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STUDENT RESEARCH PROJECTS
2005
(Summer)
 Anu
Maharjan (Merck/AAAS fellow)
"Sodium Transport through ion carriers and ion channels"
2004-2005
(Academic Year)
Lesley-Ann Giddings '05 (Honors Thesis)
"NMR Kinetic Characterization of N-trifluoroacetylazetidine"
Several methods
were used to successfully synthesize TFAZE. Now that TFAZE has been
synthesized
and isolated, kinetic data can be obtained for TFAZE in various solvents.
Temperature studies done on TFAZE in chloroform at 25 ºC, 35 ºC,
45 ºC, and 55 ºC have shown no exchange effects. Higher temperature
and a lower magnetic field (ex. 300 MHz) conditions are being explored
to determine whether under these new conditions exchange can be observed.
Theoretical calculations are currently being done on TFAZE to estimate
the energy of the rotational C-N barrier. The program Gaussian 98 runs
ab initio calculations using complex basis sets in an effort to determine
structural and electronic properties of molecules. The B3LYP/G-311+G**
(Becke-style 3-Parameter Density Functional Theory) basis set was used
to estimate the ground state energy and geometry of TFAZE. The ground
state energy was determined to be -665.06 Hartrees. In the future, a
better approximation of TFAZE’s ground state energy will be obtained
by using the Moller-Plesset perturbation theory (MP2) to calculate the
second order correction to Hartree Fock-determined energies.
In order to estimate the energy and geometry of the transition state
a lower basis set (a less polarized basis set that does not have many
functions), 6-31HG, was used. The optimized ground state energy using
this basis set was -620.45 Hartrees. The stationary point of the transition
state is in the process of being identified. With this data, the difference
between the energies of the ground state and the transition state will
enable kinetic properties, such as the activation Gibbs free energy,
entropy, and enthalpy, to be calculated.
2003-2004
(Academic Year)
Caitlin Gossett '05J (Special Study)
"Sodium flux via redox ion channel in lipid vesicles"
A
cell membrane is essential for the integrity and function of the cell.
Membrane components have many functions including providing a protective
barrier for the cell and regulating transport in and out of the cell.
The cell membrane is composed of a lipid bilayer that regulates the flow
of selected molecules in and out of the cell. Within the lipid bilayer
there are ion channels composed of complex proteins that allow permeability
to certain molecules and ions. The purpose of this project is to test
a series of ion channel analogs synthesized by Dr. Dennis Hall at the
University of Florida as potential sodium ion channels. Aida Manu ‘05
and I will prepare lipid vesicles, which mimic some of the cell membrane
behavior, and then I will use 2D Nuclear Magnetic Spectroscopy to show
ion exchange through the incorporated synthetic ion channel. I will first
test the EXSY 2D pulse sequence by measuring the internal rotation exchange
of the methyls in dimethyl formamide using 1H observation. EXSY will finally
be tested using the ion transporter monensin, which will allow to optimize
the necessary parameters to obtain a 23Na EXSY on the synthetic ion channel.
Lesley-Ann Giddings '05 (Special Study) (under the synthethic supervision
of David Bickar)
"Synthesis and purification of N-trifluoroacetyl azetidine"
In order
to investigate whether angle strain affects the internal rotational barrier
around cyclized amides, kinetic studies using NMR are being conducted
on cyclized amides of different ring sizes in gas and liquid phase. The
four-membered ring compound, trifluoroacetylazetidine (TFAZE), was synthesized
for this kinetic study. Triethylamine (0.025 mols, 3.5 mL) was added to
azetidine hydrochloride (0.0107 mols, 1.0 g), which was cooled to 273
K in an ice-bath. A dropwise addition of trifluoroacetic anhydride (1.7
mL) was made to the reaction flask. Additional trifluoroacetic anhydride
(0.30 mL) was diluted with ether in an addition funnel, which was then,
through dropwise addition, added to the reaction mixture. After the addition
was complete, the reaction mixture was slowly stirred for approximately
60 minutes. The mixture was then diluted with distilled water and the
pH of the mixture was determined to be basic. The organic layer was separated
and the aqueous layer was backwashed with ether. After all organic layers
were combined, the layers were washed with dilute HCl to neutralize any
remaining aqueous material from layer. Using a roto-vap, the ether was
evaporated from reaction mixture. Column chromatography was used to purify
the product on an alumina packed column. In order to further purify the
column eluant, the eluant was distilled under reduced pressure. Molecular
sieves were added to remove water that condensed within collection flask
of distillate. Based on 1H NMR results, the addition of azetidine to trifluoroacetic
anhydride under these conditions yielded TFAZE with other polymers even
after distillation. Therefore, further purification of TFAZE or a new
synthetic procedure is necessary in order to conduct a kinetic study on
TFAZE in both gas and liquid phases.
