Systems and methods for drug discovery based on desorption electrospray ionization

Abstract

The invention generally relates to systems and methods for drug discovery based on desorption electrospray ionization. In certain aspects, the invention provides a method of producing a modified antibiotic compound, the method comprising: directing a liquid droplet spray discharge from a sample probe onto a discrete spot comprising an antibiotic and one or more reactants so as to form a microdroplet that comprises the antibiotic and the one or more reactants and allowing a reaction to occur in the microdroplet between the antibiotic and the one or more reactants to produce a modified antibiotic; and collecting the modified antibiotic.

Description

[0001] Attorney Docket No.: PURD- 165 / 01 WO 28593 / 767

[0002] PATENT APPLICATION SYSTEMS AND METHODS FOR DRUG DISCOVERY BASED ON DESORPTION ELECTROSPRAY IONIZATION

[0003] Related Application

[0004] The present application claims the benefit of and priority to U.S. provisional patent application serial number 63 / 775,560, filed March 21, 2025, the content of which is incorporated by reference herein in its entirety.

[0005] Government Support

[0006] This invention was made with government support under TR004139, awarded by the NIH. The government has certain rights in the invention. This invention was made with government support under FA9550-21-1-0170, awarded by the AFOSR. The government has certain rights in the invention.

[0007] Field of the Invention

[0008] The invention generally relates to systems and methods for drug discovery based on desorption electrospray ionization.

[0009] Background

[0010] The rapid emergence of resistant bacteria is occurring worldwide, endangering the efficacy of antibiotics, which have transformed medicine and saved millions of lives. Many decades after the first patients were treated with antibiotics, bacterial infections have again become a threat. The antibiotic resistance crisis has been attributed to the overuse and misuse of these medications, as well as a lack of new drug development by the pharmaceutical industry due to reduced economic incentives and challenging regulatory requirements. The Centers for Disease Control and Prevention (CDC) has classified a number of bacteria as presenting urgent, serious, and concerning threats, many of which are already responsible for placing a substantial clinical and financial burden on the U.S. health care system, patients, and their families.

[0011] Coordinated efforts to implement new policies, renew research efforts, and pursue steps to manage the crisis are greatly needed.Attorney Docket No.: PURD- 165 / 01 WO 28593 / 767

[0012] PATENT APPLICATION

[0013] Summary

[0014] The invention generally relates to use of new platform that allows for rapid production of modified bioactive molecules in droplets that can be isolated, interrogated, and purified through a single high-throughput process. In specific embodiments, the invention allows for the late stage functionalization of existing antibiotics to create rapidily create an array of newly modified antibitoics that have greater killing ability than previously existing antibiotics, thereby providing new antibitoics that avoid the current antibiotic resistance crisis.

[0015] In certain aspects, the invention provides a method of producing a modified antibiotic compound, the method comprising: directing a liquid droplet spray discharge from a sample probe onto a discrete spot comprising an antibiotic and one or more reactants so as to form a microdroplet that comprises the antibiotic and the one or more reactants and allowing a reaction to occur in the microdroplet between the antibiotic and the one or more reactants to produce a modified antibiotic; and collecting the modified antibiotic. In certain embodiments, a rate of the reaction among the antibiotic and the one or more reactants in the microdroplet is accelerated as compared to a rate of the reaction among the antibiotic and the one or more reactants in the microdroplet in a bulk liquid.

[0016] In certain embodiments, prior to the collecting step, the microdroplet is directed to a mass spectrometer in which the modified antibiotic is mass separated and selected. In certain embodiments, the sampling probe is a desorption electrospray ionization probe and the liquid droplet spray discharge is a desorption electrospray ionization active discharge. In certain embodiments, the sampling probe comprises a gas source and a voltage source. In certain embodiments, the mass spectrometer is a bench-top mass spectrometer or a miniature mass spectrometer.

[0017] In certain embodiments, prior to the directing step, the method first comprises providing a substrate, the substrate comprises a plurality of discrete spots, wherein each of the plurality of discrete spots comprises a different one or more reactants and the antibiotic to thereby produce a plurality of different modified antibiotics.

[0018] In certain embodiments, the substrate is a movable substrate and the movable substrate is operably coupled to a motor that moves the substrate in an automated manner. In certain embodiments, the sampling probe is operably coupled to an movable arm and the movable arm is operably coupled to a motor that moves the sampling probe in an automated manner. In certainAttorney Docket No.: PURD- 165 / 01 WO 28593 / 767

[0019] PATENT APPLICATION

[0020] embodiments, the substrate is a first substrate and the method further comprises providing a second substrate configured to align with the first substrate such that the modified antibiotic produced in the microdroplet from the discrete location on the first substrate is landed at a corresponding location on the second substrate.

[0021] In other aspects, the invention provides a method of producing a modified antibiotic compound, the method comprising: providing a first substrate comprising a first discrete spot comprising an antibiotic and one or more reactants; directing a liquid droplet spray discharge from a sample probe onto the discrete spot so as to form a microdroplet that comprises the antibiotic and the one or more reactants and allowing a reaction to occur in the microdroplet between the antibiotic and the one or more reactants to produce a modified antibiotic; collecting the modified antibiotic at a corresponding location on a second substrate; and analyzing the modified antibiotic from the corresponding location on the second substrate. In certain embodiments, a rate of the reaction among the antibiotic and the one or more reactants in the microdroplet is accelerated as compared to a rate of the reaction among the antibiotic and the one or more reactants in the microdroplet in a bulk liquid.

[0022] In certain embodiments, prior to the collecting step, the microdroplet is directed to a mass spectrometer in which the modified antibiotic is mass separated and selected. In certain embodiments, the sampling probe is a desorption electrospray ionization probe and the liquid droplet spray discharge is a desorption electrospray ionization active discharge. In certain embodiments, the sampling probe comprises a gas source and a voltage source. In certain embodiments, the mass spectrometer is a bench-top mass spectrometer or a miniature mass spectrometer.

[0023] In certain embodiments, the first substrate comprises a plurality of discrete spots, wherein each of the plurality of discrete spots comprises a different one or more reactants and the antibiotic to thereby produce a plurality of different modified antibiotics. In certain embodiments, the substrate is a movable substrate and the movable substrate is operably coupled to a motor that moves the substrate in an automated manner.

[0024] In certain embodiments, the second substrate is configured for linear and rotary movement. In certain embodiments, the sampling probe is operably coupled to an movable arm and the movable arm is operably coupled to a motor that moves the sampling probe in an automated manner.Attorney Docket No.: PURD- 165 / 01 WO 28593 / 767

[0025] PATENT APPLICATION

[0026] Brief Description of the Drawings

[0027] FIG. 1 shows an exemplary embodiment of a novel hybrid, high-throughput automated instrumental system.

[0028] FIG. 2A shows an exemplary chemical synthesis module and FIG. 2B illustrates an exemplary workflow in such module.

[0029] FIG. 3 illustrates various different exemplary reaction types that can be performed by the chemical reaction module and various exemplary types of chemical products that may be produced by such module.

[0030] FIG. 4A illustrates a two-position DESI stage and FIG. 4B shows additional aspects of a work-flow through the system.

