<p>      Chemical reactions in micron-sized droplets under ambient conditions are often orders of magnitude faster than the equivalent bulk reactions due to the large interfacial effects. The investigation of the underlying mechanisms driving the unique surface chemistry of droplets, as well as their applications and implications in synthesis, has garnered considerable interest. This dissertation delves into three key subtopics: (1) Exploring partial solvation as a mechanism for accelerating reactions in microdroplets, (2) Investigating the spontaneous oxidation and reduction of heteroatom double bonds induced by water radical cations and anions generated from water, and (3) Examining the role of oxazolone intermediates in prebiotic peptide synthesis and the emergence of homochirality in living systems.</p> <p>      Chemical reactions can be accelerated in microdroplets but with previously unclear mechanisms. Here we report a systematic study of organic reactions of common types in microvolumes and compare their rates with those in bulk solution. The observed interfacial area effect, molecularity effect and solvent effect provided experimental evidence for partial solvation at gas/liquid interface as one of the major contributors to the observed more than 10⁴-fold acceleration in microdroplets.</p> <p>      Recent spectroscopic results as well as computations demonstrate the existence of a strong electric field at aqueous droplet surfaces, which can result in microdroplet-specific reactions, especially their intrinsic redox properties. Spontaneous oxidation or reduction without external oxidants or reductants has been reported. One explanation for the existence of active species is dissociation of the radical cation/anion pair (H₂O^+∙/ H₂O^-∙), recently argued to occur in pure bulk water, to provide the free radical cation and radical anion. In this work, we reported spontaneous oxidation of heteroatom double bonds (e.g. sulfone to sulfonic acid, ketone to carboxylic acid) in non-aqueous microdroplets containing traces of water (<1%). Meanwhile, the simultaneous oxidation and reduction of several phosphonates was discovered, supporting the radical pair as the source of reactive species in water microdroplets.</p> <p>      One implication of microdroplet chemistry lies in its connection to prebiotic synthesis. Peptide formation from amino acids is thermodynamically unfavorable but a recent study provided evidence that the reaction occurs at the air/solution interfaces of aqueous microdroplets. Here we show that (i) the suggested amino acid complex in microdroplets undergoes dehydration to form oxazolone; (ii) addition of water to the oxazolone forms the dipeptide; and (iii) reaction of oxazolone with other amino acids forms tripeptides. Furthermore, the chirality of the reacting amino acids is preserved in the oxazolone, and strong chiral selectivity is observed when converting the oxazolone to tripeptide. This last fact ensures that optically impure amino acids will undergo chain extension to generate homochiral peptides. Peptide formation in bulk by wet-dry cycling shares a common pathway with the microdroplet reaction, both involving the oxazolone intermediate.</p>

<p>The rapid analysis of complex samples using mass spectrometry (MS) provides valuable information in both point-of-care (e.g. drug testing) and laboratory-based applications, including the generation of spectral libraries for classification of biosamples, the identification of biomarkers through large-scale studies, as well as the synthesis and bioactivity assessments of large compound sets necessary for drug discovery. In all these cases, the inherent speed of MS is attractive, but rarely fully utilized due to the widespread use of sample purification techniques prior to analysis. Ambient ionization methodologies can help circumvent this drawback by facilitating high-throughput qualitative and quantitative analysis directly from the complex samples without any need for work-up. For instance, the use of swabs or paper substrates allows for rapid identification, quantification, and confirmation, of drugs of abuse from biofluids or surfaces of forensic interest in a matter of minutes, as described in the first two chapters of this dissertation. Faster analysis can be achieved using an automated desorption electrospray ionization (DESI) platform which allows for the rapid and direct screening of complex-sample microarrays with throughputs better than 1 sample per second, giving access to rich spectral information from tens of thousands of samples per day. The development of the bioanalytical capabilities of this platform, particularly within the context of drug discovery (e.g. bioactivity assays, biosample analysis), is described across most other chapters of this dissertation. The use of DESI, a contactless ambient ionization