all trimers contained the Val-oxazolidinone internal standard, Phe-oxazolidinone, and Tyr(OMe)Ac. High resolution mass spectrometry on each of the degradation mixtures corroborated the presence of the respective oxazolidinones. Characterization data for the Vai standard and monomer oxazolidinones is provided in Table 3 below.
Table 3: Characterization data for oxazolidinones
(xiii) Directed oligourethane (OU library synthesis
[00175] All sixteen OU directed (or biased) libraries were synthesized in parallel according to the general coupling procedure described in section (viii) above. OUs were synthesized on a 10 pmol scale, using 47.6 ± 0.3 mg of L-phenylalaninol 2-chlorotrityl polystyrene resin with a 0.21 mmol/g loading. Each coupling step used 297 pL of the coupling mixture described below. Instead of a discrete monomer in the coupling mixture, a mixture of monomers was used to make a pool of oligourethanes with statistically random monomer sequences. Each of the 16 directed libraries was synthesized with a unique mixture of monomers, biased towards different monomers. Table 1 and FIG. 2 show the concentration of monomers in the coupling mixtures for each directed library. The 9th coupling step, which installed Tyr(OMe)Ac as the 10th monomer, was performed twice to improve coupling efficiency. After synthesis, OUs were cleaved from the solid support and evaporated to dryness as described above, leaving a brown residue. (xiv) Directed oligourethane (OU) library synthesis
[00176] Each directed library represents a pool of oligourethanes that could contain any number of 108 different possible sequences, making it untenable to identify individual OUs on the LC-MS. To characterize these OUs, a portion of each directed library was fully chain-end degraded and the resulting R-oxazolidinone composition profile was used to estimate the average monomer composition of the OU in each directed library, according to the following procedure.
[00177] After synthesis and cleavage from the polystyrene solid support, oligourethane libraries were dissolved in 400 mL 25% PLO/MeCN and 6.25 mL of each directed library was removed and diluted with the degradation mixture described above. These aliquots of the directed libraries were fully degraded according to the method outlined above and submitted for LC-MS analysis, using the LC-MS instrumentation and method described above. Oxazolidinones were identified by searching for their [M+H]+ m/z intensities in the MSD1 total ion chromatogram (TIC) using Agilent ChemStation. The software automatically identified significant peaks in the resulting extracted ion chromatogram (EIC) and calculated the area under each curve. Oxazolidinone EIC peak signals were recorded if they could be identified above the noise by human eye and if they had a retention time that was satisfactorily close to that of the oxazolidinone standards run in the study above (summarized in Table 3).
[00178] To visualize the OU monomer composition on a heat map, oxazolidinone EIC peak areas had to be adjusted as discussed in further detail in Examples above (see also FIGS. 3A-3D). More particularly, oxazolidinone EIC peak areas were normalized by dividing by the peak area of the Val-oxazolidinone internal standard in its sample, which was added during OU degradation. Because every oligourethane in the pool begins with phenylalaninol (already present on the solid-phase support used for synthesis) Phe- oxazolidinone can be used as a proxy for the number of OU in a sample. To estimate the average monomer composition of the OU in the directed libraries, oxazolidinone EIC peak areas were also normalized by dividing by the area of the Phe-oxazolidinone EIC peak in the same library sample. Finally, some oxazolidinones were more easily ionized through the positive mode of ESI than others. For easier interpretation of results, Phe-oxazolidinone adjusted EIC peak areas were scaled from 0 to 1 (or min-max adjusted) based on the largest adjusted value for each R- group across all libraries. This facilitated improved comparison of relative monomer composition across all oxazolidinones, regardless of their signal intensities. [00179] After these adjustments, oxazolidinone values were plotted as a heatmap, comparing all directed libraries and the final combined probe library (FIG. 14). Oxazolidinones derived from monomers that were present at a greater concentration during a library synthesis have the highest adjusted values in that directed library. For example, Leu- oxazolidinone and Cha-oxazolidinone read the highest adjusted values in the aliphatic-biased library. The adjusted values for Trp-oxazolidinone, Tyr-oxazolidinone, and BnTrz- oxazolidinone are the highest in the aromatic -biased library. Despite consisting of libraries of highly monomer-biased oligourethanes, the adjusted values for most R-oxazolidinones are similar in the combined probe library. The three exceptions are Lys- and His-oxazolidinones which appear under-represented, and Asp-oxazolidinone which appears over-represented. Tyr(OMe)Ac values are fairly consistent across all samples, suggesting a similar extent of OU degradation.