[0031] FIG. 5 illustrates in an exemplary manner how the fluid handling components of the system operate together to prepare chemical products and biological molecules in the incubation molecule for a bioassay and subsequent analysis in a mass spectrometer.

[0032] FIG. 6 illustrates in another embodiment how the fluid handling components of the system operate together to prepare chemical products and biological molecules in the incubation molecule for another bioassay and subsequent analysis in a mass spectrometer.

[0033] FIG. 7 shows a multiplexed inductive nano-electrospray and micro-electrophoresis apparatus to achieve rapid in situ sample clean-up and ionization using small volume samples.

[0034] FIG. 8A illustrates a second characterization position that can optionally be coupled and operated via the systems of the invention and FIG. 8B shows additional aspects of a work-flow through the system.

[0035] FIGS. 9-10 further show the characterization and additional biological activity exploration that can be achieved via the second characterization position.

[0036] FIG. 11A shows how the data obtained from the system can be used to determine structure - bioactivity correlations and FIG. 1 IB shows final steps in the workflow using systems of the invention.

[0037] FIG. 12 illustrates an exemplary workflow in the chemical synthesis module.

[0038] FIG. 13 panels A-C show a dip-and-go multiplexed nanoESI System. Panel A) Tip loading of the nanoESI emitters from 96-well plates (10 s). Panel B) In situ electrophoretic clean-up; note that it is performed simultaneously to all the emitters (10 s). Panel C) Sequential MS analysis of the emitters (40 s). All times given are for 12 emitters.Attorney Docket No.: PURD- 165 / 01 WO 28593 / 767

[0039] PATENT APPLICATION FIG. 14 is an illustration showing an exemplary data analysis module for implementing the systems and methods of the invention in certain embodiments.

[0040] FIGS. 15A-B show late-stage functionalization of anti-fungal agent AmB.

[0041] FIG. 16 shows reaction screening of set of antibiotics showing heat maps for functionalization.

[0042] FIG. 17 shows conversion of carboxylic acid to amide in an antibiotic.

[0043] FIG. 18 shows array -to-array collection, bioassay and in situ product identification methodology.

[0044] FIG. 19 shows a demonstration of dramatic change in bioactivity of an antibiotic after chemical modification.

[0045] FIG. 20 shows P-Lactam activity is reducted by the ring-opening enzyme beta-lactamase produced by Bacillus cereus in response to presence of Amoxicillin or Ampicillin.

[0046] Detailed Description

[0047] The invention generally relates to systems and methods for drug discovery based on desorption electrospray ionization. The platform described herein provides for the utilization, in concert, of a set of capabilities allow for accelerated reactions in microdroplets; generation of microdroplets by an MS ionization method (e.g., DESI); and reaction screening (especially with use of an automated HT system) that integrates preparation of reaction mixtures and their arraying on 2D plates with a mass spectrometer which interrogates the reactant array during a DESI ionization event and then records reaction products in the secondary droplets by mass analysis while also intermittently allowing product collection by droplet deposition on a surface including in the form of a product array that is indexed to the reactant array. Products of special interest are those generated by late stage functionalization (LSF) of existing bioactive molecules, the capability to utilize LSF being conferred by the accelerated reactions that occur in the flying microdroplets. The products can be collected on spots occupied by enzymes (and their supporting media) or these can be added subsequently, to allow enzyme-based bioassays of the synthetic products with DESLMS being a particularly appropriate method of measuring bioactivity in the arrayed products because it combines MS analysis with imaging capabilities.

[0048] As an alternative to enzyme assays of bioactivity, competitive displacement assays or AST assays for microorganism kills can be used. It is noteworthy that in certain embodimentds,Attorney Docket No.: PURD- 165 / 01 WO 28593 / 767

[0049] PATENT APPLICATION

[0050] there no need to purify collected products prior to the bioassays because DESI, unlike other MS ionization methods, is little affected by phosphate buffers or enzyme-supporting media, and furthermore, because MS analysis and product collection can be alternated so that only product known to have favorable composition can be tested for bioassay.

[0051] High Throughput Platform

[0052] Certain aspects of high throughput synthesis and analysis systems and uses thereof are described for example in US 12,505,900, the content of which is incorporated by reference herein in its entirety. Additional description is also provided in WO 2024 / 118729, the content of which is incorporated by reference herein in its entirety. High throughput bioassays are described for example in US 11,397,166, the content of which is incorporated by reference herein in its entirety. In certain embodiments, current properties of the automated system include a 3 Hz rate (reaction and analysis), 50 nL sample volumes (ca. 5 ng amounts) per spot, 6,144 spot density on teflon surfaces on plates with standard microtiter dimensions. MS and MS / MS capabilities. QToF mass spectrometer.

[0053] As now described here, FIG. 1 shows an exemplary embodiment of a novel hybrid, high-throughput automated instrumental system. As shown in FIG. 1, the system may include a chemical synthesis module comprising: one or more instruments for generating arrays of discrete spots on a substrate; and a desorption electrospray ionization (DESI) source for desorbing and ionizing chemical molecules and forming a chemical product from each discrete spot. The system may also include an analysis module comprising a mass spectrometer positioned to receive the desorbed and ionized chemical product from each spot. The system may also include an incubation module configured to incubate the chemical product with one or more biological materials for conducting a biological assay. The system may also include a bioassay module configured to analyze results of the biological assay comprising the chemical product. The system may also include a control apparatus comprising: a fluid handling apparatus comprising one or more robotic instruments configured to handle and move a substrate and handle one or more fluids among the chemical synthesis module, the incubation module, and the bioassay module; and a control module comprising software to control and coordinate operation of each of the chemical synthesis module, the incubation module, and the bioassay module.Attorney Docket No.: PURD- 165 / 01 WO 28593 / 767

[0054] PATENT APPLICATION FIG. 1 also includes an optional aspect of the system, which is optionally a second characterization position that is accessible by the fluid handling apparatus of the control module and that can be operable coupled to the control module. As shown by example in FIG. 1, the second characterization position may comprise a Raman spectrometer, which is only exemplary and non-limiting of the possible options for the second characterization position.

[0055] FIG. 2A shows an exemplary chemical synthesis module and FIG. 2B illustrates an exemplary workflow in such module. The module is a high-throughput platform that combines a set of interrelated methods in which mass spectrometry methods may be used to create droplets and thin films in which accelerated reactions occur, while simultaneously or subsequently using mass spectrometry to analyze the product distribution in the droplets and / or thin films. This platform has broad applicability to many different chemical and biological systems. The fluid handling system controls sample handling and generates microwell plates of various sizes, exemplified here as a 384 well plate. The fluid handling system using a pinning device interacts with the microwell plate to generate a substrate of discrete spots, using for example a pinning device. A DESI source is integrated as part of the system and directed a DESI active spray discharge onto each of the spots sequentially. The spray discharge desorbs and ionizes analytes from each spot, which are directed into a mass spectrometer for analysis. This high-throughput system has the capability of screen reaction products of interest atl second per reaction (or less) in a system capable of 24-hour continuous operation.

[0056] As shown in FIG. 2A, in certain embodiments, the control module further comprises software that generates a heat map of the chemical product from each spot to illustrate successful formation of the chemical product for each spot. The software may then analyze the heat map and determine which chemical products should be rescanned for structural information.