Aida Manu '05 (co-supervised
with Adam Hall) (Special Study)
"Sodium flux via redox cobalticinium ion channel in lipid vesicles"
In this project, we are continuing the study undertaken last year on sodium
flux via ion channel transport, this time using cobaltocenium instead
of the ferrocenium ion channel. Cobaltocenium is a redox-active synthetically-derived
ion channel, synthesized by Dr. C. Dennis Hall. It mimics the regulatory
behavior of natural ion channels and thus only selectively allows certain
ions into and out of a cell. Caitlin Gossett and I create the lipid vesicles,
following the guidelines from Dr. Adam Hall, and those act as the cells
that will be regulated by the ion channels. The ion channels will be added
to the vesicles, and by using sodium 1D NMR, I will study whether and
how the ion channel controls the flux of sodium ions into and out of the
vesicles.
2002-2003
(Academic Year)
Anindita Hom-Choudhury '05 & Aida Manu '05 (co-supervised with
Adam Hall) (Special Study)
"Sodium flux via redox ferrocinium ion channel in lipid vesicles"
Cell membranes are highly selective barriers forming the outside of cells.
They are impenetrable to water soluble molecules and ions. However, certain
permeability does occur with the aid of complex protein molecules within
the membrane bilayer that do allow selected molecules and ions to flow
in and out of the cell. The purpose of this project is to test a series
of ion channel analogs synthesized by Dr. Dennis Hall at the University
of Florida as potential sodium ion channels. Dr. Adam Hall in the Biological
Sciences here at Smith College is in charge of creating lipid vesicles
that mimic some of the cell membrane behavior and my role is to actually
test the ion channels with the lipid vesicles using 23Na Nuclear Magnetic
Resonance (NMR) spectroscopy.
2001-2002
(Academic Year)
Molly Bowman '03 (under the supervision of David Bickar)
"Synthesis and purification of N-trifluoroacetyl pyrrolidine"
A solution of 0.1 mol (8.4 mL) pyrrolidine in 10 mL anhydrous ether was
cooled to 273 K in a salt-ice bath, and 10 mL of a solution of anhydrous
ether containing 0.05 mol trifluoroacetic anhydride was added slowly with
stirring. After the addition was complete, the reaction mixture was allowed
to warm to room temperature and left to equilibrate for 30 min before
distilling under reduced pressure. After a small forerun (~2 mL), a single
fraction (6.5 mL) distilling at 350 K (10 Torr) was collected. The distillate
contained both pyrrolidine, trifluoroacetamide and the pyrrolidinium trifluoroacetate
salt. Upon cooling, the pyrrolidinium trifluoroacetate salt crystallized,
and the remaining liquid, composed of primarily N-trifluoroacetylpyrrolidine,
was decanted and passed through a 1 x 5 cm column of dry alumina to remove
the remaining salt. The column eluant (3.5 mL, 54% overall yield) was
a clear, colorless liquid. 1H and 13C NMR confirmed the identity of the
eluant to be N-trifluoroacetyl pyrrolidine.