(xv) Characterization of combined oligourethane probe library
[00180] After characterization, all 16 directed libraries of oligourethanes were combined and dissolved in 6.3 mL of 25% ILO/MeCN, creating a slightly cloudy amber solution at an approximate concentration of 25 mM. This solution of oligourethanes was used without purification and stored at 4 °C. This mixture of biased libraries represents 16 different cross-reactive sensors for differential sensing of surfaces, which are interrogated by elution time and monomer composition. This pool of oligourethanes would be impossible to characterize by LC-MS without chain-end degradation into the oxazolidinones. Supporting that characterization, the total ion chromatogram of the pool of oligourethanes in the positive mode shows a broad peak over several minutes instead of unique peaks (FIG. 15). The 10-mer oligourethanes forming the mixture are calculated to have a range of exact masses between 1400 and 2465 amu, and a multitude of signals in this m/z range is seen in the positive mode mass spectrum between 6 and 14 minutes of the LC-MS analysis. A broad shoulder between 700 and 1230 m/z typically corresponds, without being bound by theory, to the signals for oligourethanes which have gained two positive charges during electrospray ionization.
(xvi) Synthesis of bovine serum albumin (BSA) functionalized silica
[00181] To synthesize bovine serum albumin (BSA) functionalized silica, 20.40 g of propionyl chloride-functionalized silica gel (20.40 mmol, 1 mmol/g loading, 200-400 mesh, 60 A pore size, Sigma Aldrich) and 26.47 g BSA (0.40 mmol, 59 lysine residues/molecule) were suspended in 300 mL anhydrous DMF. 1.65 mL anhydrous pyridine (20.48 mmol) was added and the suspension was shaken at 50 °C for 24 hours. Following this, 150 mL water was added to the reaction flask to improve BSA solubility and the mixture was further shaken at room temperature for 48 hours. The reaction was filtered, leaving behind a beige precipitate. The precipitate was resuspended in 500 mL DI H2O, sonicated, and filtered. The precipitate was washed with a further 2 L DI H2O, 400 mL 25% H2O in MeCN, and 900 mL acetone. The precipitate was then dried under vacuum overnight. Following this, any clumps in the precipitate were broken apart and then passed through a stainless steel 30 mesh sieve, resulting in 42.48 g of a free flowing solid. Based on the dry mass of the starting silica gel and the dried isolated product, an estimated 8 pmol/g BSA was loaded on silica. FTIR solid-phase analysis shows stretches associated with both the propionyl-functionalized silica gel and BSA in the isolated product (FIG. 16).
[00182] The lysine residues in BSA were suspected to have added the acyl chloride to form an amide bond to covalently attach the BSA to silica. However, without being bound by either theory, BSA may instead be strongly absorbed to the surface of the silica gel through non-covalent interactions. To test if BSA would leach out of the silica gel support, which would be consistent with nonco valent interactions between BSA and the silica gel, 50 mg of the isolated solid was shaken in 250 mL of water at room temperature for 16 hours. Filtration and LC-MS analysis of the aqueous filtrate shows no BSA in solution (FIG. 17).
