Rebecca
Craig-Schapiro |
Mutating
the N-terminal domain of L9 at two rationally determined positions strongly enhances
protein stability
Rebecca
Craig-Schapiro, University of Oklahoma, Burcu Anil, Jae-Hyun Cho, Daniel Raleigh,
Department of Chemistry, Stony Brook UniversityThe
effects of point mutations can be observed to gain information about protein folding.
NTL9 is the N-terminal domain of the ribosomal protein L9. This 56 residue region
contains a three stranded antiparallel beta sheet between two alpha helices and
folds via a two-state mechanism. Previous experiments have shown that mutating
Lys12 to Met and Gly34 to D-Ala confer enhanced stability, and it is hypothesized
that the combination of these mutations will lead to further stability. The double
mutant peptide was prepared by FMOC solid phase peptide synthesis and was purified
by reverse phase HPLC. The stability of the K12M G34D-A mutant was compared to
that of wild-type NTL9 and the two single mutants using thermal and chemical denaturation
experiments at pH 5.45. The thermal denaturation curve for the double mutant in
native buffer and in 4.5 M urea contained no post-transition region, and thus
the Tm could not be determined. Thermal denaturation monitored at 222 nm and 280
nm in 2 M GuHCl yielded a Tm of 91 C. The Tm of the wild-type protein in native
buffer is approximately 80 C, indicating that the double mutant has considerably
more thermal stability. GuHCl denaturation experiments yielded a ?G of unfolding
of 6.79 kcal/mol for the double mutant, compared to 4.17 kcal/mol for the wild-type,
4.80 kcal/mol for the K12M mutant, and 6.04 kcal/mol for the G34D-A mutant. From
stopped flow experiments carried out in GuHCl, the folding rate of the double
mutant was determined to be 2146 s-1, compared to 865 s-1 of the wild-type protein.
The unfolding rate for the double mutant was 0.1 s-1 and the wild-type was 0.9
s-1, indicating that the mutant folds faster and unfolds slower. Structure formation
was followed using circular dichroism and NMR. Circular dichroism in the far UV
region provides a measure of the secondary structure during folding, while the
near UV region monitors tertiary structure. The CD spectra exhibited the minima
and maxima associated with the expected mixed alpha-helix and beta-sheet structure
of NTL9. Using NOESY, TOCSY, and COSY, the alpha carbon 1H chemical shift assignments
of the wild-type and single mutants were compared to those of the double mutant.
The chemical shifts of the residues that could be assigned were close to those
of the wild-type, indicating that the structure of the protein had not changed.
Future work includes determination of stability by hydrogen-deuterium exchange
studies. |
| Kate
Edelman | Synthesis
of Trigonal Prismatic Nanostructures
Kate
Rose Edelman, Texas A&M University-Kingsville; and Andreas Mayr, Department
of Chemistry, Stony Brook University
Technological
advances in computing and electronics beyond the limits of current methodologies
will likely involve the use of molecular devices. With this in mind, a class of
single-electron devices has been designed that requires nanoscale objects to be
arranged in a particular array between nanoelectrodes. Three-dimensional nanostructures
have been proposed as a means to achieve the spatial arrangement of the nanoelectronic
system.
The focus of this work is to synthesize trigonal prismatic nanostructures
for use in single-electron devices. The prismatic nanostructures consist of triangular
faces that are connected to vertical edges by corner units. A phenanthroline molecule
bearing ethynyl groups has been chosen for use as a corner unit for triangular
faces. Efforts to prepare this compound with a palladium/copper catalyst system
were unsatisfactory. When the copper co-catalyst was used in the reaction, intermediates
with terminal ethynyl groups were undergoing undesirable side reactions. The reaction
was repeated excluding the copper co-catalyst and the results showed that the
molecule was obtainable without loss of ethynyl-substituted phenanthrolines. NMR
spectroscopy, column chromatography, and TLC were all used to isolate and characterize
the reaction products.
