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Jasbir K. Deol 

Confirming the Use of Phenyl Chloroformate as an Appropriate Addition-Elimination Standard in LFER Analyses

Jasbir K. Deol, Biological Chemistry (senior), Wesley College, Dover, DE

Mentor: Dr. Malcolm J. D'Souza, Professor of Chemistry & Director of Sponsored Research

Background: Chloroformate esters are used in peptide and related synthesis of prodrugs (D'Souza, M.J. and Kevill, D.N., 2013). Two decades ago, phenyl chloroformate (PhOCOCl) was extensively studied in a variety of solvent mixtures and an addition-elimination (A-E) mechanism with a rate-determining addition-step was proposed (Kevill, D.N. and D'Souza, M.J., 1997). Since then, it has served as a reference compound in linear free energy relationship studies (LFERs) of numerous acyl halides. In this project, Phenyloxy carbonyl tosylate (PhOCOOTs) was first synthesized and its specific rates were determined in the same set of solvents as PhOCOCl at 25.0 oC. The principal goal of the project is to determine the presence or absence of any correlation trends between the rates of reaction (energy barriers) observed and the differences in anionic leaving group abilities (between the chloride and tosyl anions).

Methods: PhOCOOTs was synthesized from equimolar additions of silver tosylate to phenyl chloroformate. A rapid precipitation of silver chloride occurred and the resulting solution was used directly as the source of the substrate. The specific rates of reactions were determined by acid-base titrations and conductometry. For the slower runs in this study, acid-base titrations were employed with lacmoid in acetone as the indicator solution, and sodium methoxide as the base. For conductometry, a computer program followed the progress of reaction by documenting the conductivity changes due to the electrical conductance of the ions produced.

Results: The specific rates of PhOCOOTs in a wide range of solvents were found to be very similar to those observed for PhOCOCl (in the same set of solvents).

Conclusions: A tosyl leaving group is a sulfonic acid derivative which is a better leaving group than the chloride anion as it considerably stabilizes the negative charge by resonance stabilization. If the leaving group played an important role in the rate-determining step, the rates of reaction of PhOCOOTs should have been much faster than those for PhOCOCl. We therefore conclude that the mechanism of reaction of PhOCOOTs is exactly the same as PhOCOCl, and hence, it is very appropriate to continue to use PhOCOCl as a standard for A-E reactions.

Acknowledgements: Research funded by National Institute of General Medical Sciences - NIGMS (8 P20 GM103446-13) from the National Institutes of Health (DE-INBRE program). 

 

Megan Durrant

Determining the mechanism of Octyl Chloroformate using acid-base titrations

Megan Durrant: Medical Technology (junior), Wesley College, Dover, DE

Mentor: Dr. Malcolm J. D'Souza, Professor of Chemistry & Director of Sponsored Research

Background: Octyl chloroformate (OctOCOCl) is used as a precursor for drugs that inhibit multiple enzymatic pathways to control obesity. The drug inhibits lipase which stops your body from breaking down and absorbing fat. Alkyl chloroformates such as OctOCOCl are important precursors for many products, and, determining the mechanism of reaction for such subtrates is extensive and tedious work. Hence, this undergraduate research project is funded through the DE-INBRE program as it trains students in the basic biomedical methodologies used to develop more effective drugs.

Methods: In this project, the acid-base titration method has been used to study the specific rates of OctOCOCl in different concentrations of aqueous acetone, aqueous ethanol (EtOH), aqueous methanol (MeOH), aqueous 1,1,1,3,3,3-Hexafluoroisoproponal (HFIP), aqueous 2,2,2, trifluoroethanol (TFE), and in mixtures of TFE-EtOH at 25.0 oC.

Results: The reaction rates of the substrate (OctOCOCl) increased with an increase in the concentration of water in the reaction solvent.

Conclusions: The initial trends observed are very similar in nature to those seen when shorter chain alkyl chloroformates were reacted in a similar set of solvents.