(xvii) Sensor Array/Column Chromatography Procedure
[00183] To have consistency between samples, a 10 mL graduated burette was used as the chromatography column. Substrates were packed as a slurry in the mobile phase (25% H20/MeCN) up to the 5.5 ± 0.2 mL mark on the burette. The oligourethane probe solution was warmed to room temperature and sonicated prior to use. 100 pL of the oligourethane probe solution was loaded onto the column with an Eppendorf pipette and washed with 100 pL of the mobile phase five times. The eluent was collected as 1 mL fractions in a 96-well plate, up to 32 fractions. Optionally, a stream of nitrogen gas was used to densely pack the stationary phase or to push the mobile phase through the column. After running the column, the fractions were evaporated to dryness under vacuum with a Genevac, revealing either a white powder or a yellow residue. [00184] Next, column fractions containing the eluted oligourethanes were degraded into the monomer-derived oxazolidinone cyclized products. Dried oligourethanes were redissolved in the sequencing mixture described elsewhere, samples were degraded according to the procedure outline above, and submitted for LC-MS analysis, using the method outline above. R-oxazolidinones were measured by identifying for their [M+H]+ m/z intensities in the MSD1 total ion chromatogram (TIC) using Agilent ChemStation. The software automatically identified significant peaks in the resulting extracted ion chromatogram (E1C) and calculated the area under each curve. Oxazolidinone EIC peak signals were recorded if they could be identified above the noise by human eye and if they had a retention time that was satisfactorily close to that of the oxazolidinone standards as shown in Table 3 above.
(xviii) Principle Component Analysis (PC A)
[00185] Code to perform PCA was written in Python, using the SciKitLeam PCA package. All graphs used to visualize PCA results were created through the Matplotlib library in Python. Code was written with assistance from ChatGPT version 3.5 and 4.0. Data related to PCA are provided in FIGS. 6, 7, 20, 21, and 22. Data related to the results of the loading plots analysis are presented in FIGS. 9 and 10.
(xix) Linear Discriminant analysis (LDA)
[00186] Code to perform LDA was written in Python, using the SciKitLearn Linear Discriminant Package. Code to perform the jackknife analysis was written in Python, using the SciKitLearn LeaveOneOut library. All graphs used to visualize LDA results were created through the Matplotlib library in Python. Code was written with assistance from ChatGPT version 3.5 and 4.0. Data related to the LDA is provided as FIG. 11.
* * *
[00187] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
REFERENCES
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Claims

CLAIMS:
1. A method of determining the identity of a surface comprising:
(A) loading the surface into a chamber; and
(B) exposing a length of the surface in the chamber to a chemical probe, wherein the chemical probe comprises a solution of one or more oligourethanes in the presence of a mobile phase;
(C) identifying the chemical probe and/or measuring a retention time of the chemical probe; and
(D) correlating the identity of the chemical probe or the retention time of the chemical probe to the identity of the surface.
2. The method of claim 1, wherein the oligourethane is a polymer comprising at least two monomer units of the formula R 0 , wherein R is an independently selected chemical group.
3. The method of claim 1, wherein the oligourethane is a polymer comprising at least two unique monomer units of the formula R O , wherein R is an independently selected chemical group.
4. The method according to any one of claims 1-3, wherein the oligourethane comprises from about three to about 50 monomer units.
5. The method of claim 4, wherein the oligourethane comprises from about 5 urethane moieties to about 15 monomer units.
6. The method of claim 5, wherein the oligourethane comprises about 10 monomer units.
7. The method according to any one of claims 1-6, wherein the chemical group comprises an amino group, an alcohol group, a C1-C12 alkyl group, a C1-C12 cycloalkyl group, a C1-C12 heterocycloalkyl group, a C2-C12 alkenyl group, a C2-C12 alkynyl group, a C1-C12 aryl group, a C1-C12 aralkyl group, a C1-C12 heteroaryl group, a C1-C12 heteroaralkyl group, a C1-C12 alkylamino group, a C2-C24 dialkylamino group, a guanidyl group, a hydroxide group, a C1-C12 alkoxide group, a C1-C12 cycloalkoxide group, a mercaptate group, a sulfate group, a phosphate group, or a C1-C12 carboxylate group, or a substituted version of any of these groups, wherein the substituted version includes a polar functional group, a halide, a hydrogen bond donating group, a hydrogen bond accepting group, an acidic group, or a basic group.