[0057] Numerous different chemical compounds and reactions can be conducted in the chemical synthesis module. FIG. 3 illustrates various different exemplary reaction types that can be performed by the chemical reaction module and various exemplary types of chemical products that may be produced by such module.

[0058] In certain embodiments, the chemical synthesis module further comprises: a two-position DESI stage wherein a first position is configured for analysis of the chemical product and is proximate an inlet of the mass spectrometer and a second position located away from the inlet of the mass spectrometer; and a switch controlled by the control module to switch between the firstAttorney Docket No.: PURD- 165 / 01 WO 28593 / 767

[0059] PATENT APPLICATION

[0060] and second position (FIG. 4A). To achieve high-throughput screening of properties, the system is configured to allow collection of the synthetized products using aDESI-spray-angle switching approach (as described herein). In short, a new DESI stage, with two positions (analysis: close to the MS inlet, and collection: away from the MS) is used. The analysis position is used for reaction screening in high-density plates, while the collection position is used to deposit the product on lower-density well-plates. With this approach, perhaps 10% of the reactions screened would yield hot-spots in a typical plate, and approximately 1-2 ng of product will be deposited.

[0061] FIG. 4B then shows additional aspects of the workflow within the systems and methods of the invention. At this point, hotspots, (chemical compounds of interest) have been identified and the system, via the control module and fluid handling apparatus and optionally instruments of the chemical module, is ready to engage the incubation module for incubating the chemical product and biological molecules for a bioassay. The incubation module can be programed via the software to run any type of incubation required for whatever biological assay is to be conducted. In exemplary embodiments, the incubation module is configurable to perform incubation of a biological assay selected from the group consisting of cytotoxicity, enzyme reactivation, antibiotic activity, binding affinity, enzyme inhibition, antiviral activity, agonism / antagonism, and / or blood / brain barrier penetration. In certain embodiments, he incubation module is operably associated with the fluid handling apparatus of the control module and the one or more fluid handling instruments of the chemical synthesis module. FIG. 4B illustrates exemplary bioassays, e g. cytotoxicity, which is described in more detail in the Examples below.

[0062] FIG. 5 illustrates in an exemplary manner how the fluid handling components of the system operate together to prepare chemical products and biological molecules in the incubation molecule for a bioassay and subsequent analysis in a mass spectrometer. FIG. 6 illustrates in another embodiment how the fluid handling components of the system operate together to prepare chemical products and biological molecules in the incubation molecule for another bioassay and subsequent analysis in a mass spectrometer.

[0063] In certain embodiments, the bioassay module comprises a multiplexed inductive nanoelectrospray and micro-electrophoresis apparatus to achieve rapid in situ sample clean-up and ionization using small volume samples. In such embodiments, the control module may control operation of the multiplexed inductive nano-electrospray and micro-electrophoresis apparatus. That is illustrated in in FIG. 7.Attorney Docket No.: PURD- 165 / 01 WO 28593 / 767

[0064] PATENT APPLICATION FIG. 8A illustrates a second characterization position that can optionally be coupled and operated via the systems of the invention and FIG. 8B shows additional aspects of a work-flow through the system. The second characterization position is accessible by the fluid handling apparatus of the control module and can be operable coupled to the control module. As shown by example in FIG. 8A, the second characterization position comprises a Raman spectrometer. FIGS. 9-10 further show the characterization and additional biological activity exploration that can be achieved via the second characterization position.

[0065] FIG. 11A shows how the data obtained from the system can be used to determine structure - bioactivity correlations and FIG. 1 IB shows final steps in the workflow using systems of the invention. Particularly, deep learning methods and neural -network-based models can be implements to uncover structure - bioactivity correlations.

[0066] Fluid Handling

[0067] Any fluid handling instrument known in the art can be used in systems of the invention. In certain embodiments, the liquid handling instrument is a Biomek liquid handler (for example I-series) as produced and sold by Beckman Coulter. Description of such liquid handler are described for example in U.S. patent numbers 10,274,505; 10,048,284; 9,910,054; 9,519,000; 9,506,943; 9,482,684; 9,446,418; 9,285,382; 9,274,132; 9,140,715; 9,046,506; 9,046,455;

[0068] 8,996,320; 8,973,736; 8,962,308; 8,956,570; 8,932,541; 8,840,848; 6,841,379; and 5,737,498, the content of each of which is incorporated by reference herein in its entirety.

[0069] Mi croel ectrophoresi s

[0070] Micro-electrophoretic probes and systems are described further for example in U.S. patent application publication number 2019 / 0019662, the content of which is incorporated by reference herein in its entirety. In certain aspects, the invention makes use of electrophoretic forces to displace interfering solution-phase ions prior to chemical analysis, i.e., move solutionphase ions to a region of the solution remote from the sprayer. In certain embodiments, the probes of the invention employ two electrodes to create an electrophoretic field by means of which ions, such as H+, Na+, K+, Br-, C1-, Acetate-, Formate-, etc., can be manipulated. Except for H+ and OH-, these solution phase ions cause deleterious effects during mass spectrometric (MS) analysis by causing a single analyte (M) to have multiple signals (e g., M + H+, M + Na+,Attorney Docket No.: PURD- 165 / 01 WO 28593 / 767

[0071] PATENT APPLICATION

[0072] and M + K+), or by suppressing analyte signals altogether. These adducts not only lower the signal of the analyte, but also result in cluster formation, congested spectra and reduced sensitivity of the MS analyzer. The electrophoretic probes of the invention remove ions from the region from which the analytical spray occurs. The removal is not permanent but persists for long periods and as such it allows mass spectrometry to be performed under normal conditions.

[0073] To perform the desalting process, a hollow conduit and an electrode are inserted into a hollow body of a mass spectrometry probe having a distal tip. The hollow conduit is electrically conductive and may be operably coupled to a power source and a reservoir that holds a liquid sample. A distal end of the hollow conduit is positioned in the hollow body to be in contact with the liquid sample that is expelled from the hollow conduit. A separate electrode is placed at the rear of the emitter, not in contact with the liquid sample but connected inductively (e.g., a distal end of the electrode is located at a different distance from the distal tip of the hollow body than a distal end of the hollow conduit, e.g., farther away)). A voltage is applied to the hollow conduit as it transports the liquid sample from the reservoir into the hollow body. The hollow conduit polarizes the liquid sample as the liquid sample flows through the hollow conduit and into the hollow body. This generates a charge body of solution which is connected via the external hollow conduit to one pole of the power supply. In this manner, the liquid sample may act as an electrode. When the appropriate electrical potentials (e.g., DC potentials) are applied to the electrode, the ions migrate to the rear of the capillary, away from the tip of the hollow body. After a short period, the electrode is turned off while voltage is still supplied to the hollow conduit, which electrical charge continues to be imparted to the liquid sample in the hollow body, even if there is not flow or liquid in the hollow conduit. The spectrum generated has signals related primarily to protonated molecules (M + H+) and minimal signal resulting from cation adducts of the molecule or from cationic salt clusters. The same experiment can be applied to negative ions using an opposite polarity on the electrodes. The mass spectrometry probe of the invention has been used to improve the analytical performance in the analysis of therapeutic pharmaceuticals, peptides, proteins, and contaminants often found in waste streams.

