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University of Tennessee, KnoxvilleTrace: Tennessee Research and CreativeExchange
Masters Theses Graduate School
12-2009
Development of a Novel Lysophosphatidic AcidActivity Probe to Identify and Characterize NewProtein TargetsLeah M. CuthriellUniversity of Tennessee - Knoxville
This Thesis is brought to you for free and open access by the Graduate School at Trace: Tennessee Research and Creative Exchange. It has beenaccepted for inclusion in Masters Theses by an authorized administrator of Trace: Tennessee Research and Creative Exchange. For more information,please contact [email protected].
Recommended CitationCuthriell, Leah M., "Development of a Novel Lysophosphatidic Acid Activity Probe to Identify and Characterize New Protein Targets." Master's Thesis, University of Tennessee, 2009.https://trace.tennessee.edu/utk_gradthes/520
To the Graduate Council:
I am submitting herewith a thesis written by Leah M. Cuthriell entitled "Development of a NovelLysophosphatidic Acid Activity Probe to Identify and Characterize New Protein Targets." I haveexamined the final electronic copy of this thesis for form and content and recommend that it be acceptedin partial fulfillment of the requirements for the degree of Master of Science, with a major in Chemistry.
Michael Best, Major Professor
We have read this thesis and recommend its acceptance:
David Baker, Frank Vogt
Accepted for the Council:Carolyn R. Hodges
Vice Provost and Dean of the Graduate School
(Original signatures are on file with official student records.)
To the Graduate Council: I am submitting herewith a thesis written by Leah M. Cuthriell entitled “Development of a Novel Lysophosphatidic Acid Activity Probe to Identify and Characterize New Protein Targets.” I have examined the final electronic copy of this thesis for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Master of Science, with a major in Chemistry. Michael Best Major Professor We have read this thesis and recommend its acceptance: David Baker Frank Vogt Accepted for the Council: Carolyn R. Hodges
Vice Provost and Dean of the Graduate School
(Original signatures are on file with official student records)
Development of a Novel Lysophosphatidic Acid Activity Probe to Identify and Characterize Protein Targets
A Thesis Presented for The Master of Science Degree
University of Tennessee, Knoxville
Leah Cuthriell December 2009
ii
Acknowledgements
First and foremost, I would like to thank God for giving me the ability and
health to complete my undergraduate and graduate career. Secondly, I would
like to thank my parents and sister. Your love and support mean the world to me.
Mom and Dad, without your hard work and sacrifice I would not be where I am
today. I am truly grateful for everything you have done to provide for Delila and
me.
Dr. Best, you are a great advisor. Thank you so much for letting me join
your group and for your patience with me and guidance, especially when
research was not going so well. To the members of the Best group, you guys are
amazing. Thank you so much for your help throughout these two and a half
years. You guys have made graduate school an enjoyable experience. I will
never forget all the fun times, laughs, and frustrations we have had together. I
wish you all the best and will miss you guys.
iii
Abstract
Lipids are categorized according to their biological function. Phospholipids,
glycolipids, and cholesterol are bulk membrane lipids that offer structure and
support to the cells and organelle membranes. Signaling lipids include
diacylglycerol, phosphatidic acid, and lysophosphatidic acid (LPA). These are
low abundance lipids that are active in various pathways. LPA acts through
GPCR receptors to activate G or beta mediated stimulation of phospholipase C
leading to phosphatidylinositol-(4,5)-bisphosphate hydrolysis, Gi mediated
inhibition of adenyl cyclase, and Gi mediated stimulation of the mitogenic Ras-
MAP kinase. To further elucidate the biological activity of LPA and potentially
identify new LPA binding receptors, activity based protein profiling (ABPP) can
be utilized to label and identify enzymes. This thesis discusses the development
of an LPA activity probe which incorporates a benzophenone group that can be
photocrosslinked to a protein via ultraviolet radiation and a secondary azide tag
to facilitate purification of the probe and bound protein.
iv
Table of Contents Chapter Page Chapter 1: Introduction ........................................................................... 1 Background and Significance................................................................................ 4
LPA and Autotaxin Relationship............................................................................ 6
LPA Signaling........................................................................................................ 7
Activity Based Protein Profiling ........................................................................... 12
Chapter 2: Research Design ................................................................ 19
Experimental Procedure......................................................................................26
References ................................................................................................ 60
v
List of Figures
Figure Page Figure 1 General lipid structures 2 Figure 2 Autotaxin production of LPA 5 Figure 3 Illustration of activity based protein profiling 13 Figure 4 Current ABPP probes 15 Figure 5 Fluorophosphonate probes 16 Figure 6 LPA activity 19 Figure 7 Initial head group synthesis 22 Figure 8 Head group synthesis 23 Figure 9 Lipid tail synthesis 24 Figure 10 Lipid synthesis 25
vi
List of Abbreviations ABPP Activity Based Protein Profiling ASPP Active Site Peptide Profiling ATX Autotaxin cAMP Cyclic Adenosine Monophosphate DCC N,N-dicyclohexylcarbodiimide DDQ 2,3-dichloro-5,6-dicyanobenzoquinone EDG Endothelial Gene Differentiation EGF Epidermal Growth Factor ENTH Epsin Amino Terminal Homology FP Fluorophosphonate GPCR G protein-coupled receptors GDP Guanosine Diphosphate GTP Guanosine Triphosphate ICAT Isotope Coded Affinity Tags LC-MS Liquid Chromatography Mass Spectrometry LPA Lysophosphatidic Acid LPC Lysophosphatidylcholine LPP Lipid Phosphate Phosphohydrolases MAPK Mitogen-Activated Protein Kinase Mud-PIT Multidimensional Protein Identification Technology PH Pleckstrin Homology PLA1 Phospholipase A1 PLD Phospholipase D PPAR Peroxisome Proliferator-Activated Receptor sPLA2 Secretory Phospholipase A2 TOP Tandem Orthogonal Proteolysis
1
Chapter 1: Introduction
Lipids compose the cell membrane and help maintain cell structure. This
was discovered by Gorter and Gendell in 1925 when they performed experiments
with chromocytes and found the cells were surrounded by a lipid layer two
molecules thick.1 For over 50 years, the biological roles of lipids were thought to
be limited to composing membrane bilayers and acting as intermediates in lipid
biosynthesis.2 However, it was later discovered that lipids play more extensive
roles in physiological function than originally thought. It is now known that lipids
play key roles in signal transduction and are intermediates in numerous important
biological pathways.