8. The method according to any one of claims 2-7, wherein at least one of the monomer units has an R group selected from among a Cl -Cl 2 aryl group, a Cl -Cl 2 heteroaryl group, a C1-C12 aralkyl group, a C1-C12 heteroaralkyl group, or a substituted version of any of these groups.
9. The method according to any one of claims 2-8, wherein at least one of the monomer units has an R group selected from among a C1-C12 alkyl group, a C1-C12 cycloalkyl group, a C1-C12 heterocycloalkyl group, or a substituted version of either of these groups.
10. The method according to any one of claims 2-9, wherein at least one of the monomer units has an R group selected from among a C2-C12 alkenyl group, a C2-C12 alkynyl group, or a substituted version of either of these groups.
11. The method according to any one of claims 2-10, wherein at least one of the monomer units has an R group comprising a positive charge.
12. The method according to any one of claims 2-11, wherein at least one of the monomer units has an R group comprising a protonated amino group, a protonated C1-C12 alkylamino group, a protonated C2-C24 dialkylamino group, a protonated guanidyl group, or a substituted version of any of these groups.
13. The method according to any one of claims 2-12, wherein at least one of the monomer units has an R group comprising a negative charge.
14. The method according to any one of claims 2-13, wherein at least one of the monomer units has an R group comprising a hydroxide group, a C1-C12 alkoxide group, a Cl- C12 cycloalkoxide group, a mercaptate group, a sulfate group, a phosphate group, or a C1-C12 carboxylate group, a substituted version of any of these groups.
15. The method according to any one of claims 2-14, wherein at least one of the monomer units has an R group comprising a protecting group.
16. The method according to any one of claims 1-15, wherein identifying the chemical probe comprises degrading the chemical probe to obtain a degraded solution.
17. The method according to claim 16, wherein the degraded solution comprises at least one oxazolidinone.
18. The method according to either claim 16 or claim 17, wherein the method further comprises identifying an oxazolidinone in the degraded solution or the composition of oxazolidinone in the degraded solution.
19. The method according to any one of claims 16-18, wherein an oxazolidinone in the degraded solution or the composition of oxazolidinone in the degraded solution is identified by spectrometry.
20. The method of claim 19, wherein the spectrometry is mass spectrometry (MS).
21. The method of claim 20, wherein the mass spectrometry is a liquid chromatography mass spectrometry (LC-MS).
22. The method according to any one of claims 1-21, wherein the identity of the chemical probe or the retention time of the chemical probe is correlated with the identity of the surface through a microprocessor.
23. The method according to any one of claims 1-22, wherein correlating the identity of the chemical probe to the identity of the surface comprises correlating the identity of an oxazolidinone in the degraded solution or the composition of oxazolidinone in the degraded solution to the identity of the surface.
24. The method according to any one of claims 1-23, wherein the identity of the oxazolidinone in the degraded solution or the composition of oxazolidinone in the degraded solution is correlated with the identity of the surface through a microprocessor.
25. The method according to any one of claims 1-24, wherein the surface is a soil sample.
26. A method of synthesizing a library of oligourethanes for identifying or detecting a surface, wherein the method comprises:
A) coupling at least two unique monomer units of the formula: in a coupling reaction mixture to provide a pool of oligourethanes, wherein: the coupling reaction mixture comprises the unique monomer units and solvent;
R is a chemical group;
Ri and R2 are each independently hydrogen or a protecting group, or Ri and R2 are taken together and are a protecting group; and
R;, is an activating group; wherein the unique monomer units in the coupling mixture do not have the same R group; and B) combining at least one pool of oligourethanes to provide the library of oligourethanes.
27. The method according to claim 26, wherein the coupling reaction mixture comprises between 2 and 50 unique monomer units.