Acknowledgements: This research project is funded by the INBRE grant (8 P20 GM103446-13) sponsored by the National institute for General Medical Sciences and the National Institute for Health.

 

 

Catherine E. Gross

Chloride (Cl-) vs Tosylate (OTs-): Leaving Group Battles at Alkoxycarbonyl Carbon

Catherine E. Gross: (BioChemistry Senior), Wesley College

Mentor: Dr. Malcolm J. D'Souza, Professor of Chemistry & Director of Sponsored Research

Background: The octyloxy carbonyl group is used in the synthesis of novel 2-oxy-bezoxazinone derivatives whose capability in the inhibition of enzyme to catalyse the hydrolysis of an ester functionality is used the treatment of obesity. In chemistry, tosylate is a better leaving group than the chloride anion since the displaced group is the resonance stabilized anion. In this projects, we compare the rates of reaction of octyloxy carbonyl tosylate to octyl chloroformate at 25.0 oC.

Methods: Octyloxy carbonyl tosylate is prepared by mixing a equimolar solution of oxyl chloroformate and silver tosylate. The silver chloride precipitate forms immediately and is suction filtered out of the solution. In one project, our research group will compare the specific rates of solvolysis of octyl chloroformate in a variety of solvents with varying ionizing abilities. My project will study octyloxy carbonyl tosylate in detail and compare the rates against octyl chloroformate. These two compounds are studied in a wide variety of aqueous alcohols, aqueous acetone, and several mixed fluoro-alcohols using the acid-base titration method, with acetone diluted lacmoid as an indicator and sodium methoxide as the base.

Results: Octyloxy carbonyl tosylate had similar reaction rates in some solvents, but reacted faster in fluoro-alcohols.

Conclusion: Solvolysis data of such organic compounds are useful to effectively prepare a pharmaceutically acceptable carrier or diluent. In n-alkoxycarbonyl esters, the leaving group has an effect on the rate determining step in SN1 reaction in some solvents.

Acknowledgements: This research was funded by the National Institute of General Medical Sciences - NIGMS (8 P20 GM103446-13) from the National Institutes of Health (DE-INBRE).

 

 

Laura Malinowski

The Application of Genetic Sequencing in the Study of the XYLT1 gene in Baratela-Scott Syndrome

Laura Malinowski: Biological Chemistry (Junior), Wesley College, Dover, DE

Mentor: Dr. Katia Sol-Church, Primary Investigator at the Biomolecular Core Laboratory of Nemours

Background: Baratela-Scott Syndrome (BSS) is a genetic disorder which causes bone deformities, lack of proper growth, and mental disabilities in the patients it affects. Previous studies, along with work done in the Nemours Biomedical Research Facility, have identified XYLT1 as being most likely responsible for the occurrence of BSS. The work done during this internship has been continuing the study of XYLT1 and its implications in BSS in order to identify the exact mutations responsible. That information could then be used to develop possible treatments for patients affected with BSS. In this project, XYLT1 was studied through the applications of techniques such as polymerase chain reactions and DNA Sanger sequencing, which allowed researchers to closely scrutinize the affected DNA sequences for single nucleotide polymorphisms (SNPs) and mutations possibly passed from the parent to the affected child.

Methods: Primers were designed and chosen in order to attach to the areas of XYLT1 where amplification of the DNA sequence was desired. The DNA of BSS patients and their families were amplified using primers that mostly focused around chromosome 16, exon 1 in the XYLT1 coding gene. Once amplified through the PCR process, the amplification was observed on a gel before the PCR samples were processed for sequencing. After sequencing, the amplified areas of DNA were uploaded to a data table and compared to the Human Genome Project data. Any noticeable SNPs or mutations were noted. The children were also compared to their parents to locate any homozygous or heterozygous sites they may have inherited from their parents.

Results: Locations were found where the children and the mothers’ or fathers’ DNA sequences were not correctly lining up or where the DNA was not strongly amplifying.