Continuing efforts with these materials will focus
on the completion of the synthesis of the triangular face, as well as the prismatic
nanostructure. Wider availability of these nanostructures will make future applications
in the blossoming fields of nanotechnology and nanoelectronics possible. This
research has been funded by the National Science Foundation Research Experiences
for Undergraduates (NSF-REU) program at Stony Brook University. |
| Jessica
Hilborn | Approaches
to the Syntheses of New Molybdenum Complexes as Models for the Active Site in
Nitrogenase Enzymes
Jessica
Hilborn, Carroll College; Edythe Maa, and Michelle Millar. Department of Chemistry
ofStony Brook University
Since
the discovery of the nitrogenase enzyme, the mechanism of its operation (the catalytic
conversion of N2 to NH3) has been pondered by biologists and chemists alike. X-ray
crystallographic and spectroscopic studies indicate that the active site of the
nitrogenase enzyme contains a unique inorganic cluster composed of one molybdenum,
seven iron, and nine sulfur atoms, as shown below. Understanding the biological
role of nitrogen fixation is crucial, as it provides a source of nitrogen used
in the building of amino acids and nucleic acids in living things. The enzyme
is also able to perform nitrogen fixation under reasonable conditions (290 K,
1 atm). In comparison, industrial processes, such as the Haber-Bosch, carry out
the conversion of N2 to NH3 for commercial use, yet require harsh conditions (700+
K, 500 atm), consuming energy and money. Thus, it is important for scientists
to figure out how the biological fixation process works so it can be applied to
other fields.
One of the questions posed by scientists is where exactly
the N2 activation site is located on the iron-molybdenum cofactor: the Fe or the
Mo. Focused studies on molybdenum coordination chemistry have produced two successful
model systems that are capable of converting N2 to NH3. The Chatt system uses
an Mo(0) catalytic center coordinated to four phosphine ligands. The Schrock system
uses an Mo(III) center coordinated to an amine and three amide ligands, creating
complexes of the form [N(N)3MoL].
The purpose of the work we are doing
is to model the active site and to mimic the catalytic properties of the nitrogenase
enzyme. To more closely represent the Fe-Mo center in the nitrogenase, which coordinates
sulfur instead of nitrogen, we have chosen to use different but analogous ligands
as Schrock to synthesize new Mo-thiolate complexes. Past studies have shown that
the ligands used must also effectively block the formation of Mo-N-N-Mo bridges
between sites, which make the site robust to reduction. We used the polydentate
ligands PS3, PS3* and NS3 to counteract this problem and form model compounds
of the formulation [Mo(PS3)L2] and [Mo(PS3)L] where L is N2 or a related molecule.
Through the synthesis and study of reactivity and spectroscopic data of these
model compounds, we will have a better insight as to the mechanisms of the coordination
and activation of N2 and other related molecules at the Mo site in enzyme.
The starting materials used were [Mo(NO)2Cl2] and [Mo(NO)2Cl4]2-, which provide
a monomeric molybdenum center possessing NO ligands. The principal reaction was
the initial synthesis of Mo(NO)2Cl2 which was acquired in high yield via a reaction
of Mo(CO)6 and NOCl. Next, the starting material was allowed to react with the
aforementioned ligands under various conditions and solvent systems to synthesize
a useful compound. Reactions were monitored with IR spectra and cation tests.
Successful X-ray crystallographic data has yet to be obtained. This work was funded
by the NSF-REU 2004 Summer Research Grant. |
| Diana
Leung | Regio-
And Stereo-Selective Rh-Catalyzed C-H Insertion Of A Carbamate Nitrogen
Diana
Leung, Binghamton University, and Kathlyn A. Parker, Department of Chemistry,
Stony Brook University
The
experiments carried out were to determine if it was possible to synthesize D-Angolosamine
Glycal, one of the building blocks needed for synthesizing kidamycin. Kidamycin
is an antitumor antibiotic. To produce D-Angolosamine Glycal C-H insertion of
a carbamate nitrogen must be done to form the precursor of D-Angolosamine Glycal.