[0074] In certain embodiments, the invention probes an electrophoretic, multi-electrode mass spectrometry probe (e.g., a nano-ESI probe) that operates to rapidly desalt matrices without the addition of any chemical modifiers. The setup, as described herein, is a small capillary which may contain a liquid, such as water. The analyte, is delivered to the tip of the capillary,Attorney Docket No.: PURD- 165 / 01 WO 28593 / 767

[0075] PATENT APPLICATION

[0076] optionally via a syringe pump (e.g., sample reservoir), connected to an insulated fused silica line (e.g., electrically conductive hollow conduit) for two minutes (4 pL / min). A high voltage connector, HV1, which delivers 1.5 kV to the front of the capillary through conduction of the solvent insulated line via the syringe pump. In certain embodiments, HV1 may be equivalent to a typical spray voltage in any spray based ionization source. The second electrode, HV2, is inside the glass capillary, but not in contact with the solvent (ca 5mm air gap). As shown, HV1 and HV2 each terminate at different distances from a distal tip of the hollow capillary. In the exemplified embodiment, HV2 terminates closer to a rear of the capillary than HV1.

[0077] The body of the mass spectrometry probe is a hollow unitary body. This is exemplary and not required in all embodiments. In the context of the invention, unitary refers to the fact that walls of the body of the mass spectrometry probe include no breaks, cuts, or disconnected regions. Rather, the walls of the probe run continuously from the distal tip of the probe to a rear of the probe without any breaks, cuts, or disjointed regions. In that manner, the mass spectrometry probes can operate without the formation of a liquid bridge or any other connector that would be required to connect to parts of a disjointed non-unitary probe body. Similarly, the methods of the invention can be performed without the need for a liquid bridge in the probe setup.

[0078] Without being limited to any particular theory or mechanism of action, in-source desalting typically occurs in three steps. For positive mode analysis, HV1 is set to (+)1.5 kV and the syringe pump is operated as described above. The data gathered during step l is a typical spectrum that contains M + H+, M + Na+ and M + K+ species. Next, a second potential, HV2, is applied inductively to a rear of the solvent meniscus, ranging from -3 kV to -5 kV. While on, the positive ion signal is depleted due to the high negative electric field which sequesters the positive ions to the rear of the capillary. After a time period, (e.g., 1 minute (exemplary time period)), voltage from HV2 is removed (voltage is terminated) and solution-phase ions proceed to the front of the emitter; however, due to mobility, the protons reach the tip of the emitter before Na+ and K+ resulting in a spectrum containing primarily protonated ions.

[0079] Desorption Electrospray Ionization

[0080] Desorption electrospray ionization (DESI) is described for example in Takats et al. (U.S. Pat. No. 7,335,897), the content of which is incorporated by reference herein in its entirety. DESIAttorney Docket No.: PURD- 165 / 01 WO 28593 / 767

[0081] PATENT APPLICATION

[0082] allows ionizing and desorbing a material (analyte) at atmospheric or reduced pressure under ambient conditions. A DESI system generally includes a device for generating a DESI-active spray by delivering droplets of a liquid into a nebulizing gas. The system also includes a means for directing the DESI-active spray onto a surface. It is understood that the DESI-active spray may, at the point of contact with the surface, include both or either charged and uncharged liquid droplets, gaseous ions, molecules of the nebulizing gas and of the atmosphere in the vicinity. The pneumatically assisted spray is directed onto the surface of a sample material where it interacts with one or more analytes, if present in the sample, and generates desorbed ions of the analyte or analytes. The desorbed ions can be directed to a mass analyzer for mass analysis, to an IMS device for separation by size and measurement of resulting voltage variations, to a flame spectrometer for spectral analysis, or the like.

[0083] In this system, a spray is generated by a conventional electrospray device. The device includes a spray capillary through which the liquid solvent is fed. A surrounding nebulizer capillary forms an annular space through which a nebulizing gas such as nitrogen (N2) is fed at high velocity. In one example, the liquid was a water / methanol mixture and the gas was nitrogen. A high voltage is applied to the liquid solvent by a power supply via a metal connecting element. The result of the fast-flowing nebulizing gas interacting with the liquid leaving the capillary is to form the DESI-active spray comprising liquid droplets. DESI-active spray also may include neutral atmospheric molecules, nebulizing gas, and gaseous ions. Although an electrospray device has been described, any device capable of generating a stream of liquid droplets carried by a nebulizing gas jet may be used to form the DESI-active spray.

[0084] The spray is directed onto the sample material which in this example is supported on a surface. The desorbed ions leaving the sample are collected and introduced into the atmospheric inlet or interface of a mass spectrometer for analysis by an ion transfer line which is positioned in sufficiently close proximity to the sample to collect the desorbed ions. Surface may be a moveable platform or may be mounted on a moveable platform that can be moved in the x, y or z directions by well-known drive means to desorb and ionize sample at different areas, sometimes to create a map or image of the distribution of constituents of a sample. Electric potential and temperature of the platform may also be controlled by known means. Any atmospheric interface that is normally found in mass spectrometers will be suitable for use in the invention. Good results have been obtained using a typical heated capillary atmospheric interface. Good resultsAttorney Docket No.: PURD- 165 / 01 WO 28593 / 767

[0085] PATENT APPLICATION

[0086] also have been obtained using an atmospheric interface that samples via an extended flexible ion transfer line made either of metal or an insulator.

[0087] Multiplexing and Inductive Charging

[0088] In certain embodiments, multiplexing on sample loading and analysis is used in the systems and methods of the invention, optionally using inductive charging for analysis. Such approaches are described for example in U.S. patent application publication number 2020 / 0381238, the content of which is incorporated by reference herein in its entirety. In certain embodiments, the induced DC nESI ionization source includes a 3D electrical controlled moving platform, emitter holder and a pogo pin holder. In such embodiments, the emitter holder is preloaded with 96 emitters and samples. The emitter holder is attached to the 3D moving stage by a 3D printed connector. The emitter holder is designed to easily attached and detached from the moving stage for convenience of sample introduction and cleaning. The front (side facing the MS inlet) of the emitter holder has 96 holes to hold 96 emitters. Inside the holes, there are 96 individual electrodes with the same length as the emitter holder. When loading the emitters into the holder, these electrodes are inserted into the emitters but do not reach the sample solution. The other ends of the electrodes go from the rear (side opposite from the MS inlet) and are soldered to a PCB with 96 holes. On the PCB, there are 96 isolated copper layers electrically in contact with the 96 electrodes by soldering. A pogo pin electrode placed behind the PCB is aligned with the MS inlet. The position of the pogo pin electrode is fixed by the pogo pin holder on a fixed arm of the 3D moving stage. The pogo pin electrode touches the PCB. When the device is running, the motion control system first goes to the top right starting point and moves in the vertical y-direction to find the first row of emitters and then moves in the horizontal x-direction to analyze samples in the first row in sequence. When an emitter is aligned with the MS inlet, the pogo pin touches the corresponding copper layer on the PCB and 2 ~ 3.5 kV volts is applied to the electrode for induced DC nESI ionization of the sample in the tip of the emitter. Note that the electrode does not contact the sample so ionization is induced. Because the flow rate in inductive nESI is very low, so there is enough time to record the high-quality MS data in spite of very small sample volume.

