46 Foundational Undergraduate Experiences in the Laboratory Improving Aminative Suzuki-Miyaura Coupling Using Microwave Irradiation

Authors:

Priscilla George, Polpum Onnuch, Dilek Dogutan Kiper

Date Created:

2025-01-01

Course Title:

Professor:

Not specified

About Paper:

A recently published paper by Professor Richard Liu’s lab at accelerates reaction times. Consequently, the microwave-assisted Harvard University’s Department of Chemistry and Chemical aminative Suzuki-Miyaura reactions should broaden the library Biology introduced an innovative method for improving the well- of substrates and afford higher yields. My group is working established Suzuki-Miyaura Coupling reaction, which transforms specifically on 4-methoxy-N-(4-(trifluoromethyl)phenyl)aniline C-C biaryls to C-N-C diaryls by adding a nitrene. This new substrate, along with commercially available aminative reagent, methodology is significant as it enables the use of the same startingdiphenylphosphinyl hydroxylamine, and trifluoromethyl phenyl materials purchased for Suzuki-Miyaura and Buchwald-Hartwig boronic acid. With the tBuBrettPhos palladium catalyst, reactions to create compounds bearing an aminated linkage, we’re prepared to evaluate our hypothesis using the microwave which are otherwise difficult to synthesize. Optimal yields were reactor. If our cross-coupling reaction is successful, we should reached using a bulky palladium catalyst, such as tBuBrettPhos. observe 4-methoxyphenyl-4-(trifluoromethyl)aniline, which can 1 13 Although this reaction is widely applicable to various groups, be confirmed using H and C nuclear magnetic resonance including aryl halides, pseudohalides, boronic acids, and esters, spectroscopy, as well as other characterization techniques such as well as many functional groups and heterocycles, it was as high-resolution mass spectroscopy (HRMS) and elemental not successful for other groups under conventional heating analysis. Overall, this would improve the aminative Suzuki- conditions. Therefore, we hypothesize that microwave irradiation Miyaura coupling, enabling the further expansion of synthetic could be effective for substrates where thermal heating was organic chemistry. unsuccessful, as it not only minimizes side reactions but also The Effect of LpxC Active-Site Mutations on Gram-negative Bacterial Proliferation Silas Nwaishienyi, Sien Verchave, Daniel Kahne Harvard College | Cabot House | Molecular and Cellular Biology | 2028 Gram-negative bacteria share an essential compound of their outer 10, and 50 μM, the bacteria’s optical density showed 0.76, 0.77, membranes called lipopolysaccharide (LPS). LpxC catalyzes LPS 0.78, and 0.72, respectively. Second, we designed primers to edit formation by deacetylating a pre-LPS component, committing the original LpxC gene with our desired mutation, changing the the cell to its production. Prior studies have revealed the catalytic base Asp-242 to Leu-242. Using primers that mutated lethality of LPS inhibition to Gram-negative bacteria, serving as athe catalytic base residue Asp-242 to Leu-242, we amplified with promising area of focus for antibiotics. This study investigates tha polymerase chain reaction to produce mutant LpxC plasmids survivability of Gram-negative E. coli with substitution mutations for insertion into E. coli and growth at 10 μM of inducer. The in the catalytic region of the LpxC gene, namely D242L. First, we results are pending, but we expect to see reduced cell density tested for ideal LpxC expression with the unmodified histidine- because a leucine substitution, while geometrically similar, cannot tagged LpxC gene to set the condition for expressing the mutant replicate the chemical interactions that aspartate has with LpxC’s gene. We cultured a storage strain of E. coli with the LpxC substrate, making the mutant dysfunctional. This would imply that and antibiotic resistance genes in growth media containing 50 cell survival requires this aspartate, indicating useful for designing μg/mL of antibiotic. We cultured these bacteria to an optical efficient enzyme inhibitors. density of 0.4, and took four samples to grow in the expression- inducer IPTG, concentrated to 0-50 μM. From inducers of 0, 5, InvestigatingtheeffectsofMicrowaveIrradiationonAminativeSuzuki-Miyaura Coupling Chris-Ander Paraison, Daniel Kahne, Dilek Dogutan Kiper Harvard College | Eliot House | Chemistry | 2028 Recent advances have demonstrated that by incorporating a nitrene ofK P3 (b4se), O-diphenylphosphinylhydroxylamine(aminating (N–R) group into Suzuki-Miyaura coupling, diaryl amines can agent), and a palladium catalyst bearing the stabilizing ancillary be successfully synthesized affording a new reaction named as ligand tBuBrettPhos Pd G3. We conducted the initial experiments aminative Suzuki-Miyaura (ASM) coupling. This process unites under two differentconditions: conventional heating ina sand bath Suzuki-Miyaura (C–C bond forming reaction) and Buchwald- at 100 C for 12 hours, and microwave irradiation under otherwise Hartwig (C–N bond forming reaction) couplings to generate C– identical conditions. Conventional heating yielded the desired N–C-linked products from common aryl electrophiles and boronic diaryl amine product in ▯10% yield, consistent with prior reports acids under only conventional heating. Suzuki-Miyaura (SM) and identifying thiazole electrophiles as challenging substrates. The Buchwald-Hartwig(BH)couplingiscommonlyusedinproduction microwave-assisted reaction is under investigation, and its results of medicines, dyes, etc. Therefore, the significance