a cyhq-linker for the controlled spatiotemporal …
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A CYHQ-LINKER FOR THE CONTROLLED SPATIOTEMPORAL STUDY OF GENE
EXPRESSION IN ZEBRAFISH
by
Kyle Harris
(Under the Direction of Timothy M. Dore)
ABSTRACT
Several hydroxyquinoline-protected acetates were synthesized to study how changes in
substituents at C-8 affect their ground-state and excited-state chemistry and photoreactivity.
Strong electron withdrawing groups form a greater proportion of the anionic form of the
quinoline at neutral pH. A photoremovable protecting group for carbonyl compounds, Bhc-
dithiol, should be more resistant to hydrolysis in the dark than Bhc-diol. The synthesis of a
protected version of Bhc-dithiol is described. A CyHQ-based linker for the activation of
morpholinos was synthesized for use in spatio-temporal studies of gene expression in developing
zebrafish embryos. This cyano-based derivative of BHQ has a three-fold higher molar
absorptivity than BHQ and higher sensitivity to one-photon excitation, which will enable more
efficient photoactivation of the morpholino than a previous BHQ-based-linker analog.
INDEX WORDS: CyHQ, Caged morpholino, XHQ-OAc, Bhc-dithiol, Photoremovable
Protecting Group, PPG
A CYHQ-LINKER FOR THE CONTROLLED SPATIOTEMPORAL STUDY OF GENE
EXPRESSION IN ZEBRAFISH
by
Kyle Harris
B.S. The University of Georgia 2006
A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment
of the Requirements for the Degree
MASTER OF SCIENCE
ATHENS, GEORGIA
2010
2010
Kyle Harris
All Rights Reserved
A CYHQ-LINKER FOR THE CONTROLLED SPATIOTEMPORAL STUDY OF
GENE EXPRESSION IN ZEBRAFISH
Major Professor: Timothy M. Dore Committee: Vladimir Popik
George Majetich Robert Phillips
Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia May 2010
iv
ACKNOWLEDGEMENTS
I would like to thank Dr. Timothy M. Dore for his guidance, generosity, and endless
support throughout my graduate career at the University of Georgia. I would also like to thank
Dr. Vladimir Popik, Dr. George Majetich, and Dr. Robert Phillips for serving on my committee.
I would like to thank my colleagues for their helpful discussions and guidance: Dr. Yue
Zhu, Dr. Matthew O’Connor, Robert, Kutlik, Duncan McLain, Adam Rea, Steven Flynn, Alex
Walker, Hunter Wilson, Banks Deal, Cullen Timmons, and James McNamara. Their support and
friendship was invaluable in my studies.
Finally, I’d like to thank my family and friends for their help and understanding in my
endeavors. Without them I would not be where I am today.
v
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS...........................................................................................................vi
CHAPTER 1
Synthesis of XHQ-caged acetate derivatives and resonance Raman characterization in
aqueous solutions ................................................................................................................1
CHAPTER 2
Development of Bhc-dithiol as a new photochemical protecting group for aldehydes and
ketones ...............................................................................................................................12
CHAPTER 3
Caging antisense reagents to control gene expression.......................................................24
CHAPTER 4
Experimental Section .........................................................................................................34
REFERENCES ................................................................................................................................76
1
CHAPTER 1
Synthesis of XHQ-caged acetate derivatives and resonance Raman characterization in aqueous
solutions
A novel photochemical protecting group, 8-bromo-7-hydroxyquinoline (BHQ),
synthesized by Dore et al. has low fluorescence and has a sufficient two-photon uncaging to be
used in biological applications.1 BHQ-OAc has previously been synthesized and studied for its
mechanism of photolysis by our lab.2 Through Stern-Volmer quenching, time-resolved infrared
(TRIR), and 18O-labeling experiments it was proposed that the photolysis of BHQ-OAc proceeds
through a solvent assisted photoheterolysis (SN1) reaction similar to that found for Bhc (Figure
1).3 Irradiation of 1 in aqueous media at pH 7.2 leads to a singlet excited state (2), which can
either undergo intersystem crossing (ISC) followed by decay back to the starting phenolate (1) or
undergo either homolytic or heterolytic cleavage of the carbon-oxygen bond followed by single
electron transfer to generate a zwitterion-like intermediate (4). Finally, trapping of the cation or
electropositive carbon by water gives the products (5 and acetate). Hydroxyquinolines are
known to be protonated on a picosecond time scale in the excited state even in basic aqueous
solutions, however the role of intermediate (6) is not currently understood.
2
Figure 1: Proposed mechanism of the BHQ photolysis reaction
8-Bromo-7-hydroxyquinoline, a derivative of 7-hydroxyquinoline, can exist in four
prototropic forms in water-containing environments: a normal neutral species (N), an enol-
deprotonated zwitterionic tautomer (T), an enol-deprotonated anion (A), and an imine-protonated
cation (C) (Figure 2). These four forms of BHQ in aqueous solutions complicate studies of its
chemistry and photochemistry. Understanding what forms BHQ and various derivatives are
found in aqueous conditions can lead to a fundamental understanding of substituent effects on
these various forms.