2000-2001
(Summer)
Jennifer Ring '03 (Merck/AAAS summer fellow)
"Actin Studies in Schizosaccharomyces pombe"
Actin is a major cellular protein that forms the structural support for
the cytoskeleton. It is responsible for maintenance of cell structure,
shape and many forms of intracellular transport. Despite the structural
nature of actin, it is a very dynamic molecule. Both in vivo and in vitro,
actin may exist in two forms, globular (G-actin) and filamentous (F-actin).
The G-actin remains as a pool of non-polymerized actin from which the
cell can utilize it as needed, whereas the F-actin is polymerized into
microfilaments. In this respect, the cell needs to be able to regulate
the actin polymerization and depolymerization as needed physiologically.
In several vertebrate cell types, a toxin from the bacterium, Clostridium
botulinum, is able to post-translationally modify G-actin by ADP-ribosylation
at the specific site, Arg-177. As a result, this modification prevents
polymerization. As actin is one of the most conserved proteins across
all eukaryotes, Arg-177 is found in all organisms for which their actin
sequence has been determined. Interestingly, however, it has been discovered
by the Scordilis lab that the fission yeast, Schizosaccharomyces pombe
, endogenously ADP-ribosylates its actin. It is unknown whether the yeast
actin is modified at Arg-177. As very little is known about this process,
it is hypothesized that S. pombe uses this modification as a regulatory
mechanism to control the available pool of polymerizable actin. The goal
of this study is to study the modification with nuclear magnetic resonance
(NMR), to develop protocols to identify and distinguish between ADP-ribose,
ATP and ADP when either bound to or associated with actin, and to establish
the exact stoichiometry of the ADP-ribosylation. This past summer besides
isolating and purifying actin from S. pombe we improved the purification
protocol by increasing the yield of the final products. NMR protocols
were established for the identification and distinction of the three nucleotides.
Further, G-actin and F-actin extracted from rabbit skeletal muscle were
used as a control proteins to develop the NMR protocols for studying actin.
Several experiments were conducted to suppress the water peak in the NMR
spectra and an excellent procedure now exists. This project will be continued
this fall semester.
2000-2001
(Academic Year)
Rachel Herzig-Marx '02 (Special Study)
" Identification of an ADPR Post-Translational Modification to Actin by
Multinuclear NMR Spectroscopy"
This study is a continuation of the work done over the summer. The ultimate
goal of the project is to be able to differentiate the bound ADP-R from
free and bound ATP and free adenosine diphosphate (ADP). Two peaks characteristic
of ADP-R at 5.3 and 5.15 ppm, corresponding to the a and b isomeric conformations
of the 1" proton on the ribose ring, were found. These two peaks do not
show up in the spectra of ADP or ATP, and as a result are an excellent
indicator of the presence of ADP-R. A 31P NMR vs. pH study was also performed
in order to obtain the best separation of the phosphate signals of ATP,
ADP and ADP-R. Best separation was achieved at neutral pH. All NMR solutions
were prepared at a pH of 7.3.
Elizabeth Nicholas '01 (Special Study)
" Synthesis and Gas-Phase DNMR Study of N-trifluoroacetyl Cyclic Compounds"
The goal of this project is to study the effect cyclic N substitution
has on the internal-rotational energy barrier of trifluoroacetamides.
N-trifluoroacetyl pyrrolidine, piperidine and morpholine will be synthesized
and their energy barriers to internal rotation compared. Solution and
gas-phase data will be used in order to elucidate the effect solvents
play in this process.
1999-2000
(Summer)
Rachel Herzig-Marx '02 (Merck/AAAS summer fellow)
" Identification of an ADPR Post-Translational Modification to Actin by
Multinuclear NMR Spectroscopy"
It is found in eukaryotic cells, that the protein G-actin does not polymerize
when attached to adenosine diphosphoribose ADP-R) through its ARG-177
residue. The purpose of this project is to determine, using Nuclear Magnetic
Resonance (NMR) spectroscopy, whether or not the same holds true with
G-actin from the yeast Schizosaccharomyces pombe. The first part
of this project was to develop an NMR protocol using a more readily available
actin: rabbit skeletal G-actin. Both 1H and 31P NMR spectroscopic studies
will be carried on the Schizosaccharomyces pombe using the rabbit
skeletal actin protocol. The protein signals of the rabbit skeletal G-actin
in an aqueous solution appeared in the region between 1.0 and 4.0 ppm
and could be assigned to the amide backbone protons from previously published
results. The second part of the project involved the characterization
of the ADP-R modification also using 1H and 31P NMR. Each molecule of
G-actin is found to be bound to one molecule of adenosine triphosphate
(ATP) and one cation, Ca2+ or Mg2+. The final part of the project, the
analysis of the G-actin extracted from the Schizosaccharomyces pombe
using the optimized NMR protocol and the 31P NMR data will be done in
the fall of 2000.