of ASM will inform whether non-conventional heating provides significant coupling is being able to access both SM and BH bond products kinetic or thermodynamic advantages. while only having to supply SM reactants and a nitrene source, Our results aim to assess whether microwave irradiation can requiring less starting reagents to be carried and lowering material costs. overcome the low reactivity of heterocyclic triflates in aminative cross-coupling. Future work includes quantifying microwave However, the potential role of microwave irradiation in enhancing yields, confirming product identity via H NMR, and replicating the isolated yields of ASM reactions has been largely unexplored. both trials to establish statistical significance. Ultimately, if To evaluate this, we synthesized 2-methylthiazole-5-carboxylate- microwave-assisted conditions are shown to enhance product trifluoromethanesulfonate (a type of heterocyclic triflate) as an yield, this could offer a more efficient protocol for synthesizing electrophile and subjected it to aminative cross-coupling with (4- structurally diverse diaryl amines, especially from otherwise trifluoromethyl)phenylboronic acid (nucleophile) in the presence unreactive electrophiles. Synthesisof2-methylthiazole-5-carboxylateN-(4-trifluoromethyl)phenyl)aniline via Aminative Suzuki-Miyaura Coupling under Microwave Irradiation using tBuBrettPhos Karolina Spiewak, Dilek Dogutan Kiper, Daniel Kahne Harvard College | Currier House | Molecular and Cellular Biology | 2028 Our work builds upon recent research by Professor Richard with potential applications in medicinal chemistry. Thus far, Y. Liu and his team from the Department of Chemistry and we performed ASMC between a 2-methylthiazole-5-carboxylate Chemical Biology at Harvard University on Aminative Suzuki- moiety and 4-trifluoromethylphenyl-substituted aniline, catalyzed Miyaura Couplings (ASMC). In their report, the insertion of by tBuBrettPhos (3.0 mol %) in 2-MeTHF (0.2 M) at 100 C for▯ a formal nitrene into the palladium (Pd)-catalyzed pathway 46 hours under microwave conditions. Microwave irradiation introduces C-N-C-linked diaryl amine products, rather than enables in situ heating and significantly accelerates the chemical traditional C-C-linked biaryls. Although the new methodology reaction. Additionally, Microwave-Assisted Aminative Suzuki- offers a new route to compounds bearing C-N-C bonds, some Miyaura (MW-ASM) reaction affords cleaner crude products due of the substituents, including five-member heteroaryl groups to fewer side reactions, making purifications easier and requiring such as thiazole, isothiazole, pyrazole, imidazole, and oxazole, less solvent, silica gel for column chromatography, and fewer were either not tried or were not given the target compounds uses of other materials and supplies. Together, these factors under conventional heating conditions, even if the bulky biaryl reduce laboratory waste and lower costs, supporting cleaner monophosphine ligand tBuBrettPhos was used. To address this and safer chemistry research overall. In conclusion, using limitation, our study uses the same ligand (tBuBrettPhos) but microwave irradiation inASM reactions willbe more effectiveand employs microwave irradiation to potentially overcome thermal efficient for industrial applications, such as drug discovery, natural heating inefficiency. Our goal is to synthesize 2-methylthiazole- product synthesis, and making new building blocks through high- 5-carboxylate N-(4-trifluoromethyl)phenyl)aniline, a compound throughput screening. 48 Foundational Undergraduate Experiences in the Laboratory Investigating the Role of His265 in Escherichia coli LpxC Catalysis: A Mutational Approach Elisabeth Maria Aleksandra Stevens, Cait Moffatt, Daniel Kahne, Sien Verschave Harvard College | Leverett House | Integrative Biology | 2028 Gram-negative bacteria are unique for their outer membrane a H265A substitution in Escherichia coli cells using PCR-based consisting of lipopolysaccharide (LPS) molecules, which dually site-directed mutagenesis. We hypothesized that alanine’s small serve as potent endotoxins and barriers to antimicrobial entry. Thisnd nonpolar nature would occlude any participation in acid-base endows them with heightened intrinsic and acquired resistance chemistry and ultimately disrupt LpxC’s catalytic activity. So far, mechanisms, posing adrasticglobalhealththreatand underscoring we have successfully purified the wild type and run an SDS-PAGE the imperativeness of developing new bactericidal therapeutics. gel to establish optimal conditions for protein production, as well One candidate of interest for targeted drug design is LpxC, a as transformed the mutant plasmids into expression cells. We also metal-dependent deacetylase that catalyzes the committed step of plan to overexpress the modified strain and visualize the bacterial LPS synthesis. An important residue in the protein’s active site isolates using electron microscopy to discern overall membrane is His-265, which serves as a general acid by protonating the integrity. Future work will explore how these functional and 2-amino leaving group of UDP-(3-O-(R-3-hydroxymyristoyl))-N- structural insights can inform the development of broad spectrum acetylglucosamine, LpxC’s natural substrate. To better quantify LpxC antibiotics beyond current clinically-ineffective alternatives howessentialthisaminoacidisintheturnoverofproductbyLpxC, like hydramate-based inhibitors. and thus the overall formation of LPS, we decided to perform Harvard Stem Cell Institute Internship Program