28. The method according to claim 27, wherein the coupling reaction mixture comprises between 5 and 15 unique monomer units.
29. The method according to claim 28, wherein the coupling reaction mixture comprises 10 unique monomer units.
30. The method according to any one of claims 26-29, wherein the library comprises between 5 and 20 pools of oligourethanes.
31. The method according to any one of claims 26-30, wherein the library comprises about 16 pools of oligourethanes.
32. The method according any one of claims 26-31, wherein the coupling mixture for each pool of oligourethanes has a different molar ratio of monomer units.
33. The method according to any one of claims 26-32, wherein at least one of the coupling mixtures comprises at least a first unique monomer unit at a higher concentration than at least a second unique monomer unit.
34. The method according to claim 33, wherein between 5 and 20 of the coupling mixtures comprise at least a first unique monomer unit at a higher concentration than at least a second unique monomer unit.
35. The method according to either claim 33 or claim 34, wherein 15 of the coupling mixtures comprise at least a first unique monomer unit at a higher concentration than at least a second unique monomer unit.
36. The method according to any one of claims 33-35, wherein the first unique monomer unit has a concentration between about 2 and about 30 times higher than concentration of the second unique monomer unit.
37. The method according to any one of claims 33-36, wherein the first unique monomer unit has a concentration between about 4 and about 30 times higher than the concentration of the second unique monomer unit.
38. The method according to any one of claims 33-37, wherein the first unique monomer unit has a concentration about 4, about 5, about 6, or about 7 times higher than the concentration of the second unique monomer unit.
39. The method according to any one of claims 33-38, wherein the R group of the first monomer unit is selected from among -NH2, -OH, a C1-C12 alkyl group, a C1-C12 cycloalkyl group, a C1-C12 heterocycloalkyl group, a C1-C12 alkoxy group, a C2- C12 alkenyl group, a C2-C12 alkynyl group, a C1-C12 acyl group, a C1-C12 aryl group, a C1-C12 aralkyl group, a C1-C12 heteroaryl group, a C1-C12 heteroaralkyl group, a C1-C12 alkylamino group, a C2-C24 dialkylamino group, a guanidyl group, a hydroxide group, a C1-C12 alkoxide group, a C1-C12 cycloalkoxide group, a mercaptate group, a sulfate group, a phosphate group, a C1-C12 carboxylate group or a substituted version of any of these groups, wherein the substituted version includes a polar functional group, a halide, a hydrogen bond donating group, a hydrogen bond accepting group, an acid group, or a basic group.
40. The method according to any one of claims 33-39, wherein the R group of the first monomer unit is selected from among a C1-C12 aryl group, a C1-C12 heteroaryl group, a C1-C12 aralkyl group, a C1-C12 heteroaralkyl group, or a substituted version of any of these groups.
41. The method according to any one of claims 33-39, wherein the R group of the first monomer unit is selected from among a C1-C12 alkyl group, a C1-C12 cycloalkyl group, a C1-C12 heterocycloalkyl group, or a substituted version of either of these groups.
42. The method according to any one of claims 33-39, wherein the R group of the first monomer unit is selected from among a C2-C12 alkenyl group, a C2-C12 alkynyl group, or a substituted version of either of these groups.
43. The method according to any one of claims 33-39, wherein the R group of the first monomer unit comprises a positive charge.
44. The method according to any one of claims 33-39, wherein the R group of the first monomer unit comprises an amino group, a C1-C12 alkylamino group, a C2-C24 dialkylamino group, a guanidyl group, or a substituted version of any of these groups.
45. The method according to any one of claims 33-39, wherein the R group of the first monomer unit comprises a negative charge.
46. The method according to any one of claims 33-39, wherein the R group of the first monomer unit comprises a hydroxide group, a C1-C12 alkoxide group, a C1-C12 cycloalkoxide group, a mercaptate group, a sulfate group, a sulfoxide group, a phosphate group, or a carboxylate group, a substituted version of any of these groups.