Elisabeth Nicholas '01 (Schultz Foundation (summer fellow)
" Study of the Biodegradation of 4-chlorophenol by the Microorganism Pseudomonas
by NMR Spectroscopy"
There is great interest in the use of NMR spectroscopy to study biomolecules
and cellular systems. NMR spectroscopy is an analytical tool that can
provide information on the chemical nature of metabolites, intermediates,
and if performed in vivo, can also provide non-invasive information on
cellular environments and metabolism. NMR studies are now addressing the
biodegradation of halogenated organic compounds by microorganisms. In
recent years, detailed NMR protocols have been developed to study biodegradation
of aromatic and cyclic contaminants, for example acenaphthene and morpholine.1-3
Monserrate and Häggblom in 1997 studied the dehalogenation and biodegradation
of brominated phenols under diverse conditions using High Pressure Liquid
Chromatography (HPLC) and ion chromatography techniques.4 This research
proposal will address the application of NMR techniques to study the metabolic
pathway that chlorinated benzoic acids undergo during biodegradation.
The project will involve the selection of an effective microorganism or
activated sediment, and the development of an NMR protocol to detect both
the chlorinated benzoic acid and its biodegraded intermediates and products.
Sarah Thorpe '01 (Schultz Foundation summer fellow)
" Study of the Biodegradation of 4-chlorophenol by the Microorganism Pseudomonas
by NMR Spectroscopy"
There is great interest in the use of NMR spectroscopy to study biomolecules
and cellular systems. NMR spectroscopy is an analytical tool that can
provide information on the chemical nature of metabolites, intermediates,
and if performed in vivo, can also provide non-invasive information on
cellular environments and metabolism. NMR studies are now addressing the
biodegradation of halogenated organic compounds by microorganisms. In
recent years, detailed NMR protocols have been developed to study biodegradation
of aromatic and cyclic contaminants, for example acenaphthene and morpholine.1-3
Monserrate and Häggblom in 1997 studied the dehalogenation and biodegradation
of brominated phenols under diverse conditions using High Pressure Liquid
Chromatography (HPLC) and ion chromatography techniques.4 This research
proposal will address the application of NMR techniques to study the metabolic
pathway that chlorinated phenols undergo during biodegradation. The project
will involve the selection of an effective microorganism or activated
sediment, and the development of an NMR protocol to detect both the chlorinated
phenol and its biodegraded intermediates and products.
1999-2000
(Academic Year)
Sare Arecanli '00 (Honors thesis)
" Conformational DNMR Studies:A Chemical and Philosophical Analysis"
This thesis is composed of two sections: a chemical component on dynamic
nuclear magnetic resonance spectroscopy (DNMR) kinetics studies on conformational
changes, and a philosophical component on seeing with the NMR, the relationship
between theory and instrumentation, and the role of instrumentation in
the progress of science.
Chemical Component: DNMR can be used to study conformational
processes in molecules. Molecules must possess enough energy to overcome
an energetic barrier in order for conformational change to take place.
This energy can be calculated by analyzing the rates of conformational
change at a series of temperatures. The goal of the chemical component
was twofold. In the first section, it was to develop a protocol for analyzing
conformational processes in simple systems, more specifically to study
the internal rotation around the amide partial double bond in diisopropylacetamide.