Abstract:

A recently published paper by Professor Richard Liu’s lab at accelerates reaction times. Consequently, the microwave-assisted Harvard University’s Department of Chemistry and Chemical aminative Suzuki-Miyaura reactions should broaden the library Biology introduced an innovative method for improving the well- of substrates and afford higher yields. My group is working established Suzuki-Miyaura Coupling reaction, which transforms specifically on 4-methoxy-N-(4-(trifluoromethyl)phenyl)aniline C-C biaryls to C-N-C diaryls by adding a nitrene. This new substrate, along with commercially available aminative reagent, methodology is significant as it enables the use of the same startingdiphenylphosphinyl hydroxylamine, and trifluoromethyl phenyl materials purchased for Suzuki-Miyaura and Buchwald-Hartwig boronic acid. With the tBuBrettPhos palladium catalyst, reactions to create compounds bearing an aminated linkage, we’re prepared to evaluate our hypothesis using the microwave which are otherwise difficult to synthesize. Optimal yields were reactor. If our cross-coupling reaction is successful, we should reached using a bulky palladium catalyst, such as tBuBrettPhos. observe 4-methoxyphenyl-4-(trifluoromethyl)aniline, which can 1 13 Although this reaction is widely applicable to various groups, be confirmed using H and C nuclear magnetic resonance including aryl halides, pseudohalides, boronic acids, and esters, spectroscopy, as well as other characterization techniques such as well as many functional groups and heterocycles, it was as high-resolution mass spectroscopy (HRMS) and elemental not successful for other groups under conventional heating analysis. Overall, this would improve the aminative Suzuki- conditions. Therefore, we hypothesize that microwave irradiation Miyaura coupling, enabling the further expansion of synthetic could be effective for substrates where thermal heating was organic chemistry. unsuccessful, as it not only minimizes side reactions but also The Effect of LpxC Active-Site Mutations on Gram-negative Bacterial Proliferation Silas Nwaishienyi, Sien Verchave, Daniel Kahne Harvard College | Cabot House | Molecular and Cellular Biology | 2028 Gram-negative bacteria share an essential compound of their outer 10, and 50 μM, the bacteria’s optical density showed 0.76, 0.77, membranes called lipopolysaccharide (LPS). LpxC catalyzes LPS 0.78, and 0.72, respectively. Second, we designed primers to edit formation by deacetylating a pre-LPS component, committing the original LpxC gene with our desired mutation, changing the the cell to its production. Prior studies have revealed the catalytic base Asp-242 to Leu-242. Using primers that mutated lethality of LPS inhibition to Gram-negative bacteria, serving as athe catalytic base residue Asp-242 to Leu-242, we amplified with promising area of focus for antibiotics. This study investigates tha polymerase chain reaction to produce mutant LpxC plasmids survivability of Gram-negative E. coli with substitution mutations for insertion into E. coli and growth at 10 μM of inducer. The in the catalytic region of the LpxC gene, namely D242L. First, we results are pending, but we expect to see reduced cell density tested for ideal LpxC expression with the unmodified histidine- because a leucine substitution, while geometrically similar, cannot tagged LpxC gene to set the condition for expressing the mutant replicate the chemical interactions that aspartate has with LpxC’s gene. We cultured a storage strain of E. coli with the LpxC substrate, making the mutant dysfunctional. This would imply that and antibiotic resistance genes in growth media containing 50 cell survival requires this aspartate, indicating useful for