47. The method according to claim 33, wherein the R group of a monomer unit comprises a protecting group.
48. The method according to any one of claims 33-47, wherein at least one of the coupling mixtures comprises at least a first monomer unit and a third monomer unit at a higher concentration than at least a second monomer unit.
49. The method according to claim 48, wherein between 5 and 20 of the coupling mixtures comprise at least a first monomer unit and a third monomer unit at a higher concentration than at least a second monomer unit.
50. The method according to either claim 48 or claim 49, wherein 15 of the coupling mixtures of the pools of oligourethanes of the chemical probe comprises at least a first monomer unit and a third monomer unit at a higher concentration than at least a second monomer unit.
51. The method according to any one of claims 48-50, wherein the R group of the third monomer unit is selected from among -NH2, -OH, a C1-C12 alkyl group, a C1-C12 cycloalkyl group, a C1-C12 heterocycloalkyl group, a C1-C12 alkoxy group, a C2- C12 alkenyl group, a C2-C12 alkynyl group, a C1-C12 acyl group, a C1-C12 aryl group, a C1-C12 aralkyl group, a C1-C12 heteroaryl group, a C1-C12 heteroaralkyl group, a C1-C12 alkylamino group, a C2-C24 dialkylamino group, a guanidyl group, a hydroxide group, a C1-C12 alkoxide group, a C1-C12 cycloalkoxide group, a mercaptate group, a sulfate group, a phosphate group, a C1-C12 carboxylate group or a substituted version of any of these groups, wherein the substituted version includes a polar functional group, a halide, a hydrogen bond donating group, a hydrogen bond accepting group, an acid group, or a basic group.
52. The method according to any one of claims 48-51, wherein the R group of the third monomer unit is selected from among a C1-C12 aryl group, a C1-C12 heteroaryl group, a C1-C12 aralkyl group, a C1-C12 heteroaralkyl group, or a substituted version of any of these groups.
53. The method according to any one of claims 48-51, wherein the R group of the third monomer unit is selected from among a C1-C12 alkyl group, a C1-C12 cycloalkyl group, a C1-C12 heterocycloalkyl group, or a substituted version of either of these groups.
54. The method according to any one of claims 48-51, wherein the R group of the third monomer unit is selected from among a C2-C12 alkenyl group, a C2-C12 alkynyl group, or a substituted version of either of these groups.
55. The method according to any one of claims 48-51, wherein the R group of the third monomer unit comprises a positive charge.
56. The method according to any one of claims 48-51, wherein the R group of the third monomer unit comprises an amino group, a C1-C12 alkylamino group, a C2-C24 dialkylamino group, a guanidyl group, or a substituted version of any of these groups.
57. The method according to any one of claims 48-51, wherein the R group of the third monomer unit comprises a negative charge.
58. The method according to any one of claims 48-51, wherein the R group of the third monomer unit comprises a hydroxide group, a C1-C12 alkoxide group, a C1-C12 cycloalkoxide group, a mercaptate group, a sulfate group, a phosphate group, or a carboxylate group, a substituted version of any of these groups.
59. The method according to claim 48, wherein the R group of a monomer unit comprises a protecting group.
60. The method according to any one of claims 48-50, wherein: the R group of the first monomer unit comprises an aliphatic group; and the R group of the third monomer unit comprises an aromatic group.
61. The method according to any one of claims 48-50 and 60, wherein: the R group of the first monomer unit is selected from among a Cl -C 12 alkyl group, a Cl -Cl 2 cycloalkyl group, a Cl -Cl 2 heterocycloalkyl group, or a substituted version of either of these groups; and the R group of the third monomer unit is selected from among a Cl -Cl 2 aryl group, a C1-C12 heteroaryl group, a C1-C12 aralkyl group, a C1-C12 heteroaralkyl group, or a substituted version of any of these groups.