A complete temperature study was performed and the data was used to test
a Microsoft Excel Spreadsheet that was designed to analyze simple (A3X)
spin systems. Preliminary data shows that the DG‡
obtained with this spreadsheet is in the same order of magnitude as the
literature value. The goal of the second section of the chemical component
is to begin the study of the kinetics of more complicated spin systems,
in particular the chair-to-chair interconversion in six-membered rings.
First, a study of the effect of sulfur in a heterocyclic ring was studied
using thiomorpholine. Then, the effect of chlorine substitution on cyclohexane
was studied with chlorocyclohexane. These compounds were selected to be
studied given their vapor pressures and their potential energy barriers.
This thesis includes a complete temperature study of solution chlorocyclohexane
(in ethanol d6) and solution thiomorpholine, and preliminary data on gas
chlorocyclohexane.
Philosophical Component: The philosophical component includes
sections on what constitutes seeing with instruments, the relationship
between theory and instrumentation, and assessing the progress of science
by tracking the progress of instrumentation. In the section on seeing
with instruments, the issue of seeing with the NMR is discussed against
the backdrop of the debates on seeing with microscopes, and then a new
definition of seeing is proposed. In the next section first the debates
on the relationship between theory and instrumentation are outlined, and
then a new relationship is proposed. The final section, first debates
on assessing the progress of science are outlined, then the development
of the NMR is provided as an example of the role of instrumentation in
the progress of science, and finally, the relationship between theory
and instrumentation that had been proposed in the second section is evaluated
with respect to the history of the NMR.
1998-1999
(Summer)
Sare Arecanli '00 (Schultz foundation fellow)
" Conformational DNMR Studies:A Chemical and Philosophical Analysis"
The partial double bond between the carbonyl oxygen, carbon, and the nitrogen
in diisopropyl acetamide (DIPA) hinders the rotation around the amide
C-N bond. Solvent effects have a significant influence on the rate of
rotation. The solvent effects on the rotational barrier of DIPA were studied
using dynamic Nuclear Magnetic Resonance Spectroscopy. Standard 1D and
a 2D COSY spectra of DIPA were used to completely characterize its chemical
shifts and coupling constants, and determine how protons were coupled
to one another. After testing a number of solvents to find polar, non-polar,
protic, and aprotic solvents which show some form of slow exchange, coalescence,
and fast exchange of DIPA in the temperature range 0-100°C, acetic-d3
acid-d, toluene-d8, deuterium dioxide, and DMSO-d6 were chosen. Toluene
was also selected for a low temperature (0 to -20°C) study. The experimental
data corresponding to the methyl region of the DIPA spectrum was imported
from the JEOL NMR spectrometer, and loaded into a customized Microsoft
Excel spreadsheet. This Excel spreadsheet calculates a simulated NMR lineshape
assuming the DIPA methyl resonances to be an uncoupled A3B3 two-site exchange
system. A non-weighed linear-square regression analysis is performed on
the difference between the experimental and simulated data and a best-fit
rate constant is obtained at each temperature. Using the Eyring equation,
DG‡s for the barrier to the C-N internal rotation
of DIPA were calculated. The following experimental DG‡s
were obtained: DIPA in D2O: 16.8 kcal/mol; DIPA in toluene-d8: 16.3 kcal/mol;
DIPA in DMSO-d6: 16.3 kcal/mol. These values were found to be in good
agreement with literature one. A theoretical calculation (Geometry optimization-AM1)
was also performed and found to be lower than our experimental values
which is in agreement with published values.