Diacylglycerol is the first known lipid second messenger. Its role as a
second messenger was proved by Hokin and Hokin in 1950 when they
discovered that acetyl or carbamylcholine caused an early turnover of inositol
phospholipids.2 This discovery, along with Nishizuka uncovering that membrane
lipids activate cyclic nucleotide-independent kinase activity, and the revelation
that negatively charged phospholipids replaced membrane components as
kinase activators, indicated that lipids are more involved in eukaryotic cell
biochemistry than previously thought.2-3 Furthermore, lipids are involved in
organelle biogenesis and can regulate intracellular protein dynamics by binding
protein domains. General lipid structures are given in Figure 1.
2
.
Lipids can be broken down into two main categories: bulk membrane lipids
and signaling lipids. Three major types of membrane lipids are phospholipids,
glycolipids, and cholesterol. Examples of phospholipids are phosphatidylcholine,
phosphatidylinositol, phosphatidylserine, and phosphatidylethanolamine. These
lipids, along with cholesterol, compose the bulk of the cell membrane. Low
abundance signaling lipids such as phosphatidic acid, diacylglycerol, the
phosphatidylinositol polyphosphates, and lysophosphatidic acid function as
signaling lipids that regulate biological processes.4 A key role of these lipids is to
recruit peripheral proteins to the cell membrane in a spatiotemporally specific
manner.5
Membranes of cellular components are not uniformly composed of lipids.
Lipid type and concentration are dependent on local lipid metabolism and
regulated lipid transport.5a For example, plasma membranes, endosomes, and
trans-Golgi network membranes maintain transbilayer asymmetry by actively
transporting particular types of lipids from one opposing leaflet to another.5a
Originally, it was thought that specific localization of peripheral proteins would
3
require protein–protein interactions because protein–lipid interactions were not
expected to be sufficiently specific.5a However, this idea was refuted when it was
determined that subcellular localization behaviors of peripheral proteins can best
be elucidated by analyzing the kinetics and energetics of their binding to model
membranes, as long as the membranes mimic the cellular membrane to which it
is targeted.5a
Peripheral proteins are composed of one or more modular domains
specialized in lipid binding. These include pleckstrin homology (PH), epsin
amino terminal homology (ENTH), and Tubby domains along with many others.6
Some peripheral proteins, such as phospholipase A2, do not contain separate
protein binding domains, but instead utilize their own molecular surface or an
amphipathic secondary structure.5a Other proteins are post-translationally
modified through the attachment of lipid anchors that embed them in the lipid
bilayer. A prominent example involves the Ras GTPases, which have been
implicated in cancer. Initial protein–lipid binding is mediated by electrostatic
interactions between anionic lipid surfaces and cationic protein surfaces.5a In
addition to the role of recruiting proteins to the plasma membrane, lipids such as
lysophospholipids act as intercellular mediators. Intercellular lipid mediators act
via membrane receptors or nuclear receptors known as corticosteroids in order to
effect biological changes.7
4
Background and Significance
The initial indication that lysophosphatidic acid 6a plays a more extensive
role than just being an intermediate in intracellular lipid biosynthesis came when
it was found to induce smooth muscle contraction.8 LPA research began in the
1990s with the discovery that LPA is mitogenic and signals through various G
protein-coupled receptors (GPCR).9 LPA is now known to act as an extracellular
lipid mediator that produces growth factor-like responses in a wide range of cells.
LPA is a lysolipid. Lysolipids possess the distinguishing characteristic in
that they only contain one acyl chain located at either the sn-1 or sn-2 position.
LPA is composed of a single acyl chain located at the sn-1 position, a glycerol
backbone, and a phosphate headgroup (Figure 2). It is the smallest and
structurally simplest glycerophospholipid.10 For a long time, the origins of
extracellular LPA production were unclear until it was determined that autotaxin
(ATX) and phospholipase D (PLD) were the same enzyme that catalyzed LPA
production.10-11 Autotaxin (ATX) was discovered in melanoma cell culture and
was found to increase melanoma cell motility.12 PLD and ATX cleave the choline
group from lysophosphatidylcholine (LPC), producing LPA (Figure 2).
LPA is produced by several cell types including mature neuron cells,
Schwann cells, adipocytes, and fibroblasts.13 Production of the lysolipid by these
cells is the source of LPA found in serum, saliva, and follicular fluid.9 In addition,
LPA is also produced in ovarian cancer cells, glioblastomas, and melanoma
cells, indicating that this lipid may play a role in tumor survival and metastasis.
5
LPA is synthesized in the aforementioned cell lines by two methods: the removal
of a fatty acid acyl chain from phosphatidic acid by phospholipase A1 (PLA1) or
phospholipase A2, or the removal of a choline group from phosphatidylcholine,
as shown in Figure 2.8
In phospholipase A1 production of LPA, PLA1 releases the fatty acid chain
from the sn-1 position of membrane phospholipids, producing sn-2 and
polyunsaturated isoforms of LPA that interact with LPA receptor 3.14 Type II
secretory phospholipase A2 (sPLA2) removes an acyl chain from the sn-2
position but is not capable of producing hydrolyzed lipids in intact cell
membranes, resulting in low levels of LPA in the plasma. However, sPLA2 is
capable of selectively hydrolyzing lipids in damaged cells and microvesicles that
are released during apoptosis and by cancer cells, and are found in large
numbers in malignant fluids leading to high levels of LPA expressed in cancer
cells.8
6
LPA and Autotaxin Relationship
The protein autotaxin (ATX) was first discovered in melanoma cell culture
and has been found to stimulate cancer cell motility, angiogenesis, and cell
proliferation, indicating that it may be pivotal in cancer biology.15 It is a member
of the nucleotide pyrophosphatase/phosphodiesterase family of enzymes.12a
ATX is a type II integral membrane protein that contains a single transmembrane
domain, two somatomedin B-like domains, and a catalytic domain.16 The highest
levels of ATX/PLD mRNA are expressed in kidney, brain, lung, intestine, and
ovary cells.