62. The method according to any one of claims 48-50, wherein: the R group of the first monomer unit comprises an aliphatic group; and the R group of the third monomer unit comprises an unsaturated group.
63. The method according to any one of claims 48-50 and 62, wherein: the R group of the first monomer unit is selected from among a Cl -Cl 2 alkyl group, a Cl -Cl 2 cycloalkyl group, a Cl -Cl 2 heterocycloalkyl group, or a substituted version of either of these groups; and the R group of the third monomer unit is selected from among a C2-C12 alkenyl group, a C2-C12 alkynyl group, or a substituted version of either of these groups.
64. The method according to any one of claims 48-50, wherein: the R group of the first monomer unit comprises an aliphatic group; and the R group of the third monomer unit comprises a positive charge.
65. The method according to any one of claims 48-50 and 64, wherein: the R group of the first monomer unit is selected from among a Cl -Cl 2 alkyl group, a Cl -Cl 2 cycloalkyl group, a Cl -Cl 2 heterocycloalkyl group, or a substituted version of either of these groups; and the R group of the third monomer unit is selected from among a Cl -Cl 2 alkylamino group, a C2-C24 dialkylamino group, a guanidyl group, or a substituted version of any of these groups.
66. The method according to any one of claims 48-50, wherein: the R group of the first monomer unit comprises an aliphatic group; and the R group of the third monomer unit comprises a negative charge.
67. The method according to any one of claims 48-50 and 66, wherein: the R group of the first monomer unit is selected from among a Cl -Cl 2 alkyl group, a Cl -Cl 2 cycloalkyl group, a Cl -Cl 2 heterocycloalkyl group, or a substituted version of either of these groups; and the R group of the third monomer unit is selected from among hydroxide group, a C1-C12 alkoxide group, a C1-C12 cycloalkoxide group, a mercaptate group, a sulfate group, a phosphate group, or a carboxylate group, a substituted version of any of these groups.
68. The method according to any one of claims 48-50, wherein: the R group of the first monomer unit comprises an aromatic group; and the R group of the third monomer unit comprises an unsaturated group.
69. The method according to any one of claims 48-50 and 68, wherein: the R group of the first monomer unit is selected from among a Cl -Cl 2 aryl group, a C1-C12 heteroaryl group, a C1-C12 aralkyl group, a C1-C12 heteroaralkyl group, or a substituted version of any of these groups; and the R group of the third monomer unit is selected from among a C2-C12 alkenyl group, a C2-C12 alkynyl group, or a substituted version of either of these groups.
70. The method according to any one of claims 48-50, wherein: the R group of the first monomer unit comprises an aromatic group; and the R group of the third monomer unit comprises a positive charge.
71. The method according to any one of claims 48-50 and 70, wherein: the R group of the first monomer unit is selected from among a Cl -Cl 2 aryl group, a C1-C12 heteroaryl group, a C1-C12 aralkyl group, a C1-C12 heteroaralkyl group, or a substituted version of any of these groups; and the R group of the third monomer unit is selected from among a Cl -Cl 2 alkylamino group, a C2-C24 dialkylamino group, a guanidyl group, or a substituted version of any of these groups.
72. The method according to any one of claims 48-50, wherein: the R group of the first monomer unit comprises an aromatic group; and the R group of the third monomer unit comprises a negative charge.
73. The method according to any one of claims 48-50 and 72, wherein: the R group of the first monomer unit is selected from among a Cl -Cl 2 aryl group, a C1-C12 heteroaryl group, a C1-C12 aralkyl group, a C1-C12 heteroaralkyl group, or a substituted version of any of these groups; and the R group of the third monomer unit is selected from among hydroxide group, a C1-C12 alkoxide group, a C1-C12 cycloalkoxide group, a mercaptate group, a sulfate group, a phosphate group, or a carboxylate group, a substituted version of any of these groups.
74. The method according to any one of claims 48-50, wherein: the R group of the first monomer unit comprises an unsaturated group; and the R group of the third monomer unit comprises a positive charge.