[0089] To solve the problem of sample introduction presented by the traditional nESI work flow, we have developed a “dip and go” strategy using a multiplexed system. In such an approach, 96Attorney Docket No.: PURD- 165 / 01 WO 28593 / 767

[0090] PATENT APPLICATION

[0091] emitters with 20-micron tip size are preloaded into the emitter holder. The size of the holder is designed to correspond to the size of the standard 96-well plate and the position of each emitter corresponds to the position of each well in the 96-well plate. To load the sample, one holds the emitter holder and lets the side with emitters face the 96-well plate, lowers the holder and allows every emitter to be immersed into sample solution for 10 seconds and then lifts the holder. This procedure can be done manually or with a robot. The amount of sample solution introduced into emitter is ca. 100 nL. Sample loading amounts can be varied by using different loading times. Induced electrophoretic cleaning (“desalting”) can be applied to the samples on the emitters prior to sample analysis to achieve better analytical performance for samples with a complex matrix. By applying voltage (e.g., more than 5 kV, with either the same or opposite polarity to that used for nESI analysis) to the electrodes simultaneously, the high electrical field induced in the sample in the emitter tip will cause electrophoresis. Ions with large ionic mobility such as anions and cations from simple salts in the solution will migrate towards the two ends of the solution, leaving substances with small ionic mobility such as peptides will remain essentially in their original positions and will be subject to selective ionization.

[0092] To perform offline electrophoretic cleaning one holds the emitter holder and allows the copper layer of the PCB touch a copper plate connected to the high voltage output of a power supply. At 0.5 to 1 cm distance from the emitter tip, another copper plate which is grounded is placed so as to set up a large potential change in the sample solution to initiate electrophoresis. The electrophoresis is maintained for 10 seconds and then the emitter holder is re-installed onto the back to the 3D moving stage platform. Following the same steps described in section A one records spectra of the cleaned samples. This method is more convenient but slightly slower (because cleaning slightly slows the rate of motion used for ionization).

[0093] The alternative to offline cleaning is to perform online cleaning using one HV supply for cleaning and a second one for ionization. To perform online electrophoretic cleaning, the emitter holder is attached to the moving stage. When performing the cleaning, the moving stage allows the emitter holder to move from left to right. The left pogo pin on a pogo pin holder is supplied with -6 kV volts to induce electrophoretic cleaning of the sample that points towards the grounded counter electrode. Subsequently, after cleaning, the emitter moves and is aligned with the MS inlet at which point the right pogo pin electrode with 2 to 3.5 kV volts applied to theAttorney Docket No.: PURD- 165 / 01 WO 28593 / 767

[0094] PATENT APPLICATION

[0095] pogo pin holder initiates inductive nESI analysis of sample in the emitter by the same process described in A. This method is faster and the sample screening rate can be maximized.

[0096] As mentioned above, inductive charging can be useful in a multiplex analysis setting. Inductive charging is further described for example in U.S. patent number 9,184,036, the content of which is incorporated by reference herein in its entirety. In inductive charging the probe includes a spray emitter and a voltage source and the probe is configured such that the voltage source is not in contact with the spray emitter or the spray emitted by the spray emitter. In this manner, the ions are generated by inductive charging, i.e., an inductive method is used to charge the primary microdroplets. This allows droplet creation to be synchronized with the opening of the sample introduction system (and also with the pulsing of the nebulizing gas). Inductive nESI can be implemented for various kinds of nESI arrays due to the lack of physical contact.

[0097] Examples include circular and linear modes. In an exemplary rotating array, an electrode placed ~2 mm from each of the spray emitters in turn is supplied with a 2-4 kV positive pulse (10-3000 Hz) giving a sequence of ion signals. Simultaneous or sequential ions signals can be generated in the linear array using voltages generated inductively in adjacent nESI emitters. Nanoelectrospray spray plumes can be observed and analytes are detected in the mass spectrum, in both positive and negative detection modes. In the electrophoretic clean-up working mode, direct current voltage source (1.5-6 kV) was used to induce nanoelectrospray. Different from the previous example induced by alternating current voltage, the induced electrical field keeps the same direction in this mode, which ensures efficient electrophoretic cleaning performance.

[0098] Ion traps and mass spectrometers

[0099] Any ion trap known in the art can be used in systems of the invention. Exemplary ion traps include a hyperbolic ion trap (e.g., U.S. patent number 5,644, 131, the content of which is incorporated by reference herein in its entirety), a cylindrical ion trap (e.g., Bonner et al., International Journal of Mass Spectrometry and Ion Physics, 24(3):255-269, 1977, the content of which is incorporated by reference herein in its entirety), a linear ion trap (Hagar, Rapid Communications in Mass Spectrometry, 16(6): 512— 526, 2002, the content of which is incorporated by reference herein in its entirety), and a rectilinear ion trap (U.S. patent number 6,838,666, the content of which is incorporated by reference herein in its entirety).Attorney Docket No.: PURD- 165 / 01 WO 28593 / 767

[0100] PATENT APPLICATION

[0101] Any mass spectrometer (e.g., bench-top mass spectrometer of miniature mass spectrometer) may be used in systems of the invention and in certain embodiments the mass spectrometer is a miniature mass spectrometer. An exemplary miniature mass spectrometer is described, for example in Gao et al. (Anal. Chem. 2008, 80, 7198-7205.), the content of which is incorporated by reference herein in its entirety. In comparison with the pumping system used for lab-scale instruments with thousands of watts of power, miniature mass spectrometers generally have smaller pumping systems, such as a 18 W pumping system with only a 5 L / min (0.3 m3 / hr) diaphragm pump and a 11 L / s turbo pump for the system described in Gao et al. Other exemplary miniature mass spectrometers are described for example in Gao et al. (Anal. Chem., 2008, 80, 7198-7205.), Hou et al. (Anal. Chem., 2011, 83, 1857-1861.), and Sokol etal. (Int. J. Mass Spectrom., 2011, 306, 187- 195), the content of each of which is incorporated herein by reference in its entirety.

[0102] The control system of the Mini 12 (Linfan Li, Tsung-Chi Chen, Yue Ren, Paul I.