designing μg/mL of antibiotic. We cultured these bacteria to an optical efficient enzyme inhibitors. density of 0.4, and took four samples to grow in the expression- inducer IPTG, concentrated to 0-50 μM. From inducers of 0, 5, InvestigatingtheeffectsofMicrowaveIrradiationonAminativeSuzuki-Miyaura Coupling Chris-Ander Paraison, Daniel Kahne, Dilek Dogutan Kiper Harvard College | Eliot House | Chemistry | 2028 Recent advances have demonstrated that by incorporating a nitrene ofK P3 (b4se), O-diphenylphosphinylhydroxylamine(aminating (N–R) group into Suzuki-Miyaura coupling, diaryl amines can agent), and a palladium catalyst bearing the stabilizing ancillary be successfully synthesized affording a new reaction named as ligand tBuBrettPhos Pd G3. We conducted the initial experiments aminative Suzuki-Miyaura (ASM) coupling. This process unites under two differentconditions: conventional heating ina sand bath Suzuki-Miyaura (C–C bond forming reaction) and Buchwald- at 100 C for 12 hours, and microwave irradiation under otherwise Hartwig (C–N bond forming reaction) couplings to generate C– identical conditions. Conventional heating yielded the desired N–C-linked products from common aryl electrophiles and boronic diaryl amine product in ▯10% yield, consistent with prior reports acids under only conventional heating. Suzuki-Miyaura (SM) and identifying thiazole electrophiles as challenging substrates. The Buchwald-Hartwig(BH)couplingiscommonlyusedinproduction microwave-assisted reaction is under investigation, and its results of medicines, dyes, etc. Therefore, the significance of ASM will inform whether non-conventional heating provides significant coupling is being able to access both SM and BH bond products kinetic or thermodynamic advantages. while only having to supply SM reactants and a nitrene source, Our results aim to assess whether microwave irradiation can requiring less starting reagents to be carried and lowering material costs. overcome the low reactivity of heterocyclic triflates in aminative cross-coupling. Future work includes quantifying microwave However, the potential role of microwave irradiation in enhancing yields, confirming product identity via H NMR, and replicating the isolated yields of ASM reactions has been largely unexplored. both trials to establish statistical significance. Ultimately, if To evaluate this, we synthesized 2-methylthiazole-5-carboxylate- microwave-assisted conditions are shown to enhance product trifluoromethanesulfonate (a type of heterocyclic triflate) as an yield, this could offer a more efficient protocol for synthesizing electrophile and subjected it to aminative cross-coupling with (4- structurally diverse diaryl amines, especially from otherwise trifluoromethyl)phenylboronic acid (nucleophile) in the presence unreactive electrophiles. Synthesisof2-methylthiazole-5-carboxylateN-(4-trifluoromethyl)phenyl)aniline via Aminative Suzuki-Miyaura Coupling under Microwave Irradiation using tBuBrettPhos Karolina Spiewak, Dilek Dogutan Kiper, Daniel Kahne Harvard College | Currier House | Molecular and Cellular Biology | 2028 Our work builds upon recent research by Professor Richard with potential applications in medicinal chemistry. Thus far, Y. Liu and his team from the Department of Chemistry and we performed ASMC between a 2-methylthiazole-5-carboxylate Chemical Biology at Harvard University on Aminative Suzuki- moiety and 4-trifluoromethylphenyl-substituted aniline, catalyzed Miyaura Couplings (ASMC). In their report, the insertion of by tBuBrettPhos (3.0 mol %) in 2-MeTHF (0.2 M) at 100 C for▯ a formal nitrene into the palladium (Pd)-catalyzed pathway 46 hours under microwave conditions. Microwave irradiation introduces C-N-C-linked