ATX production of LPA is of biological importance because it plays key
roles in various pathways. ATX utilizes LPC as a substrate, which exists at high
concentrations in many bodily fluids. The resulting product, LPA, stimulates
cellular responses such as malignant cell proliferation and LPA autocrine control
of adipose tissue. The fact that autotaxin stimulation in malignant cell
proliferation is increased in the presence of LPC provides evidence that LPA is a
signaling component in cell proliferation. The proposed mechanism of cancer
cell motility involves activation through GPCR LPA1.12a In addition to the role it
plays in cell proliferation, ATX-induced LPA production also results in the
differentiation of adipocytes and adipocyte cell lines. ATX has been shown to be
upregulated in genetically obese mice. These two facts, along with studies
showing that LPA exists in extracellular fluid of adipose cells and is released in
7
vitro by adipocytes, imply that LPA paracrine control of adipose tissue could be
mediated by ATX.17
LPA signaling
LPA activity is receptor-mediated with the lipid interacting with at least
three G protein-coupled receptors that belong to the endothelial differentiation
gene subfamily, which traverses the membrane seven times.9 The first receptor
to be discovered was LPA1, which is the most widely expressed LPA receptor
with high mRNA levels in the colon, intestine, pancreas, small intestine, and
heart.8-9 High levels of this receptor are also present in the cerebral cortical
ventricular zone during neurogenesis and in adult oligodendrocytes and
Schwann cells.8
After its discovery, two other LPA receptors known as LPA2 and LPA3
were identified. LPA2 and LPA3 are not as widely distributed as LPA1 but are
overexpressed in cancer cells, indicating their role in the disease. Recently,
three other LPA receptors were identified.18 One receptor known as LPA4 is
unique in the fact that it does not belong to the EDG family, but is most similar to
the purinergic family, which means LPA may have a greater biochemical role
than previously thought.9 While there is not enough evidence to support LPA4
activation of phopholipase C and adenylyl cyclase, circumstantial evidence
suggests that the receptor may tentatively mediate the activity of these enzymes.
LPA receptors are known to couple to three subfamilies of G proteins
known as Gα, Gi, and G12/13.9 These three major receptors share high homology
8
in amino acid sequences in all areas except for the C-terminal region.9 The
extent of carboxyl tail homology between the LPA1 and LPA2 receptors is 27%
with the LPA2 and LPA3 receptors having only 17% homology.19 Since all other
domains have the same homology, the ability of the receptors to effect biological
change must stem from the different amino acid sequences located in the
carboxyl terminal region.19-20 Evidence to prove the specificity of the carboxyl
terminal region has been found from studies published by Xu et al., in which a
yeast two-hybrid screen was utilized to identify TRIP6 as a LPA2 receptor
interacting protein.19
Major signaling routes for LPA include: G or beta mediated stimulation of
phospholipase C leading to phosphatidylinositol-(4,5)-bisphosphate hydrolysis, Gi
mediated inhibition of adenyl cyclase, Gi mediated stimulation of the mitogenic
Ras-MAP kinase, Gα and G12/13 mediated activation of small GTPase RhoA,
which regulates cytoskeletal movement, and finally Gi activation of
phosphoinositide-3-kinase, Rac, and PKB/Akt to promote cell movement and
suppress cell death.9 LPA also signals through second messenger pathways
and activates the Ras and Rho family of GTPases.9 The Ras and Rho GTPase
cycles include GDP and GTP bound states. GTP binding is promoted by specific
GDP–GTP exchange factors in which GTP bound forms can react with various
downstream effectors producing changes in the cellular environment.8
All mammalian cells, tissues, and organs with the exception of the liver
express LPA receptor subtypes of the EDG family, which indicates that LPA
9
signaling occurs in a cooperative manner. However, the identity of the LPA
receptor subtype that couples each known G protein-coupled receptor is
unknown. All three receptors can mediate PLC activation, inhibition of cyclic
adenosine monophosphate (cAMP) accumulation, and activation of mitogen-
activated protein kinase (MAPK) pathway, but different isoforms of LPA have a
different efficacy and potency for each receptor, thus triggering downstream
events. LPA2 exhibits the highest affinity of all the receptors for LPA and
couples more efficiently to neovascularizing factors with LPA1 having a bigger
effect on cell motility events.8
Ras MAP Kinase Signaling
LPA-induced activation of Ras involves the GES, SOS, and intermediate
protein tyrosine kinase dependent activity. According to studies, Ras activation
is not a direct linear pathway such as Gi-tyrosine kinase-Ras-GTP, but instead it
involves exchange factors and cell-type dependent, mutually opposing, GTPase
activating proteins.9 One proposed method of signaling suggests that LPA and
other GPCR agonists act through the epidermal growth factor receptor to trigger
mitogenic signaling. In this transactivation model, a GPCR activated
metalloprotease cleaves the epidermal growth factor (EGF) precursor at the cell
surface to stimulate EGFR signaling. However, the transactivation model is not
widely accepted since most of the studies were conducted with pharmacological
inhibitors.9
10
Rho/Rac signaling
Cytoskeletal organization is mediated through Rho GTPases. Three well
studied GTPases are RhoA, Cdc42, and Rac. LPA activation of RhoA proceeds
via G12/13 subunits that bind to three distinct Rho-specific guanine nucleotide
exchange factors, promoting RhoA-GTP accumulation and activation of
downstream Rho-Kinase, which initiates cell rounding, retraction, and endothelial
tight junction opening.9
LPA as an intracellular messenger
It is currently well known that LPA is an intermediate in phospholipid
biosynthesis, which takes place in the endoplasmic reticulum. During this
process, LPA is formed from glycerol-3-phosphate by acyl-CoA and then further
acylated to phosphatidic acid. Evidence may suggest that LPA has another role
in the body besides just being an intermediate in lipid biosynthesis. High
concentrations of LPA can compete with synthetic lipid found within the cell to
bind to the nuclear transcription factor peroxisome proliferator-activated receptor
gamma (PRAR-γ). However, it is unclear how extracellular LPA could cross the
membrane, since it would be rapidly dephosphorylated by membrane-bound lipid
phosphatases. Furthermore, if extracellular LPA found its way to the cytosol, it is
still unclear how the lipid would be able to reach the nucleus. Thus, new studies
need to be conducted in order to determine whether LPA acts as an intracellular
messenger.9
11
LPA Degradation
Logically, LPA must be degraded in mammalian cells in order to maintain
cell functionality. Extracellular LPA is degraded by phosphate group removal to
produce inactive monoacylglycerol.9 In addition, a family of membrane ecto
enzymes has been identified whose members mediate dephosphorylation of LPA
and other lipid phosphatases. This group of enzymes is known as lipid
phosphate phosphohydrolases (LPP). LPPs belong to a superfamily of
hydrolases that includes bacterial acid phosphatases and DGPPase that contain
three highly conserved domains. Unlike phospholipase C, LPP does not
dephosphorylate water-soluble phosphate esters and appears to have a
substrate similar to phosphatidic acid, indicating that it competes with
phosphatidic acid (PA) for LPA dephosphorylation. However, more studies are
needed in order to confirm their ability to degrade lysophosphatidic acid.