[0103] Hendricks, R. Graham Cooks and Zheng Ouyang “Miniature Ambient Mass Analysis System” Anal. Chem. 2014, 862909-2916, DOI: 10.1021 / ac403766c; and 860. Paul I. Hendricks, Jon K. Dalgleish, Jacob T. Shelley, Matthew A. Kirleis, Matthew T. McNicholas, Linfan Li, Tsung-Chi Chen, Chien-Hsun Chen, Jason S. Duncan, Frank Boudreau, Robert J. Noll, John P. Denton, Timothy A. Roach, Zheng Ouyang, and R. Graham Cooks “Autonomous in-situ analysis and real-time chemical detection using a backpack miniature mass spectrometer: concept, instrumentation development, and performance” Anal. Chem., 2014, 86 2900-2908 DOI:

[0104] 10.1021 / ac403765x, the content of each of which is incorporated by reference herein in its entirety), and the vacuum system of the Mini 10 (Liang Gao, Qingyu Song, Garth E. Patterson, R. Graham Cooks and Zheng Ouyang, “Handheld Rectilinear Ion Trap Mass Spectrometer”, Anal. Chem., 78 (2006) 5994-6002 DOI: 10.1021 / ac061144k, the content of which is incorporated by reference herein in its entirety) may be combined to produce the miniature mass spectrometer shown in FIG. 9. It may have a size similar to that of a shoebox (H20cm x W25cm x D35cm). In certain embodiments, the miniature mass spectrometer uses a dual LIT configuration, which is described for example in Owen et al. (U.S. patent application serial number 14 / 345,672), and Ouyang et al. (U.S. patent application serial number 61 / 865,377), the content of each of which is incorporated by reference herein in its entirety.Attorney Docket No.: PURD- 165 / 01 WO 28593 / 767

[0105] PATENT APPLICATION

[0106] System Architecture

[0107] In certain embodiments, the systems and methods of the invention can be carried out using automated systems and computing devices. Specifically, aspects of the invention described herein can be performed using any type of computing device, such as a computer, that includes a processor, e.g., a central processing unit, or any combination of computing devices where each device performs at least part of the process or method. In some embodiments, systems and methods described herein may be controlled using a handheld device, e.g., a smart tablet, or a smart phone, or a specialty device produced for the system.

[0108] Systems and methods of the invention can be performed using software, hardware, firmware, hardwiring, or combinations of any of these. Features implementing functions can also be physically located at various positions, including being distributed such that portions of functions are implemented at different physical locations (e.g., imaging apparatus in one room and host workstation in another, or in separate buildings, for example, with wireless or wired connections).

[0109] Processors suitable for the execution of computer program include, by way of example, both general and special purpose microprocessors, and any one or more processor of any kind of digital computer. Generally, a processor will receive instructions and data from a read-only memory or a random access memory or both. The essential elements of computer are a processor for executing instructions and one or more memory devices for storing instructions and data. Generally, a computer will also include, or be operatively coupled to receive data from or transfer data to, or both, one or more mass storage devices for storing data, e.g., magnetic, magneto-optical disks, or optical disks. Information carriers suitable for embodying computer program instructions and data include all forms of non-volatile memory, including by way of example semiconductor memory devices, (e.g., EPROM, EEPROM, solid state drive (SSD), and flash memory devices); magnetic disks, (e.g., internal hard disks or removable disks); magnetooptical disks; and optical disks (e.g., CD and DVD disks). The processor and the memory can be supplemented by, or incorporated in, special purpose logic circuitry.

[0110] To provide for interaction with a user, the subject matter described herein can be implemented on a computer having an I / O device, e.g., a CRT, LCD, LED, or projection device for displaying information to the user and an input or output device such as a keyboard and a pointing device, (e.g., a mouse or a trackball), by which the user can provide input to theAttorney Docket No.: PURD- 165 / 01 WO 28593 / 767

[0111] PATENT APPLICATION

[0112] computer. Other kinds of devices can be used to provide for interaction with a user as well. For example, feedback provided to the user can be any form of sensory feedback, (e.g., visual feedback, auditory feedback, or tactile feedback), and input from the user can be received in any form, including acoustic, speech, or tactile input.

[0113] The subject matter described herein can be implemented in a computing system that includes a back-end component (e.g., a data server), a middleware component (e.g., an application server), or a front-end component (e.g., a client computer having a graphical user interface or a web browser through which a user can interact with an implementation of the subject matter described herein), or any combination of such back-end, middleware, and frontend components. The components of the system can be interconnected through network by any form or medium of digital data communication, e.g., a communication network. For example, the reference set of data may be stored at a remote location and the computer communicates across a network to access the reference set to compare data derived from the female subject to the reference set. In other embodiments, however, the reference set is stored locally within the computer and the computer accesses the reference set within the CPU to compare subject data to the reference set. Examples of communication networks include cell network (e g., 3G or 4G), a local area network (LAN), and a wide area network (WAN), e.g., the Internet.

[0114] The subject matter described herein can be implemented as one or more computer program products, such as one or more computer programs tangibly embodied in an information carrier (e.g., in a non-transitory computer-readable medium) for execution by, or to control the operation of, data processing apparatus (e.g., a programmable processor, a computer, or multiple computers). A computer program (also known as a program, software, software application, app, macro, or code) can be written in any form of programming language, including compiled or interpreted languages (e.g., C, C++, Perl), and it can be deployed in any form, including as a stand-alone program or as a module, component, subroutine, or other unit suitable for use in a computing environment. Systems and methods of the invention can include instructions written in any suitable programming language known in the art, including, without limitation, C, C++, Perl, Java, ActiveX, HTML5, Visual Basic, or JavaScript.

[0115] A computer program does not necessarily correspond to a fde. A program can be stored in a file or a portion of file that holds other programs or data, in a single file dedicated to the program in question, or in multiple coordinated files (e.g., files that store one or more modules,Attorney Docket No.: PURD- 165 / 01 WO 28593 / 767

[0116] PATENT APPLICATION

[0117] sub-programs, or portions of code). A computer program can be deployed to be executed on one computer or on multiple computers at one site or distributed across multiple sites and interconnected by a communication network.

[0118] A file can be a digital file, for example, stored on a hard drive, SSD, CD, or other tangible, non-transitory medium. A file can be sent from one device to another over a network (e.g., as packets being sent from a server to a client, for example, through a Network Interface Card, modem, wireless card, or similar).

[0119] Writing a file according to the invention involves transforming a tangible, non-transitory computer-readable medium, for example, by adding, removing, or rearranging particles (e.g., with a net charge or dipole moment into patterns of magnetization by read / write heads), the patterns then representing new collocations of information about objective physical phenomena desired by, and useful to, the user. In some embodiments, writing involves a physical transformation of material in tangible, non-transitory computer readable media (e.g., with certain optical properties so that optical read / write devices can then read the new and useful collocation of information, e.g., burning a CD-ROM). In some embodiments, writing a file includes transforming a physical flash memory apparatus such as NAND flash memory device and storing information by transforming physical elements in an array of memory cells made from floatinggate transistors. Methods of writing a file are well-known in the art and, for example, can be invoked manually or automatically by a program or by a save command from software or a write command from a programming language.

[0120] Suitable computing devices typically include mass memory, at least one graphical user interface, at least one display device, and typically include communication between devices. The mass memory illustrates a type of computer-readable media, namely computer storage media. Computer storage media may include volatile, nonvolatile, removable, and non-removable media implemented in any method or technology for storage of information, such as computer readable instructions, data structures, program modules, or other data. Examples of computer storage media include RAM, ROM, EEPROM, flash memory, or other memory technology, CD-ROM, digital versatile disks (DVD) or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, Radiofrequency Identification tags or chips, or any other medium which can be used to store the desired information and which can be accessed by a computing device.Attorney Docket No.: PURD- 165 / 01 WO 28593 / 767

[0121] PATENT APPLICATION

[0122] As one skilled in the art would recognize as necessary or best-suited for performance of the methods of the invention, a computer system or machines of the invention include one or more processors (e.g., a central processing unit (CPU) a graphics processing unit (GPU) or both), a main memory and a static memory, which communicate with each other via a bus.