diaryl amine products, rather than enables in situ heating and significantly accelerates the chemical traditional C-C-linked biaryls. Although the new methodology reaction. Additionally, Microwave-Assisted Aminative Suzuki- offers a new route to compounds bearing C-N-C bonds, some Miyaura (MW-ASM) reaction affords cleaner crude products due of the substituents, including five-member heteroaryl groups to fewer side reactions, making purifications easier and requiring such as thiazole, isothiazole, pyrazole, imidazole, and oxazole, less solvent, silica gel for column chromatography, and fewer were either not tried or were not given the target compounds uses of other materials and supplies. Together, these factors under conventional heating conditions, even if the bulky biaryl reduce laboratory waste and lower costs, supporting cleaner monophosphine ligand tBuBrettPhos was used. To address this and safer chemistry research overall. In conclusion, using limitation, our study uses the same ligand (tBuBrettPhos) but microwave irradiation inASM reactions willbe more effectiveand employs microwave irradiation to potentially overcome thermal efficient for industrial applications, such as drug discovery, natural heating inefficiency. Our goal is to synthesize 2-methylthiazole- product synthesis, and making new building blocks through high- 5-carboxylate N-(4-trifluoromethyl)phenyl)aniline, a compound throughput screening. 48 Foundational Undergraduate Experiences in the Laboratory Investigating the Role of His265 in Escherichia coli LpxC Catalysis: A Mutational Approach Elisabeth Maria Aleksandra Stevens, Cait Moffatt, Daniel Kahne, Sien Verschave Harvard College | Leverett House | Integrative Biology | 2028 Gram-negative bacteria are unique for their outer membrane a H265A substitution in Escherichia coli cells using PCR-based consisting of lipopolysaccharide (LPS) molecules, which dually site-directed mutagenesis. We hypothesized that alanine’s small serve as potent endotoxins and barriers to antimicrobial entry. Thisnd nonpolar nature would occlude any participation in acid-base endows them with heightened intrinsic and acquired resistance chemistry and ultimately disrupt LpxC’s catalytic activity. So far, mechanisms, posing adrasticglobalhealththreatand underscoring we have successfully purified the wild type and run an SDS-PAGE the imperativeness of developing new bactericidal therapeutics. gel to establish optimal conditions for protein production, as well One candidate of interest for targeted drug design is LpxC, a as transformed the mutant plasmids into expression cells. We also metal-dependent deacetylase that catalyzes the committed step of plan to overexpress the modified strain and visualize the bacterial LPS synthesis. An important residue in the protein’s active site isolates using electron microscopy to discern overall membrane is His-265, which serves as a general acid by protonating the integrity. Future work will explore how these functional and 2-amino leaving group of UDP-(3-O-(R-3-hydroxymyristoyl))-N- structural insights can inform the development of broad spectrum acetylglucosamine, LpxC’s natural substrate. To better quantify LpxC antibiotics beyond current clinically-ineffective alternatives howessentialthisaminoacidisintheturnoverofproductbyLpxC, like hydramate-based inhibitors. and thus the overall formation of LPS, we decided to perform Harvard Stem Cell Institute Internship Program

Source:

Harvard / Ronni Fleming, Cait Moffatt, Sien Verschave, Daniel Kahne / 2025

Topics:

reaction, microwave, lpxc, suzuki, aminative, coupling, using, irradiation, heating, product, chemistry, group

Co-authors:

@priscillageorge193 , @polpumonnuch194 , @dilekdogutankiper195