Activity Based Protein Profiling
As evidenced by the information obtained from the text above, LPA has
extensive biological and pathophysiological roles. Much remains to be learned
about the pathways in which LPA is active and the proteins that it targets. With
the advent of genome sequencing, an array of information about the proteins
present in eukaryotic organisms has been obtained. Genomic techniques such
as chromosomal translocation, transcriptional profiling, and RNA interference–
gene silencing have given insights into the physiological roles of proteins.21
12
However, genomic techniques cannot convey other important information
about protein function, including regulation caused by small molecule binding and
post-translational protein modification. To efficiently elucidate protein function at
this level, a range of sophisticated proteomics have been developed. Within the
field of proteomics, basic techniques such as gel electrophoresis and mass
spectrometry have been extended to devise powerful techniques such as
multidimensional protein identification technology (MudPIT) and isotope coded
affinity tags (ICAT), yeast two–hybrid systems, and protein microarrays that
generate more information about quantitative differences in protein abundance
and protein interactions.22 However, these methods are not able to probe
proteins in vivo, which is disadvantageous because variation in activity caused by
small molecule binding and post-translational modification cannot be detected.22b
Probes have been developed that specifically target enzyme families. In addition
to binding already characterized enzyme classes, ABPP probes used in
conjunction with streptavidin chromatography, gel separation, and mass
spectrometry analysis can identify and assign biological function to novel
proteins.22a
The strategy of activity based protein profiling (ABPP) was developed in
order to characterize proteins based on activity rather than abundance. ABPP is
a chemical proteomic method that utilizes active site-directed probes to visualize
changes in protein activity located in cells and tissues. Although recent
advances in genomic sequencing have brought more attention to the utility of
13
ABPP, the first stages of ABPP began over a century ago.21 ABPP is ideal for
targeting an array of enzyme families since probes can be constructed that
incorporate mechanism-based inhibitors, protein-reactive natural products, and
general electrophiles.21 Currently, probes have been developed for more than a
dozen enzyme classes, including proteases, kinases, and phosphatases.
General probe design incorporates a reactive group by which the probe is
selectively cross-linked to targets, as well as a secondary tag bound by a linker.
Common reporter tags are rhodamine, azide, alkyne, and biotin. An illustration of
activity based protein profiling is given in Figure 3.
14
The principal aim of ABPP is the synthesis of small probes that can
covalently label enzyme active sites. Ideally, probes must be able to target a
wide array of enzymes, but since numerous nucleophilic residues without
catalytic activity exist within the proteome, cross-reactivity with such residues
must be avoided in order to prevent jeopardizing ABPP studies.23 This can be
achieved by combining reactive and binding groups that target conserved
mechanistic or structural features in enzyme active sites.23 Reactive groups can
be either electrophilic and modify active sites or photoaffinity groups that label
targets upon UV irradiation.23
Once the probe has labeled target proteins via reaction with active site
residues, purification of the successfully labeled proteins is facilitated by the
secondary reporter tag on the probe. Reporter tags such as alkynes and azides
can undergo click reactions that allow the probe to be biotinylated and then
purified via a streptavidin column. Examples of probes that have been currently
utilized in activity based protein profiling are given in Figure 4. In addition to the
probes drawn in Figure 4, Cravatt and colleagues have designed a series of
probes that target the serine hydrolases. These probes, termed
fluorophosphonate reagents (shown in Figure 5) have been derivatized with a
number of linkers and a reporter tag such as rhodamine or biotin in order to
visualize and characterize catalytically active serine hydrolases in proteomes.22b
These probes have shown great selectivity for labeling active serine hydrolases
but not the inactive inhibitor bound forms.
15
O O
OO
N3
O
N3
PO
OO
N
O
Bifunctional lipid probe
Benzophenone photoaffinity tag
Azide secondary tag
16
S
HNNH
O
OHN
O
HN
ONH O
N
O OH
ONH
PO
OO
OHO
Arylphosphonate probe
O OO
N
NO
HN
HN
OP
O
FO
Fluorophosphonate probe
Rhodamine tag
The great reactivity of these probes stems from an electrophilic center that
is primed for covalent reaction with a highly nucleophilic oxygen atom of serine
and the near-tetrahedral structure of the fluorophosphonate (FP) groups, which
mimic the enzyme substrate tetrahedral intermediate. Craik and Mahrus
synthesized a series of probes with complementary reactivity to the
fluorophosphonate probes.22b These probes incorporate a diphenyl phosphonate
electrophile and substrate mimicking recognition groups that promote selective
recognition.