[0123] In an exemplary embodiment shown in FIG. 14, system 200 can include a computer 249 (e.g., laptop, desktop, or tablet). The computer 249 may be configured to communicate across a network 209. Computer 249 includes one or more processor 259 and memory 263 as well as an input / output mechanism 254. Where methods of the invention employ a client / server architecture, steps of methods of the invention may be performed using server 213, which includes one or more of processor 221 and memory 229, capable of obtaining data, instructions, etc., or providing results via interface module 225 or providing results as a file 217. Server 213 may be engaged over network 209 through computer 249 or terminal 267, or server 213 may be directly connected to terminal 267, including one or more processor 275 and memory 279, as well as input / output mechanism 271.

[0124] System 200 or machines according to the invention may further include, for any of I / O 249, 237, or 271 a video display unit (e.g., a liquid crystal display (LCD) or a cathode ray tube (CRT)). Computer systems or machines according to the invention can also include an alphanumeric input device (e.g., a keyboard), a cursor control device (e.g., a mouse), a disk drive unit, a signal generation device (e.g., a speaker), a touchscreen, an accelerometer, a microphone, a cellular radio frequency antenna, and a network interface device, which can be, for example, a network interface card (NIC), Wi-Fi card, or cellular modem.

[0125] Memory 263, 279, or 229 according to the invention can include a machine-readable medium on which is stored one or more sets of instructions (e.g., software) embodying any one or more of the methodologies or functions described herein. The software may also reside, completely or at least partially, within the main memory and / or within the processor during execution thereof by the computer system, the main memory and the processor also constituting machine-readable media. The software may further be transmitted or received over a network via the network interface device.

[0126] Accelerated reactions in dropletsAttorney Docket No.: PURD- 165 / 01 WO 28593 / 767

[0127] PATENT APPLICATION

[0128] Acceleration of reactions in droplets is described for example in US 12,125,693, the content of which is incorporated by referenece herein in its entirety. Briefly, with the system described here, the reaction and screening process can be combined into a single work-flow using a single instrument that performs both the reaction product synthesis and the reaction screening. The system takes advantage of the fact that chemical reactions can be accelerated in a liquid droplet spray discharge. In that manner, the liquid droplet spray discharge can be used to rapidly conduct reactions from reagents at different locations on a substrate. The reaction occurs in the liquid droplet spray discharge as the spray discharge leaves the substrate surface toward an analysis device, such as a mass spectrometer. The formed reaction product is instantly analyzed in an automated manner without requiring any manual transfer of a reaction product from a synthesis instrument to a screening instrument. The substrate is under automated control, so that a standard combinatorial library can be generated and instantly screened without operator intervention.

[0129] The invention recognizes that acceleration of the rates of ordinary reactions occurs in droplets, and in some instances by large factors. Without being limited by any particular theory or mechanism of action, it is believed that the acceleration is partly the result of solvent evaporation and the resulting increase in reagent concentrations. There is also evidence of intrinsic reaction acceleration at the surfaces of droplets, so that the increased surface to volume ratio of microdroplets plays a significant role in reaction acceleration. Without being limited by any particular theory or mechanism of action, it is believed that the distance of travel of droplets in a spray correlates roughly with the extent of reaction, suggesting that evaporation which creates smaller droplets also increases reaction rates.

[0130] Incorporation by Reference

[0131] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure, including to the Supplementary. The Supplementary, and all other such documents are hereby incorporated herein by reference in their entirety for all purposes.

[0132] Equivalents

[0133] The invention may be embodied in other specific forms without departing from the spiritAttorney Docket No.: PURD- 165 / 01 WO 28593 / 767

[0134] PATENT APPLICATION

[0135] or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein.

[0136] EXAMPLES

[0137] Example 1 : Application accelerated reaction (derivatization) to bioactive compounds

[0138] An antifungal compound, Amphotericin B (AmB), was subjected to successful reaction in microdroplets using a series of reagents that caused epoxidation, boronic ester formation, carbdiimide coupling, addition to an arylformyl dehy de, S\Ar reaction with aN-methylpyridinium salt, and Katritzky transamination with pyrylium salts. The experiments were performed in 1 : 1 stoichiometries in acetonitrile under ambient conditions and on the low microgram scale using spray deposition onto paper in times of seconds for each reaction. The functionalized olefin, cis-diol, carboxylic acid, hydroxyl or amino, hydroxy and amino groups, gave new compounds that were characterized by tandem mass spectrometry. Yields have not yet been determined but conversion ratios (intensity product ion vs. reagent + product) ranged from 23 to 90%. FIGS. 15A-B illustrates the chemistry and shows typical MS / MS data of collected materials.

[0139] FIG. 15A shows that modification of amphotericin B (AmB) was achieved by creating microdroplets from a solution containing a 1 : 1 mixture of AmB and one of the reagents listed below, each in 1 mM concentration and in 50 nL total volume. DESI was used to create the microdroplets which were directed towards the QToF mass spectrometer of the HT DESI-MS platform. Reactions were examined at a rate of 1 Hz to record full scan mass spectra. The m / z ratios corresponding to products of interest were mass-selected and the same sample was reexamined by DESI to record the product ion MS / MS spectra. These data were recorded in 3 sec with results for 4 identical spots being averaged. The total available sample (not all used) was 50 ng / spot. Reactions 1 to 8 were performed in this way. The reactions were epoxidation with m-chloroperoxybenzoic acid (1), boronic ester formation with phenylboronic acid (2) and 4-borono-N,N,N-trimethylbenzenaminium (3), coupling with l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (4), addition with 4-formyl-N,N,N-trimethylbenzenaminium (5), SNAr with 2-chloro-l -methylpyridinium (6), and Katritzky transamination with pyrylium (7) and 2,4,6-trimethylpyrylium (8) salts. The conversion ratio (CR), a rough measure of the extentAttorney Docket No.: PURD- 165 / 01 WO 28593 / 767

[0140] PATENT APPLICATION

[0141] of reaction that does not account for differences in ionization efficiencies, was calculated as the ion intensity ratio of the product relative to the sum of the product and reactant ion intensities. The values for these reactions were as follows: (1) 26%, (2) 29%, (3) 99%, (4) 64%, (5) 25%, (6) 67%, (7) 71% (8) 80%.

[0142] FIG. 15B shows typical product ion MS / MS spectra for SNAr and Katritzky reaction products. Both products show expected loss of water and also loss of the macrocyclic ring (mass 760 Da, C12H19N2O3) with hydrogen transfer to the acetal O in the amino-sugar to give the major fragments at m / z 255 and 268 respectively. This SNAr product fragments further by dehydration (to give m / z 237) and decarboxylation (m / z 211) while the Katritzky fragment dissociates further by loss of the aminosugar to give the protonated trimethylpyridine, m / z 122. The only other significant ions are due to loss of water and CO2 (SNAr derivative) and, also for this compound, ions at m / z 313 and 267 which are not assigned.