A four-step scheme was done to produce a chiral carbamate 4 for C-H insertion
of a carbamate nitrogen. After each reaction was completed a flash chromatography
was done to obtain the product, and the use of thin layer chromatography and 1H-NMR
were used to confirm the production of the desired compound in each step.
From
previous studies done by a lab member, C-H insertion of a carbamate nitrogen 2
occurred next to the double bond. It was hypothesized that C-H insertion of a
carbamate 4 would occur next to the triple bond because the carbamate 2 insertion
occurred next to the double bond. Instead of producing the precursor for the production
of D-Angolosamine, an interesting discovery was made about the stereo-selectivity
of carbamate nitrogen for the oxygen instead of the triple bond. With such an
interesting discovery, further studies need to be conducted on why the Rh-catalyst
favored the insertion on the C-H attacted to -OTBDPS, instead of the C-H next
to the triple bond. Also, experiments need to be carried out to find a better
catalyst for producing a C-H insertion of a carbamate nitrogen to the C-H next
to the triple bond. This study was supported by grants from Chemistry REU, IBRP,
NSF, NIH, and the National Cancer Institute. |
| Robin
Padilla | Preparation
of a Terminal Diacetylene for the Supramolecular Synthesis of Diacetylene Polymers
Robin
Padilla, John Markiewicz, Frank W. Fowler and Joseph W. Lauher, Department of
Chemistry, Stony Brook University
The
discovery and development of conducting polymers in the 1970's generated much
excitement and opened new doors in chemistry, physics, and materials science in
fact their significance was enough to warrant the Chemistry Nobel Prize in 2000.
These polymers are comprised of polyacetylenes; repeating units of conjugated
carbon-carbon double bonds. The research focus of this group is on a closely related
set of compounds, polydiacetylenes. The conjugated bonds present in these polymers
account for their interesting electrical and conductive properties but also present
an interesting problem for synthetic chemists. For a 1,4-polymerization to occur,
the diacetylene monomer units within the polymer need to have a spacing of ~4.9?.
Supramolecular chemistry, the discipline concerned with non-covalent bonding interactions
and the assembly of macromolecular structures (e.g. DNA), provides a solution
to the preparation of diacetylene polymers via a host-guest strategy. It is possible
to impose the required spacing conditions for a topochemical polymerization by
using a host molecule that forms a hydrogen bond array with the desired repeat
distance. The host, in turn, imposes this same spacing on the guest molecule (which
contains the monomer unit) via hydrogen bonding, thereby providing optimal conditions
for a polymerization reaction to occur. Previous work done by this group has shown
the host-guest method to be highly effective for the design and synthesis of polydiacetylenes
with a wide variety of host and guest molecules being used. The focus of this
project was the synthesis of one such guest molecule, nicotinic acid penta-2,4
diynyl ester. The molecular structure of this compound, in particular the pyridine
moiety, makes it capable of forming strong hydrogen bonds as well as - stacking,
and thus is an ideal guest molecule for the preparation of diacetylenes polymers.
The target molecule was prepared through a four step synthesis, with the first
step being an allylic bromination of propargyl alcohol (HOCH2C CH) which was followed
by a Cadiot-Chodiewicz coupling of the propargyl group to triethylsilyl protected
acetylene (TES-C CH). The resulting diacetylene was then reacted with nicotinic
acid chloride, forming the nicotinic diacetylene ester. In the final step, the
TES group was removed from the molecule by a reaction with tetrabutylammonium
fluoride (TBAF) to yield the target molecule. This project was made possible by
funding from NSF Grant CHE 0300008. |
| Scott
Peslak | Analysis
of Integrin-Specific Binding and Fluorescently Labeled Fertilinß Polymer
Endocytosis in K562 Cells
Scott
Peslak, University of Scranton; Kenny Roberts and Nicole S. Sampson, Department
of Chemistry, Stony Brook University. Extracellular
membrane-bound proteins on sperm known as ADAMs (A Disintegrin and A Metalloprotease)
and membrane-bound receptors on egg cells known as integrins play important roles
in the process of mammalian fertilization. The fertilinß ADAM protein and
the ?9?1 and ?6?1 integrin receptors are particularly significant in the fertilization
process. Fertilin? on the sperm is thought to bind to both the ?9 and ?6 receptors
on egg cells, inducing binding and eventual fusion of the sperm.