Once probes have been synthesized, they can be utilized to study protein
interactions through a variety of different methods. One platform that exists is gel
electrophoresis. This method incorporates the use of fluorescent probes in
conjunction with biotinylated probes. Fluorescent probe labeled proteomes are
distinguished by 1D or 2D polyacrylamide gel electrophoresis. Then, biotinylated
probes can be used along with streptavidin affinity chromatography, gel
separation, and mass spectrometry in order to purify the labeled probes. Another
17
method that offers higher resolution than gel electrophoresis is liquid
chromatography–mass spectrometry (LC–MS).23
LC–MS can be divided into two categories. The first category consists of
experiments that analyze protein targets of probes. The second deals with those
that specifically analyze probe-labeled peptides derived from these targets. The
first method is essentially a combination of ABPP and MudPIT. The procedure
involves incubating biotinylated probes with streptavidin beads to enrich probe
labeled proteins. Incubation is then followed by trypsin digestion and the
enriched proteins are analyzed by LC–MS. This process is capable of detecting
50–100+ enzyme activities but does not offer a straightforward way of identifying
probe labeled peptides of enzyme targets. To overcome this disadvantage,
active site peptide profiling (ASPP) can be used. ASPP involves digesting probe
treated proteomes with trypsin prior to incubation. Probe labeled peptides are
enriched using either streptavidin or antibody resins followed by analysis with
LC–MS.21
In order to obtain the information that ABPP and ASPP offers, Speers and
Cravatt developed tandem orthogonal proteolysis–ABPP (TOP–ABPP).21 Even
though advancements in resolution and information gleaned from ABPP have
occurred, methods that involve LC–MS are still not desirable due to the large
sample volume that is required in order to run an analysis. Ideally, an ABPP
procedure will eventually be produced that offers the high resolution of LC–MS
along with the minimal sample requirement of gel electrophoresis. Progress has
18
been made towards this goal by combining capillary electrophoresis coupled with
laser-induced fluorescence. However, more work needs to be done in order to
achieve a method which produces high resolution with minimal sample
requirement.21
ABPP is currently utilized in biological experiments such as target
discovery, inhibitor discovery, and enzyme active site characterization. ABPP is
a suitable method for target discovery because it can account for protein
regulation via small molecule binding and post-translational modification.
Inhibitor discovery involves utilizing the probe as a competitor in which inhibitors
are then identified by their ability to block probe labeling of enzymes. This
strategy negates the need for recombinant expression and purification because
enzyme tests can be conducted within the proteome. In addition, the probe acts
as a substitute for substrate analysis resulting in the ability to detect enzymes
that may not be detectable via other methods. Finally, with the ability to label
new enzyme active sites, ABPP is an avenue to characterize new enzymes that
have not yet been documented.21
19
Chapter 2: Research Design
The main focus of my research has been the development of the LPA
activity probe shown in Figure 6. The synthetic route chosen for production of
the activity probe involved the incorporation of a benzophenone group and an
azide tag. Proteins that bind the LPA backbone can be cross-linked to the probe
through irradiation of the photoaffinity group with ultra-violet light. The azide can
then be used to label cross-linked adducts via click chemistry. The probe, along
with bound proteins, can then be purified via a streptavidin column. The activity
probe 23 in Figure 6 was synthesized from (S)-glycerolacetonide 1 in 16 steps.
HO OO
O
NHO
HNO
O
N3
POOH
OH
Benzophenone group
Azide tag
23
Figure 6. LPA activity probe.
20
The first synthetic route (shown in Figure 7) to the target compound began
by protecting the free alcohol of (S)-glycerolacetonide with tert-butyldiphenylsilyl
chloride to produce 2 (Figure 7). After acetal deprotection with 90% acetic acid
to 3, a monomethoxytrityl protecting group was selectively installed on the
primary hydroxyl group to yield 4. This was followed by incorporation of a p-
methoxybenzyl chloride protecting group to generate 5. However, 6 was not
obtained in high yield, and reproducibility was problematic. A better synthetic
route (shown in Figure 8) was then developed in which the order of protecting
group installation was switched. The first step of the synthesis involved
protecting the free alcohol of 1 with a p-methoxybenzyl protecting group, followed
by acetal deprotection to yield 9. After this step, the primary alcohol of 9 was
selectively protected by monomethoxytrityl chloride (MMT) followed by
incorporation of a tert-butyldiphenylsilyl protecting group to yield 11. Finally, the
MMT group was deprotected producing the free alcohol 12.
Incorporation of the benzophenone cross-linking group and azide tag
(shown in Figure 9) began with the coupling of 4-benzoylbenzoic acid to Z-N-
Boc-L-lysine methyl ester to produce 14. Trifluoroacetic acid deprotection yielded
the free amine 15, which was converted into azide 16 using 0.01 M copper
sulfate solution, 0.804 M potassium carbonate solution and imidazole-1-sulfonyl
azide. Once the azide was purified, the methyl ester was hydrolyzed using 2 M
sodium hydroxide and methanol to yield 17. Coupling of the acid to methyl 6-
aminohexanoate yielded 19 upon ester hydrolysis with 2 M sodium hydroxide
21
and ethanol (Figure 10). Following this reaction, 19 and 12 underwent a coupling
reaction to produce 20. After purification, the benzyl-protected phosphoramidite
was installed following p-methoxybenzyl deprotection with DDQ to yield 22. The
free phosphate was then produced with trimethylsilyl bromide. Finally, the lipid
intermediate was deprotected with tetrabutylammonium fluoride to produce the
target LPA probe 23.
Conclusion
The best synthethic route to produce a novel LPA activity probe involved
protecting the free alcohol of (S)-glycerolacetonide with p-methoxybenzyl
chloride. After acetal deprotection, MMT and TBDPS groups were selectively
installed. This was necessary in order to produce 12 with a free alcohol at the
sn-1 position. The next synthetic strategy involved producing an acyl chain
which incorporated a benzophenone cross-linking group and an azide label that
would facilitate purification of the probe and bound protein. Synthesis of the
derivatized acyl chain began with a lysine analog and was achieved in seven
steps yielding 19. Next, 12 and 19 were coupled together to produce 20. After
installing a dibenzyl protected phospharamidite, 23 was achieved in 61% yield
following TMSBr and TBAF deprotection to produce an LPA activity probe that
can be utilized to identify and characterize new protein targets.
22
23
OMMTHO
PMBOTBDPSClimidazole
N
OMMTTBDPSO
PMBO
OHTBDPSO
PMBOCSA
MeOH,CH2Cl210 11 12
99% 31%
24
OHN
O
N3
O
O
N N SO
ON3
CuSO4, K2CO3MeOH
1675% over two steps
NaOHMeOH
HOHN
O
N3
O
O
17
99%
25
TBDPSO OO
O
NHO
HNO
O
N3
NH
HN
O
N3
O
O
DCC, DMAP
CH2Cl2TBDPSO OH
OO
12 19
20
O
HO
99%
Figure 10. Lipid Synthesis
NH
HN
O
N3
O
O
O
OH2N
DMAP, EDCl,NMM, CH2Cl2
18
O
O
88%
O
NaOHEtOH
19
NH
HN
O
N3
O
O
HO
O
93%
26
27
TBDPSO OO
HO
NHO
HNO
O
N3
TBDPSO OO
O
NHO
HNO
O
N3
POO
OPO
ON
m-CPBACH2Cl2
21 22
Figure 10. Lipid synthesis continued.