[0143] Example 2: Functionalization of Antibiotics and Heat Maps of Conversion Ratios

[0144] FIG. 16 shows DESLMS is used to launch microdroplets from the reactant array of antibiotics mixed with the indicated reagents for N-alkylation, Katritzky transamination, Nucleophilic aromatic substitution, sulfonation and an ene reaction. Reactions give products of adequate S.N in 67% of the ca. 300 reactions examined.

[0145] Example 3 : Carbamylation of Cirofloxacin (CIP) Antibiotic

[0146] FIG. 17 shows conversion of carboxylic acid to amide in antibiotic using accelerated reactions with a carbodiimide in microdroplets and on-line measurement of conversion ratios. Reaction proceeds via the intermediate addition product before the nucleophilic substitution step to give the amide. The DESI mass spectrum shown is taken online at a rate of 1 Hz, and the product ion MS / MS spectrum of the amide is recorded in a scan of x seconds. The characteristic loss of diethylamine is likely associated with cyclization to give the protonated oxazoline product. There are no obvious trends in reactivity.

[0147] Example 4: Array -to-array collection of modified bioactive compounds and their bioassay FIG. 18 shows array -to-array collection, bioassay and in situ product identification methodology. DESLMS is used to launch microdroplets from the reactant array where samplesAttorney Docket No.: PURD- 165 / 01 WO 28593 / 767

[0148] PATENT APPLICATION

[0149] are present as spots of some 5 ng to 500 ng of material. Spray times are typically just 1 s for MS identification but up to 45 sec to collect enough material for bioassay. Reaction which occurs only during the very short (ca. 10’s ms) flight time per droplet. Deposition is onto paper which was subsequently analyzed by DESI imaging (as illustrated by the bright spots seen at right). The spots are either cut out manually to perform bioassays e.g. for chemically modified antibiotics by spot punching follow by AST disk diffusion assays. The distribution of collected products can also be determined using DESI in the imaging mode as show on the lower left. These images represent mass spectra of the collected material and they can be used to confirm the nature of the chemical transformation. Two examples are shown, S3A7 and S4A9 where S indicates sulfonation reagent and A indicates antibiotic in a large array.

[0150] Example 5: Changes in Bioactivity on Chemical Modification in Microdroplet Arrays FIG. 19 shows a demonstration of dramatic change in bioactivity of an antibiotic after chemical modification. The AST test was used to characterize unmodified Ampicillin and its Katritzky transamination product. The order of magnitude change in the diameter of the E. coli inhibition zone is an underestimate of the loss in activity given that the product was not purified of unreacted starting material. Changes in molecular structure that remove activity are less interesting than those that increase it.

[0151] Example 6: Quantitative Assays of Antibiotic Activity using Reaction Kinetics to Follow Resistance and Inhibition

[0152] FIG. 20 shows P-Lactam activity is reducted by the ring-opening enzyme beta-lactamase produced by Bacillus cereus in response to presence of Amoxicillin or Ampicillin. Inactivation (ring opening) occurs on the 2 h time scale. The effects of an inhibitor of beta-lactamase in maintaining activity are evident in the plot at right.

Claims

Attorney Docket No.: PURD- 165 / 01 WO 28593 / 767PATENT APPLICATIONWhat is claimed is:

1. A method of producing a modified antibiotic compound, the method comprising:directing a liquid droplet spray discharge from a sample probe onto a discrete spot comprising an antibiotic and one or more reactants so as to form a microdroplet that comprises the antibiotic and the one or more reactants and allowing a reaction to occur in the microdroplet between the antibiotic and the one or more reactants to produce a modified antibiotic; and collecting the modified antibiotic.

2. The method of claim 1, wherein prior to the collecting step, the microdroplet is directed to a mass spectrometer in which the modified antibiotic is mass separated and selected.

3. The method of claim 1, wherein the sampling probe is a desorption electrospray ionization probe and the liquid droplet spray discharge is a desorption electrospray ionization active discharge.

4. The method of claim 1, wherein the sampling probe comprises a gas source and a voltage source.

5. The method of claim 2, wherein the mass spectrometer is a bench-top mass spectrometer or a miniature mass spectrometer.

6. The method to claim 1, wherein a rate of the reaction among the antibiotic and the one or more reactants in the microdroplet is accelerated as compared to a rate of the reaction among the antibiotic and the one or more reactants in the microdroplet in a bulk liquid.

7. The method to claim 1, wherein prior to the directing step, the method first comprises providing a substrate, the substrate comprises a plurality of discrete spots, wherein each of the plurality of discrete spots comprises a different one or more reactants and the antibiotic to thereby produce a plurality of different modified antibiotics.Attorney Docket No.: PURD- 165 / 01 WO 28593 / 767PATENT APPLICATION8. The method of claim 7, wherein the substrate is a movable substrate and the movable substrate is operably coupled to a motor that moves the substrate in an automated manner.

9. The method of claim 7, wherein the sampling probe is operably coupled to anmovable arm and the movable arm is operably coupled to a motor that moves the sampling probe in an automated manner.

10. The method of claim 6, wherein the substrate is a first substrate and the method further comprises providing a second substrate configured to align with the first substrate such that the modified antibiotic produced in the microdroplet from the discrete location on the first substrate is landed at a corresponding location on the second substrate.

11. A method of producing a modified antibiotic compound, the method comprising:providing a first substrate comprising a first discrete spot comprising an antibiotic and one or more reactants;directing a liquid droplet spray discharge from a sample probe onto the discrete spot so as to form a microdroplet that comprises the antibiotic and the one or more reactants and allowing a reaction to occur in the microdroplet between the antibiotic and the one or more reactants to produce a modified antibiotic;collecting the modified antibiotic at a corresponding location on a second substrate; analyzing the modified antibiotic from the corresponding location on the second substrate.

12. The method of claim 11, wherein prior to the collecting step, the microdroplet is directed to a mass spectrometer in which the modified antibiotic is mass separated and selected.

13. The method of claim 11, wherein the sampling probe is a desorption electrospray ionization probe and the liquid droplet spray discharge is a desorption electrospray ionization active discharge.

14. The method of claim 11, wherein the sampling probe comprises a gas source and aAttorney Docket No.: PURD- 165 / 01 WO 28593 / 767PATENT APPLICATIONvoltage source.

15. The method of claim 12, wherein the mass spectrometer is a bench-top mass spectrometer or a miniature mass spectrometer.

16. The method to claim 11, wherein a rate of the reaction among the antibiotic and the one or more reactants in the microdroplet is accelerated as compared to a rate of the reaction among the antibiotic and the one or more reactants in the microdroplet in a bulk liquid.

17. The method to claim 11, wherein the first substrate comprises a plurality of discrete spots, wherein each of the plurality of discrete spots comprises a different one or more reactants and the antibiotic to thereby produce a plurality of different modified antibiotics.

18. The method of claim 17, wherein the substrate is a movable substrate and the movable substrate is operably coupled to a motor that moves the substrate in an automated manner.

19. The method of claim 17, wherein the second substrate is configured for linear and rotary movement.

20. The method of claim 17, wherein the sampling probe is operably coupled to an movable arm and the movable arm is operably coupled to a motor that moves the sampling probe in an automated manner.

Images