The purpose
of the research project was first to confirm the presence of the integrin receptors
through the specific binding of antibodies to integrins on K562 cells, and then
to determine if a fluorescently labeled fertilin? polymer, bound to ?9 or ?6 integrin
receptors, undergoes binding or endocytosis into the cells at varying temperatures.
To accomplish the first goal, three types of erythroleukemia human cells were
cultured - K562 wild type, K562-?9, and K562-?6. These mimic cells were used in
place of in vitro mouse egg fertilization to allow isolation of and specific experimentation
on the ?9 and ?6 subunits. The monoclonal antibody Y9A2 was added to bind to K562-?9
cells and the monoclonal antibody GoH3 was added to bind to K562-?6 cells. Alexa
568 antibodies were then added to stain the cells for imaging. Successful binding
of the antibodies to the surface of the K562-?6 and K562-?9 cells was observed
through fluorescence microscopy, while surface staining of the wild type cells
was not observed. These results confirm the presence of the ?9?1 and ?6?1 integrin
subunits on the K562 cells.
Binding and endocytosis of the fertilin? polymer,
the second goal of the research project, was attempted by adding the reduced form
of the fluorescent polymer to K562 wild type, K562-?6, and K562-?9 cells at 10
°C or 37 °C. Two types of polymer were used in the experiment - an experimental
10-mer with the ECDVT (Glu-Cys-Asp-Val-Thr) fertilin? amino acid sequence and
a control 10-mer with the scrambled CTEVD (Cys-Thr-Glu-Val-Asp) sequence. The
ECDVT fertilin? polymer was expected to bind at 10 °C and endocytose at 37
°C, mimicking the binding of sperm to eggs. Thus far, conclusive evidence
of binding or endocytosis of the fluorescent ECD polymer has not been obtained,
nor has endocytosis of the integrin receptors been observed. Future studies with
the fluorescent polymer will further explore the interaction between possible
receptors and the ADAM fertilin? protein and will determine their roles in mammalian
fertilization. This research was funded by the National Science Foundation and
the National Institute of Health Grant HD-38579.
|
| Kathryn
Pietsch | Design
and Synthesis of New Biphenol-Proline Based Phosphoramidite Ligands for Catalytic
Asymmetric Synthesis
Kathryn
Pietsch, Concordia College; Bruno Chapsal, Iwao Ojima, Department of Chemistry,
Stony Brook University
The synthesis of enantiomerically pure compounds
is essential in chemical technology, especially in pharmaceutical production.
Since biological systems identify each enantiomer of a chiral compound as different
molecules, the body can react in two dramatically different ways. This stresses
the importance of discovering techniques and reactions that yield 100% enantiomeric
excess (ee).
Catalytic asymmetric synthesis is one of the means of obtaining
enantiomerically pure compounds. During the asymmetric reaction, chirality is
induced in the substrate when a chiral ligand is paired with a metal catalyst.
Rather than developing one universal chiral ligand that yields high ee for every
reaction, it is considered more practical to develop libraries of chiral ligands
specifically adapted for various types of reactions, e.g., hydrogenation, allylic
alkylation, 1,4-addition, etc. Thus it is necessary to continue searching for
more chiral ligand possibilities.
The biphenol framework with axial chirality
has proven to be an effective chiral unit for the development of new chiral ligands.