50%
28
TBDPSO OO
O
NHO
HNO
O
N3
POO
O HO OO
O
NHO
HNO
O
N3
POOH
OH1. TMSBrCH2Cl2
2. TBAFTHF
22 23
Figure 10. Lipid synthesis continued.
61%
29
Experimental Procedures
General. All reagents were purchased from Aldrich, Acros, or ChemImpex and
used as received. Dry solvents were obtained from a Pure Solv solvent delivery
system purchased from Innovative Technology, Inc. Column chromatography
was performed using 230–400 mesh silica gel purchased from Sorbent
Technologies. NMR spectra were obtained using a Varian Mercury 300
spectrometer and a Bruker Avance 400 spectrometer. Mass spectra were
obtained with a MALDI-TOF spectrometer. Optical rotation values were obtained
using a Perkin-Elmer 241 polarimeter.
(S)-4-((4-methoxybenzyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (8)
Sodium hydride (518 mg, 12.1 mmol) was dissolved in 40 mL N,N-
dimethylformamide at 0 °C under nitrogen. Compound 7 (1 mL, 8.1 mmol) was
dissolved in 40 mL N,N-dimethylformamide and added slowly over 10 minutes to
the sodium hydride solution. After complete addition of 7, the reaction was
stirred at 0 °C for 1 h, then p-methoxybenzyl chloride (1.64 mL, 12.1 mmol) was
added. The mixture was stirred for 18 h at room temperature. After the reaction
was complete, water (50 mL) was added to quench the reaction. The mixture
was then extracted with dichloromethane and saturated sodium chloride (2 x 50
mL). The organic phase was then dried with magnesium sulfate, filtered, and
30
then concentrated. Column chromatography on silica gel and a gradient solvent
system of 10–50% ethyl acetate/hexanes afforded 8 as a clear oil (1.51 g, 74%).
NMR data matched that in the literature.24
(R)-3-(4-methoxybenzyloxy)propane-1,2-diol (9)
Compound 8 (1.51 g, 5.9 mmol) was dissolved in methanol (59 mL), and p-
toluenesulfonic acid (181 mg, 0.95 mmol) was added to the solution. The
reaction was stirred at room temperature for 24 h. The reaction was quenched
with sodium bicarbonate (110 mg, 1.3 mmol) and extracted with water and
dichloromethane (2 x 50 mL). The organic phase was dried with magnesium
sulfate, filtered, and concentrated. Column chromatography on silica gel and a
gradient solvent system of 50% methanol/ethyl acetate and 100% methanol
produced 9 (661 mg, 52%) as a clear oil.
NMR data matched that in the literature.24
(S)-1-((4-methoxybenzyl)oxy)-3-((4-methoxyphenyl)diphenylmethoxy)
propan-2-ol (10)
Diol 9 (661 mg, 3.1 mmol) was dissolved in pyridine (31 mL) under nitrogen. 4-
methoxytrityl chloride (1.91 g, 6.2 mmol), 4-dimethylaminopyridine (381 mg, 0.31
mmol), and molecular sieves were added. The reaction was stirred at room
31
temperature for 24 h. After the reaction was complete, the mixture was filtered
over Celite to remove the molecular sieves and concentrated. Column
chromatography on silica gel and a gradient solvent system of 10–50% ethyl
acetate/hexanes and 100% ethyl acetate afforded 10 as a clear oil (1.33 g, 88%).
NMR data matched that in the literature.24b
(S)-tert-butyl((1-((4-methoxybenzyl)oxy)-3-((4-
methoxyphenyl)diphenylmethoxy)propan-2-yl)oxy)diphenylsilane (11)
Alcohol 10 (408 mg, 0.84 mmol) was dissolved in pyridine (8 mL) under nitrogen.
To the mixture was added imidazole (114 mg, 1.7 mmol), t-butyldiphenyl
chlorosilane (437 µL, 1.7 mmol), and molecular sieves. The reaction was stirred
at room temperature for 18 h. The mixture was then filtered over celite to remove
the molecular sieves and concentrated. Chromatography on silica gel with a
gradient solvent system of 10–25% ethyl acetate/hexanes afforded 11 (600 mg,
99%). NMR data matched that in the literature.24b
32
(R)-2-((tert-butyldiphenylsilyl)oxy)-3-((4-methoxybenzyl)oxy)propan-1-ol
(12)
Compound 11 (599 mg, 0.83 mmol) was dissolved in methanol:dichloromethane
(33 mL 2:1). Camphorsulfonic acid (96 mg, 0.414 mmol) was added. The
reaction was stirred at room temperature for 3.5 h after which it was neutralized
by triethylamine and concentrated. Chromatography on silica gel with a gradient
solvent system of 25–35% ethyl acetate/hexanes afforded 12 (113 mg, 31%).
NMR data matched literature.24b
(S)-methyl2-(4-benzoylbenzamido)-6-((tert-
butoxycarbonyl)amino)hexanoate (14)
Compound 13 (400 mg, 1.0 mmol) was dissolved in methanol (11 mL), and 10%
palladium hydroxide (40 mg) by weight was added. The mixture was placed
under hydrogen and stirred for 24 h at room temperature. After the 24-h period,
the reaction was filtered over Celite. The filtrate was concentrated, and the
remaining traces of solvent were removed. The residue was then dissolved in
33
N,N-dimethylformamide (6.7 mL) and placed under nitrogen. N-
methylmorpholine (390 µL, 3.5 mmol), along with 4-benzylbenzoic acid (229 mg,
1.1 mmol), EDCl (233 mg, 1.2 mmol), and 4-dimethylaminopyridine (149 mg, 1.2
mmol) was added. The reaction was stirred for 5 h at room temperature. After
completion, the reaction was concentrated. Column chromatography on silica
gel with a solvent gradient system of 50% ethyl acetate/hexanes afforded 14
(351 mg, 74%). NMR data matched that in the literature.25
OHN
O
NHO
O
O
OHN
O
N3
O
O
N N SO
ON3
CuSO4, K2CO3MeOHO
2.
1. TFACH2Cl2
(S)-methyl 6-amino-2-(4-benzoylbenzamido)hexanoate (16)
Compound 14 (343 mg, 0.71 mmol) was dissolved in dichloromethane (5.9 mL).