75. The method according to any one of claims 48-50 and 74, wherein: the R group of the first monomer unit is selected from among a C2-C12 alkenyl group, a C2-C12 alkynyl group, or a substituted version of either of these groups; and the R group of the third monomer unit is selected from among a Cl -Cl 2 alkylamino group, a C2-C24 dialkylamino group, a guanidyl group, or a substituted version of any of these groups.
76. The method according to any one of claims 48-50, wherein: the R group of the first monomer unit comprises an unsaturated group; and the R group of the third monomer unit comprises a negative charge.
77. The method according to any one of claims 48-50 and 76, wherein: the R group of the first monomer unit is selected from among a C2-C12 alkenyl group, a C2-C12 alkynyl group, or a substituted version of either of these groups; and the R group of the third monomer unit is selected from among hydroxide group, a C1-C12 alkoxide group, a C1-C12 cycloalkoxide group, a mercaptate group, a sulfate group, a phosphate group, or a carboxylate group, a substituted version of any of these groups.
78. The method according to any one of claims 48-50, wherein: the R group of the first monomer unit comprises a positive charge; and the R group of the third monomer unit comprises a negative charge.
79. The method according to any one of claims 48-50 and 78, wherein: the R group of the first monomer unit is selected from among a Cl -Cl 2 alkylamino group, a C2-C24 dialkylamino group, a guanidyl group, or a substituted version of any of these groups; and the R group of the third monomer unit is selected from among hydroxide group, a C1-C12 alkoxide group, a C1-C12 cycloalkoxide group, a mercaptate group, a sulfate group, a phosphate group, or a carboxylate group, a substituted version of any of these groups.
80. The method according to any one of claims 33-79, wherein the total concentration of between about 10% and about 60% of the unique monomer units in the coupling mixture is about 2, about 3, about 4, about 5, about 6, about 7, or about 8 times higher than the total concentration of the other unique monomer units in the coupling mixture.
81. The method according to any one of claims 33-80, wherein the sum of the concentrations of about 30% of the unique monomer units in the coupling mixture is about 2, about 3, about 4, about 5, about 6, about 7, or about 8 times higher than the total concentration of the other unique monomer units in the coupling mixture.
82. A biased library of oligourethanes for detecting or identifying a surface, wherein the biased library comprises at least one pool of oligourethanes synthesized according to the method of any one of claims 33-81.
83. A method of determining the presence of a chemical analyte in a sample comprising: (A) loading the sample into a chamber; and (B) exposing a length of the sample in the chamber to a chemical probe, wherein the chemical probe comprises a solution of one or more unique oligourethanes, in the presence of a mobile phase;
(C) identifying the chemical probe and/or measuring a retention time of the chemical probe; and
(D) correlating the identity of the chemical probe or the retention time of the chemical probe to the identity of the substrate in the sample or to the identity of the sample.
84. A method of determining the identity of a substrate in a sample or the identity of the sample comprising:
(A) loading the sample into a chamber; and
(B) exposing a length of the sample in the chamber to a chemical probe, wherein the chemical probe comprises a solution of one or more unique oligourethanes, in the presence of a mobile phase;
(C) identifying the chemical probe and/or measuring a retention time of the chemical probe; and
(D) correlating the identity of the chemical probe or the retention time of the chemical probe to the identity of the sample.
85. A method of determining the identity of a substrate in a sample or the identity of the sample comprising:
(A) loading the sample into a chamber; and
(B) exposing a length of the sample in the chamber to a chemical probe, wherein the chemical probe comprises a solution of one or more unique oligourethanes, in the presence of a mobile phase, wherein the oligourethane comprises one or more distinct sequences;
(C) identifying the chemical probe and/or measuring a retention time of the chemical probe; and
(D) correlating the identity of the chemical probe or the retention time of the chemical probe to the identity of the substrate in the sample or to the identity of the sample.
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