The interest of such chiral ligands relies on its easy synthesis, but most of
all on their fine-tuning capabilities (Figure 1). Such an aspect is crucial as
these ligands could then be applied to a wide variety of asymmetric
catalytic
reactions.
We are planning to expand the phosphoramidite library (R3 =
NR), especially with proline derivatives. Proline is a commercially available
amino acid and its carbonyl functional group can be easily manipulated to produce
various derivatives. Following preliminary observation of the metallic complex
on a computer simulation program (Spartan), we hypothesized that the cyclic structure
of proline could limit the rotational freedom of the amine moiety, thus freezing
the configuration of the ligand. Furthermore, proline's side chain, which is capable
of being altered, would introduce bulkiness near the metal center and could exert
a substantial effect on the coordination of the prochiral substrate to the metal.
Phosphoramidite ligands with these properties are promising candidates for the
library.
First,
we carried out the synthesis and resolution of enantiomerically pure 3,3'-di-tert-butyl-5,5',6,6'-tetramethyl-1,1'-biphenyl-2,2'-diol
to serve as the basic framework for our ligands. We successfully synthesized other
chiral biphenol units by modifying the R1 group to hydrogen and to methyl groups.
(-)-2-(1-Methoxy-1-methylethyl) pyrrolidin (R2 = R3 = Me) is the first derivative
that we have successfully synthesized from L-proline. The designed phosphoramidite
ligand (R1 = H and R2 = R3 = Me) is in the process of being synthesized and will
be subjected to various asymmetric catalytic reactions with an overall goal of
achieving high enantioselectivity in those reactions.Acknowledgment:
This research was funded by the National Science Foundation's Research Experience
for Undergraduates at SUNY Stony Brook.
Ojima, Iwao In Catalytic Asymmetric Synthesis, 2nd Ed.; Ojima, I. (Ed.); Wiley-VCH,
Inc.: New York, 2000.
2 Hua, Z.; Vassar, V.C.; Choi, H.; Ojima, I. PNAS. 2004,
101, 5411-5416.
|
| Kunil
Raval | The
Synthesis of an Artificial Glucose Receptor.
Kunil Raval, Michigan State
University, Dale Drueckhammer, and Yang Yongliang. Department of Chemistry, Stony
Brook University The
aim of our research is to develop a "speedometer" for monitoring glucose
levels in the blood. Just as a car's speedometer continuously measures our rate
of travel, displaying all fluctuations so that we can keep our speed within a
reasonable limit, diabetic patients also need such a device, so that they are
able to know and thus control their glucose levels at every moment. Our technique
for continuous glucose monitoring uses the position of equilibrium in the reaction
of a glucose-specific receptor and the glucose molecule (glucose + receptor glucose-receptor)
to calculate the concentration of glucose. The more glucose present, the more
receptors will be converted to the glucose complex. The receptor we synthesize
must 1) have a convenient way to measure its binding with glucose and 2) bind
only d-glucose. All other molecules must be ignored by the receptor so that the
measurement is specific for glucose. To meet requirement one, we use the inherently
florescent aryl boronic acid moiety, Ar-B(OH)2, as the part that binds to the
alcohol functionalities on glucose.
Example of how the reactive moiety of
the receptor binds with glucose
Upon capturing glucose, the florescence
of the moiety decreases; thus, by measuring the intensity of florescence, we can
locate the position of the equilibrium. To meet the strict specificity requirement,
we designed a molecule with two aryl boronic acid groups precisely positioned
to makes a good fit with the 1,2 and 4,6 diols on one glucopyranose molecule.
The necessary location of the boronic acid groups was defined for us by vectors
generated from the Computer Assisted Vector Evaluation and Target design (CAVEAT)
program. Any molecule that could align the boronic acid groups to the necessary
vector conformation without excessive strain on the structure was considered a
candidate. In this project, we worked toward the synthesis of potential receptors.