Trifluoroacetic acid (5.9 mL) was added, and the solution was stirred at room
temperature for 2.5 h. After completion, the solvent was removed, and the
residue (15) was placed under vacuum for 24 h. The residue was then dissolved
in methanol (7.5 mL), and imidazole-1-sulfonyl azide hydrochloride (177 mg, 0.8
mmol), 0.804 M potassium carbonate solution (1.4 mL, 1.2 mmol), and 0.01 M
copper(II) sulfate pentahydrate solution (707 µL, 0.007 mmol) were added. The
reaction was stirred at room temperature for 3.5 h. After completion, the reaction
34
was extracted with ethyl acetate and 2 M hydrochloric acid. The organic phase
was dried with magnesium sulfate and concentrated. Column chromatography
on silica gel with a solvent gradient of 50% ethyl acetate/hexanes afforded 16
(208 mg, 75%). NMR data matched that in the literature.25
(S)-6-azido-2-(4-benzoylbenzamido)hexanoic acid (17)
Compound 16 (276 mg, 0.7 mmol) was dissolved in methanol (12.3 mL), and 2 M
sodium hydroxide (12.3 mL) was added. The reaction was stirred at room
temperature for 1 h. After completion, the reaction was extracted with 2 M
hydrochloric acid. The organic layer was dried with magnesium sulfate and
concentrated to yield 17 (266 mg, 99%). The residue was passed on without
further purification. NMR data matched that in the literature.25
(R)-methyl 6-(6-azido-2-(4-benzoylbenzamido)hexanamido)hexanoate (18)
Compound 17 (239 mg, 0.628 mmol) was dissolved in dichloromethane (11.4
mL), placed under nitrogen, and 4-dimethylaminopyridine (99 mg, 0.817 mmol)
35
was added. Methyl 6-aminohexanoate hydrochloride (148 mg, 0.817 mmol) was
dissolved in dichloromethane (11.4 mL) and N-methylmorpholine (691 mL, 6.28
mmol) was added. The solution was combined with 17 dissolved in
dichloromethane, and EDCl (156 mg, 0.817 mmol) was added. The reaction was
stirred at room temperature for 18 h. Column chromatography on silica gel with a
solvent gradient of 35–75% ethyl acetate/hexanes afforded 18 (273 mg, 88%).
1H NMR (300 MHz, CDCl3 ) δ 1.37 (dd, J = 14.94, 7.91 Hz, 2H), 1.61 (m, 8H),
1.83 (d, J = 7.32 Hz, 1H), 2.0 (m, 1H), 2.30 (t, J = 7.32 Hz, 2H), 3.28 (m, 4H),
3.65 (s, 3H), 4.68 (q, J = 7.04 Hz, 1H), 6.62 (m, 1H), 7.50 (t, J = 7.46 Hz, 2H),
7.62 (t, J = 7.40 Hz, 1H), 7.78 (d, J=8.30 Hz, 2H), 7.83 (d, J=8.12 Hz, 2H), 7.94
(dd, J = 8.17 Hz, 2H); 13C NMR (300 MHz CDCl3) δ 191.58, 170.86, 168.26,
166.05, 166.01, 136.55, 132.54, 129.71, 129.67, 128.35, 128.07, 126.73, 53.11,
51.14, 50.71, 38.96, 33.34, 31.88, 28.58, 28.55, 28.16, 25.84, 25.82, 23.91,
22.37; MALDI-HRMS [M + Na]+ calcd: 530.2374 found: 530.2394; [α]D296K =
+0.21 (c 6.5, 1:4 methanol/chloroform)
(R)-6-(6-azido-2-(4-benzoylbenzamido)hexanamido)hexanoic acid (19)
Compound 18 (303 mg, 0.598 mmol) was dissolved in ethanol (3 mL) and 2 M
sodium hydroxide (3 mL) was added. The solution was stirred at room
36
temperature for 1 h. Once the reaction was complete, the solution was extracted
with dichloromethane and 2 M hydrochloric acid (2 x 30 mL). The organic phase
was dried with magnesium sulfate and concentrated to yield 19 (272 mg, 93%).
The residue was used in the next reaction.
Compound 12 (320 mg, 0.6 mmol) was dissolved in dichloromethane (11 mL)
under nitrogen. N,N-dicyclohexylcarbodiimide (173 mg, 0.84 mmol) and 4-
dimethylaminopyridine (103 mg, 0.84 mmol) were added. The mixture was
stirred at room temperature for 20 minutes. Compound 19 (396 mg, 0.84 mmol)
was then added in 10 mL dichloromethane, and the reaction was stirred at room
temperature for 24 hours. Once the reaction was complete, the mixture was
concentrated. Column chromatography on silica gel with a solvent gradient
system of 35–75% ethyl acetate/hexanes afforded 20 as a clear oil (596 mg,
99%).
1H NMR (300 MHz, CDCl3) δ 1.04 (s, 9H), 1.28 (m, 2H), 1.51 (m, 8H), 1.94 (m,
2H), 2.11 (m, 2H), 3.26 (m, 4H), 3.40 (d, J = 4.86 Hz, 2H), 3.79 (s, 3H), 4.11 (m,
3H) 4.69 (s,2H), 6.74 (s, 1H), 6.82 (d, J = 8.30 Hz, 2H), 7.11 (d, J = 8.35 Hz, 2H),
37
7.40 (m, 6H), 7.50 (d, J=7.90 Hz, 2H), 7.59 (d, J=7.61 Hz, 1H), 7.65 (t, J = 7.68
Hz, 4H), 7.78 (d, J =8.30 Hz, 2H), 7.83 (d, J=8.12 Hz, 2H), 7.94 (d, J = 8.17 Hz,
2H); 13C NMR (300 MHz CDCl3) δ 195.80, 173.25, 171.18, 168.59, 166.37,
146.02, 140.31, 136.84, 135.80, 135.78, 133.61, 132.87, 130.06, 129.61, 129.15,
127.63, 127.51, 127.47, 127.09, 113.62, 72.80, 70.86, 70.19, 66.06, 55.21,
53.45, 51.04, 39.38, 33.67, 32.28, 29.00, 28.51, 26.80, 26.24, 24.16, 22.71,
19.25; MALDI-HRMS [M + Na]+ calcd: 948.4338 found: 948.4330; [α]296KD =
+0.041 (c 1.9, 1:4 methanol/chloroform)
TBDPSO OO
OO
NHO
HNO
O
N3
TBDPSO OO
HO
NHO
HNO
O
N3
DDQCH2Cl2
21
Compound 20 (147 mg, 0.159mmol) was dissolved in dichloromethane (5 mL)
and 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) (90 mg, 0.398 mmol) was
added. Following completion (4.5 h), the reaction was extracted with 10%
sodium carbonate and dichloromethane (2 x 30 mL). The organic phase was
38
dried with magnesium sulfate and concentrated. Column chromatography on
silica gel with a solvent gradient of 40–75% ethyl acetate/hexanes and 100%
ethyl acetate/hexanes afforded 21 as a clear oil (91.7 mg, 78%).