method developed in our laboratory and whose 20 years of history are overviewed in the introduction of this document, provides an additional advantage as the secondary microdroplets generated through the DESI process act as reaction vessels that can accelerate organic reactions by up to six orders of magnitude, facilitating on-the-fly synthesis of new compounds from arrays of starting materials. Unique implications of this microdroplet chemistry in the prebiotic synthesis of peptides and spontaneous redox chemistry at air-solution interfaces, together with its practical applications to the synthesis of new drug molecules, are also overviewed. The success obtained with the first automated DESI-MS system, developed within the DARPA Make It program, led to increased interest in a new-generation platform which was designed over the past year, as overviewed in the last section of this dissertation, and which is currently being installed for validation prior to the transfer of the technology to NCATS, where we anticipate it will make a significant impact through the consolidation and acceleration of the early drug discovery workflow.</p>

<p dir="ltr">Mass spectrometry (MS) has long been recognized as a technology for bioanalysis. However, this thesis focuses on exploiting mass spectrometry for chemical reactions. The work described here covers the (a) investigation of chemistry at interfaces by MS, (b) utilization of MS to accelerate drug discovery processes, and (c) applications of MS techniques for organic synthesis. MS techniques are used to scrutinize the distinctive chemistry and super acidity mechanisms at the gas/liquid interfaces by reacting carbon dioxide (gas phase) with amines (solution, in droplets). The intriguing trace water effect in creating this unique environment at the interfaces is described. A systematic survey of reactions promoted by glass microspheres at liquid/solid interfaces is conducted, revealing that glass surface can act as strong base to speed up reactions. Additionally, the ability of glass surface to degrade biomolecules is revealed, which has implications for bioanalysis. Desorption electrospray ionization (DESI), an ambient ionization method, can be used as a rapid analytical technique for the direct analysis of complex reaction mixtures or bioassays without sample workup. Moreover, DESI can also be used as a small-scale synthetic tool due to accelerated reactions in generated microdroplets. These characteristics make DESI a core technology for high-throughput (HT) experimentation that prioritizes speed to achieve three major roles. <b>(i) HT reaction screening</b> leverages the reaction acceleration phenomenon for rapid chemical space exploration, especially for the late-stage diversification of drug molecules. The entire process, from sampling the reaction mixture by droplets to on-the-fly chemical transformation during millisecond timescales to analysis by MS, achieves an overall throughput of one reaction per second in an integrated fashion. Diverse chemical transformations for various functional groups were achieved, with over 10⁴ reactions explored and over 10³ analogs identified within three hours. <b>(ii) HT synthesis</b> is achieved using an automated homebuilt array-to-array transfer system. The synthetic system uses DESI microdroplets for transferring reaction mixtures from a precursor array to products on a product array. High conversions of diverse reactions with synthetic throughput of 0.2-0.02 Hz and scale of ng-µg (pmole-nmole) in a spatially resolved manner are demonstrated. Hundreds of modified bioactive molecules are generated in an array format, and the spatial distribution of the products is visualized by mass spectrometry imaging. <b>(iii) HT bioassays</b> are demonstrated by combining the label-free nature of MS with the high-speed analysis of DESI. The contactless feature, with high tolerance towards complex mixtures, allows direct bioassays with minimal sample preparation. An opioid receptor binding assay is described with an evaluation of the binding affinity of synthesized opioid analogs. An on-surface enzymatic assay is developed for measuring the bioactivity of deposited molecules <i>in situ</i>. The consolidation of (i) HT reaction screening, (ii) HT synthesis, and (iii) HT bioassays by a single but versatile technique, HT-DESI, can expedite the early drug discovery process. For applications, MS technologies are utilized to probe reactive intermediates and the reaction mechanisms of palladium-catalyzed coupling reactions. MS is also used to explore chemical reactions for natural products, rapidly generating analogs for bioactivity evaluation and benefiting bioanalysis through the discovery of derivatization reactions. HT tandem MS is demonstrated to be powerful for structural elucidation and reaction site identification.</p>