1998-1999
(Academic Year)
Elise Hui '99 (Honors thesis)
"NMR Studies of the Diels-Alder Reaction"
NMR spectroscopy was used to monitor four Diels-Alder reactions in order
to determine whether the size, shape or chain length of the reactants
(i.e. the diene or dieneophile) affect the thermodynamic and kinetic properties
of the Diels-Alder reaction. The NMR spectra for the liquid reaction samples
indicated that the Diels-Alder reactions between cyclopentadiene and cyclopentadiene,
cyclopentadiene and acrylonitrile, and (E)-1,3-pentadiene and acrylonitrile
did proceed to form the product. No observable results were obtained for
the reaction between cyclopentadiene and (E)-1,3- pentadiene during the
period of examination. Values of Q and Keq were not obtained for all the
reactions so it was not possible to make comparisons between the four
reactions in terms of thermodynamic properties. Vapour pressure of the
compounds cyclopentadiene, (E)-1,3-pentadiene and acrylonitrile were measured
using a vacuum line and manometer. The experimental results were +322.3
mmHg for cyclopentadiene, +339.4 mmHg for E)-1,3-pentadiene and +91.2
mmHg for acrylonitrile. The discrepancies between the experimental and
theoretical vapour pressure values ranged from 6.2 mmHg to 58.8 mmHg.
NMR spectroscopy was also used to determine the rate of reaction for the
dimerization of cyclopentadiene in both the liquid and gaseous phases.
The results which were obtained indicate that the dimerization reaction
occurred at a faster rate in the liquid phase than in the gaseous phase.
1997-1998
(Academic year)
Dawn Kirnon '98 (Special Study)
"DNMR Studies of Dimethylacetamide"
Over the course of the fall, 1997 semester, I have endeavored to become
familiar with the nuclear magnetic resonance (NMR) spectrometer and how
the spectra of different molecules can be altered by altering the dynamic
properties within them. More specifically, I observed the changes in the
spectra of N, N-dimethylacetamide (DMA) as I increased the temperature
at which the spectra were taken, facilitating the rate of internal rotation
around the C-N bond. All other parameters were held constant, such as
the solvent in which DMA was dissolved (dimethylsulfoxide) and the physical
parameters of the spectrometer. I observed that the spectra of DMA contained
four distinct peaks when taken at 298K, 318K and 338K. Two of the peaks,
at approximately 3.22 and 3.39 ppm, were attributed to the two methyl
groups bonded to the nitrogen. At 358K, these two peaks began to coalesce,
and at 368K, the coalescence was even more marked. At lower temperatures,
the rotation about the C-N bond is inhibited by a partial double bond
between the O-C-N nuclei. The two methyls are therefore exhibiting different
magnetic environments although chemically equivalent. As the temperature
increases, more of the molecules have enough energy to overcome the rotation
barrier, and the NMR spectrometer does not distinguish between the two
methyls. One sharp peak is observed in the spectrum consistent with that
of a rotation about a single bond. The temperature range was not high
enough in this experiment to observe complete coalescence.
1996-1997
(Academic Year)
Sarah Cureton '97 (Mount Holyoke Honors thesis)
"Dynamic Nuclear Magnetic Resonance Spectroscopy of Diazoesters"
Chemical exchange processes in different molecules can be studied by dynamic
nuclear magnetic resonance spectroscopy (DNMR). A system undergoing a
conformational change must have enough energy to overcome a potential
energy barrier characterizing the isomerization, called the activation
energy. The exchange rate for this conformational process is directly
proportional to temperature. Lineshape analysis of the NMR signal at different
temperatures yields information about the rate process. By studying the
lineshape as a function of temperature, exchange rates can be calculated
for the system and the barrier to internal rotation can be elucidated.
Our goal is to study the effects solvents have on the internal rotation
about the C-C bond in diazoesters by comparing gas and liquid phase DNMR
data. The presence of two nitrogens in diazo compounds produces both electronic
and steric effects in the conformational process. Previous studies of
diazoketones in the liquid phase have provided information about the exchange
rates of cis-trans isomerization and the potential energy barriers to
rotation about the C-C bond. In this study diazoesters were found to have
a lower barrier than other ketones, so we are interested in pursuing a
variable temperature study on a series of these molecules. Maximum solvent
effects are felt by a molecule in solution. In addition, different solvents
yield unique values for the energy barrier. Studies in the gas phase exhibit
minimum solvent effects, whereas theoretical calculations eliminate solvent
effects altogether. A series of diazoesters will be studied in the liquid
and gas-phases and the data obtained will then be compared to results
from theoretical calculations.
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