1H NMR (300 MHz, CDCl3) δ 1.07 (s, 9H), 1.29 (m, 2H), 1.52 (m, 8H), 1.78 (m,
1H), 1.97 (m, 1H), 2.15 (t, J = 7.05, 2H), 2.43 (s, 1H), 3.27 (m, 4H), 3.53 (d, J =
3.66 Hz, 2H), 3.91 (m, 1H), 4.10 (m, 3H), 4.64 (m, 1H), 7.39 (m, 6H), 7.50 (d,
J=7.90 Hz, 2H), 7.59 (d, J=7.61 Hz, 1H), 7.65 (d, J = 7.68 Hz, 4H), 7.78 (d, J
=8.30 Hz, 2H), 7.83 (d, J=8.12 Hz, 2H), 7.94 (d, J = 8.17 Hz, 2H) 13C NMR (300
MHz CDCl3) δ 145.80 145.86, 136.98, 136.84, 136.64, 135.49, 132.69, 129.89,
128.22, 127.59, 127.45, 126.88, 71.25, 53.22, 50.86, 33.47, 32.06, 28.30, 26.66,
22.51, 19.05, 16.62 MALDI-HRMS [M + Na]+ calcd: 828.3766, found 828.3763
[α]296KD = +0.095 (c 0.41, 1:4 methanol/chloroform).
39
TBDPSO OO
HO
NHO
HNO
O
N3
TBDPSO OO
O
NHO
HNO
O
N3
POO
OPO
ON
m-CPBACH2Cl2
22
Compound 21 (100 mg, 0.124 mmol) was dissolved in dichloromethane (10 mL).
Dibenzyl diisopropylphosphoramidite (122 µL, 0.373 mmol) and 1H-tetrazole
(828 µL, 0.373 mmol) were added. The reaction was stirred at room temperature
under nitrogen for 24 h. Then the reaction was cooled to 0 °C and mCPBA (64
mg, 0.373 mmol) was added. The reaction was stirred for 1 h at room
temperature. After completion, the solvent was concentrated and the residue
was purified via chromatography on silica gel with a solvent gradient of 30–75%
ethyl acetate/hexanes and 100% ethyl acetate to yield 22 (14.1 mg, 50%).
1H NMR (300 MHz, CDCl3) δ 1.03 (s, 9H), 1.27 (m, 2H), 1.50 (m, 8H), 1.79 (m,
1H), 1.94 (m, 1H), 3.23 (m, 4H), 3.97 (m, 5H), 4.71 (m, 1H), 4.93 (s, 4H), 7.30
40
(m, 14H), 7.48 (m, 3H), 7.62 (m, 6H), 7.78 (m, 4H), 7.96 (m, 2H) 13C NMR (300
MHz CDCl3) δ 195.97, 173.68, 171.49, 171.48, 166.40, 140.36, 135.89, 133.21,
133.06, 130.98, 130.16, 128.68, 128.54, 127.965, 127.949, 127.859, 127.799,
127.28, 69.78, 69.65,, 69.49, 69.39, 67.74, 64.31, 60.50, 53.56, 51.19, 39.52,
33.79, 32.51, 29.12, 28.66, 28.79, 26.89, 26.39, 24.42, 22.81, 21.15, 19.36,
14.30, MALDI-HRMS [M + Na]+ calcd: 1088.4365, found 1088.4398 [α]296KD = -
1.16 (c 0.26, 1:4 methanol/chloroform).
23
Compound 22 (0.033 mmol) was dissolved in dichloromethane under nitrogen,
and trimethylsilyl bromide (309 µL) was added, and the reaction was run until
completion. After the disappearance of starting material, the solvent was
41
removed, and the residue was dissolved in methanol (5 mL). The reaction was
stirred at room temperature for 1 h. The methanol was then removed. After
residue solvent had been removed, the product was dissolved in 5.0 mL
tetrahydrofuran. Tetrabutylammonium fluoride trihydrate (0.327 mmol) was
added, and the reaction was allowed to run overnight. The next day the solvent
was removed, and the product was purified via a reversed-phase column with a
solvent gradient of 90–10% methanol to produce 23 (12.1 mg, 61%).
1H NMR (300 MHz, CDCl3) δ 1.05 (m, 9H), 1.29 (m, 7H), 1.65 (m, 1H), 1.95 (m,
1H), 2.33 (m, 1H), 3.35 (m, 4H), 3.68 (m, 1H), 4.12 (m, 1H), 7.39 (m, 2H), 7.59
(m, 1H), 7.73 (m,2H), 7.80 (m, 2H), 8.00 (m, 2H) 13C NMR (300 MHz, CDCl3)
196.44, 196.41, 173.86, 171.71,140.62, 137.25, 135.18, 133.53, 130.46, 130.45,
128.90, 127.86, 127.74, 63.96, 59.15, 53.80, 39.70, 34.43, 33.36, 32.54, 29.65,
29.14, 27.61, 24.85, 24.23, 20.07, 13.82, MALDI-HRMS [M+ Na]+ calcd:
691.2040, found: 691.1531
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
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66
Vita
Leah M. Cuthriell was born in Opp, Alabama in 1984 and grew up in the
Flat Creek community near Samson, AL. She is the daughter of Peadon and
Barbara Cuthriell. In May of 2003, she graduated from Samson High School in
Samson, AL. After graduation, she attended Huntingdon College in Montgomery,
AL where she received a B.S. degree in chemistry in 2007. She completed a
Master of Science degree in chemistry in December 2009.