Conclusion: The DNA is unable to be fully sequenced in certain places due to poor amplification by the primers. These places may be areas where the parents have passed down the SNPs or mutations responsible for BSS and must be further studied to eventually amplify them correctly and identify a positive area of shared genetic malformations.

Acknowledgements: This research was funded by the National Institute of General Medical Sciences - NIGMS (8 P20 GM103446-13) from the National Institutes of Health (DE-INBRE).

Katelyn Null

Trends Observed In Solvent Studies With Primary Alkyl Chloroformate Esters

Katelyn Null: Biological Chemistry (sophomore), Wesley College, Dover, DE

Mentor: Dr. Malcolm J. D’Souza, Professor of Chemistry & Director of Sponsored Research

Background: Primary alkyl chloroformates are precursors used in the synthesis of a variety of alcohols, amines, esters, and, for small and long carbon chain containing molecules. The substrate, 2-ethylhexyl chloroformate, is used in bioasssays for small molecule antagonists for several hormone receptors and inhibitors such as thyroid and estrogen. This project documents accurate and reproducible kinetic data for 2-ethylhexyl chloroformate at 25.0 oC. Such reaction progress information should be useful to those working in pharmaceutical and biomedical fields. A primary goal of my project is to compare the specific rates of solvolysis of 2-ethyl-hexyl chloroformate to other primary alkyl chloroformates studied at 25.0 oC, and observe any trends based on type of primary alkyl group (normal or branched) present.

Methods: We followed the specific rates of reaction of 2-ethyl-hexyl chloroformate in a constant temperature water-bath (kept at 25.0 oC). Using the acid-base titration method we studied it’s alcoholysis in a variety of pure and mixed aqueous alcohols. The indicator used was lacmoid (resorcinol blue) in acetone.

Results: In a number of pure and mixed aqueous alcohol mixtures, the rates of reaction increased as the solvent ionizing ability increased. We also found that in some solvents, the specific rates of reaction of 2-ethyl-hexyl chloroformate were very similar to its isomer, n-octyl chloroformate.

Conclusions: The present results extend our knowledge of the reactions of the primary alkyl chloroformates in pure and mixed alcohols.

Acknowledgements: Research funded by National Institute of General Medical Sciences - NIGMS (8 P20 GM103446-13) from the National Institutes of Health (DE-INBRE program).

Alora Wilson

New Experimental and Kinetic Data for 2-Ethylhexyl Chloroformate

Alora Wilson: Biological Chemistry (sophomore), Wesley College, Dover, DE

Mentor: Dr. Malcolm J. D’Souza, Professor of Chemistry & Director of Sponsored Research

Background: 2-Ethylhexyl chloroformte is important in the Quantitative High Throughout Screening (qHTS) assay of hormone receptor inhibitors and activators since it identifies the hormones that catalyze multiple enzymatic pathways. Additionally, 2-ethylhexyl chloroformate is important for the qHTS assay for small molecule activators of the heat shock response signaling pathway (PubChem).

Methods: Utilizing lacmoid as the indicator in acid-base titrations, the specific rates of reaction are studied in various aqueous acetone, and aqueous fluoroalcohol concentrations. Here, 2-ethylhexyl chloroformates is studied in aqueous acetone, aqueous 2,2,2-trifluoroethanol (TFE), and aqueous 1,1,1,3,3,3-hexafluoro-2-propanol mixtures.

Results: Initial results suggest that compound reactivity is strongly dependent on type of solvent used. In aqueous acetone, TFE, and HFIP, the more water present, the faster the reaction rate.

Conclusions: Since this trend is observed in the highly ionizing fluoroalcohols, it suggests that the mechanism of reaction is strongly dependent on solvent nucleophilicity.

Acknowledgements: Grant Support: National Institute of General Medical Sciences - NIGMS (8 P20 GM103446-13) from the National Institutes of Health (DE-INBRE)