Our most promising synthetic target is the receptor drawn below.
A potential
glucose receptor
One can imagine a small internal medical device reporting
the florescence intensity of a packet of receptor molecules, possibly bound to
a porous polymer bead, continuously exposed to blood with its binding equilibrium
shifting as glucose levels rise and fall. Just as we do not expect automobile
drivers to distract themselves from the road to take a speed measurement, our
research, with the financial support of NIH Grant DK059568, will ease the life
of diabetic patients, so that they do not have to be distracted from their lives
to take glucose measurements.
|
| Luciano
Santillan | Ultrafiltration
Of Oilywastewater By Electrospun Composite Membranes
Luciano Santillan,
Texas Southern University, Benjamin Hsiao, and Kyunghwan Yoon, Department of Chemistry,
Stony Brook University A
growing concern in today's society is the dramatic increase of water pollution.
Large amounts of oily wastewater are generated daily by a variety of industrial
sources which include factories, refineries, and large ships. A 300-Man Navy ship,
for example, can produce as much as 6,000 gallons per day of oily wastewater alone.
Ultrafiltration is increasingly being applied for the treatment of oily waste.
It is a pressure-driven process that uses porous membranes for the separation
of materials in the 1 nm to 50 ?m size range or compounds with molecular masses
over 5000 Daltons. The membrane acts as a selective barrier allowing small molecules
to pass through and rejecting larger macromolecules. The aim of the research is
to produce a highly porous membrane that is able to get higher flux than commercial
membranes with comparable oil rejection percent. The membrane used in this process
is 3-layered made up of polyester, PAN (polyacrylonitrile), and chitosan. The
PAN is electrospun onto the polyester commercial membrane support and later coated
with chitosan. Electrospinning is a process used to produce a small diameter of
fibers by applying electric fields to polymer solution. Chitosan coating is used
for the top layer because chitosan is hydrophilic and has a good film-forming
property. The chitosan layer acts as a hydrophilic surface reducing fouling and
also gives a good mechanical property when coupled to PAN. Various surface densities
of PAN nanofibrous membranes using 2 ~ 12 weight percent PAN solution were made
by electrospinning onto the polyester membrane support. The chitosan layers were
prepared by dip-coating technique using 0.5 ~ 1.7 weight percent chitosan solution.
The filtration experiments were done by a cross-flow filtration apparatus. The
pressure difference between inlet and outlet was used to calculate flux. This
study was supported by a training grant from the Office of Naval Research (N000140310932).
|
| Elizabeth
Sutton | Menaquinone
Biosynthesis as a Drug Target in Mycobacterium tuberculosis
Elizabeth Sutton,
State University of NY at Oneonta, and Jacque Zwahlen
Mycobacterium tuberculosis
(Mtb) infects approximately 2 billion people, killing an estimated 2 million every
year. Novel and more effective drugs are needed in order to stop the increasing
threat of the tuberculosis epidemic. A possible drug target currently being investigated
is the menaquinone biosynthetic pathway. Menaquinone is the sole quinone utilized
in oxidative phosphorylation. This pathway begins with the central metabolite
chorismate. Putative homologues of the pathway were shown to be mandatory for
cell growth (Sassetti et. al). The pathway's initial step involves conversion
of chorismate to isochorismate through the use of an enzyme annotated as MenF.
The Mtb genome was searched for a MenF homologue and two candidates EntC and MbtI
were identified. MbtI is part of the ten gene mycobactin operon, suggesting the
open reading frame (ORF) annotated as EntC is the isochorismate synthase used
in menaquinone biosynthesis. Unsuccessful attempts were made to express EntC in
E. coli. EntC was then cloned into the M. smegmatis acetamide inducible vector.
Once soluble protein is obtained, chorismate analogues shown to inhibit other
chorismate binding enzymes will be tested against Mtb EntC. These substituted
cyclohexanes will serve as a template for drug design.
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