3
Figure 2: Four forms of XHQ-OAc derivatives in aqueous solutions
The pKa of 7-HQ is 9.04 whereas pKa = 6.8 for BHQ-OAc.1 This difference indicates that
the distribution of the four forms of BHQ-OAc in neutral aqueous solutions is likely to change
noticeably with the addition of the bromine atom at the 8-position. Indeed, ground states of the
different forms of BHQ-OAc were characterized in both acetonitrile and water-rich
environments at varying pH values (pH 6-7 in neutral conditions and pH 11-12 in basic
conditions). This work allowed for analysis of the different forms of BHQ-OAc in various
environmental conditions. Substantial amounts of the neutral and anionic forms of BHQ-OAc
were found in neutral conditions, with a minute amount of the tautomer present as well.5 These
results differed drastically from previous work done on 7-hydroxyquinoline, which indicated a
much larger concentration of the tautomeric form (29%) as opposed to the anionic form (1%).6
The large substituent effect of the BHQ derivative found in neutral solution could be due to steric
and/or electronic effects of the bromine atom at the 8-position. The bulky bromine atom could
be sterically blocking tautomer formation, and the electron withdrawing nature of the bromine
atom can hinder protonation of the nitrogen atom in the quinoline ring. Another possibility is the
4
competitive hydrogen bonding between the bromine atom and water molecules in solution that
inhibit formation and transfer of a BHQ-OAc-water cyclic complex (Figure 3).5
Figure 3: Formation of XHQ-OAc-cyclic water complex
Replacing the bromine with an electron-withdrawing group that does not facilitate ISC
should improve quantum efficiency (Qu), because ISC competes with the photo-uncaging
reaction and diminishes the effectiveness of the chromophores. To this end, CyHQ-OAc was
synthesized and its photochemical properties assessed. CyHQ-OAc has a three-fold increase in
molar absorptivity over BHQ-OAc, yet the quantum efficiency of CyHQ (0.31) was only slightly
higher than that of BHQ-OAc (0.29).7 Whereas the one-photon cross section of CyHQ-OAc was
higher than BHQ-OAc, the two-photon cross section, δu, was much lower for CyHQ-OAc (0.17)
than BHQ-OAc (0.59). It is unclear why there is such a large discrepancy between the two
compounds. One hypothesis is that CyHQ-OAc fluoresces brightly relative to the other
chromophores, indicating that decay of the singlet excited state through fluorescence could
compete with uncaging, thereby limiting the quantum efficiency. Competing pathways in
photolysis, such as ISC and fluorescence, can diminish quantum efficiency significantly.
Therefore, we sought to characterize a number of derivatives of BHQ-OAc, in hopes of gaining a
deeper understanding of their ground state properties with the ultimate goal of understanding
how changing substituents at the 8-position affects quantum efficiency and the sensitivity of
these compounds to undergo a photochemical reaction by two-photon excitation. Studies into
the excited states of these compounds are also planned.
5
Several 8-substituted-7-hydroxy-quinolinyl derivatives have been synthesized and their
photochemical properties studied.7,8 To further understand the underlying chemical effects of the
different substituents, near gram scale quantities of the 8-substituted chloro, cyano, and nitro
derivatives were synthesized using previously developed methods (Figures 4, 5, and 6).7 A slight
modification to each procedure was made in which the deprotection of the MOM protecting
group on the phenol was removed using TFA instead of conc. HCl as had previously been used.
The milder conditions enabled selective deprotection of the MOM group while leaving the
acetate intact. These three compounds were then shipped to Dr. David Lee Phillips at the
University of Hong Kong for resonance Raman spectroscopy experiments armed at elucidating
what species of each compound is present in various aqueous solutions and acetonitrile
Substituents at C-8 of the quinoline are expected to strongly influence the pKa of the
phenol group and the distribution of the four protropic species. The CyHQ-OAc derivative, with
its strongly electron-withdrawing cyano group, may show significant differences in ground-state
distributions due to changes in pKa of the cyano derivative relative to BHQ-OAc. CHQ-OAc
may form more of the tautomer form because of the smaller size of the chlorine atom than
bromine, as well as less of an electron-withdrawing effect and less possibility of hydrogen
bonding, making a cyclic CHQ-OAc-water complex easier to form. Note that the nitro-
substituted HQ derivative has not yet been studied using resonance Raman spectroscopy due to
complications in understanding ground state chemistry of the compound. More experiments will
be necessary to understand the chemistry of this derivative.
6
Figure 4: Large scale synthesis of CHQ-OAc (a) NCS, CH2Cl2, 72%; (b) MOMCl, Et3N, THF, 93%; (c) SeO2, p-dioxanes, 80 ˚C, 74%; (d) NaBH4, MeOH, 96%; (e) Ac2O, DMAP, pyridine, CHCl3, 83%; (f) TFA, MeOH, 70%
Figure 5: Large scale synthesis of CyHQ-OAc (a) CHCl3, NaOH, 100 ˚C, 72%; (b) NH2OH/HCl, aq 5 M NaOH, 95 ˚C, 81%; (c) Ac2O, 70 ˚C, then 50% NaOH, then 1 M HCl, 77%; (d) MOMCl, Et3N, THF, 91% (c) SeO2, p-dioxanes, 80 ˚C, 78%; (d) NaBH4, MeOH, 90%; (e) Ac2O, DMAP, pyridine, CHCl3, 97%; (f) TFA, MeOH, 64%
7
Figure 6: Large scale synthesis of NHQ-OAc (a) HNO3, H2SO4, 64%; (b) MOMCl, Et3N, THF, 86%; (c) SeO2, p-dioxanes, 80 ˚C, 62%; (d) NaBH4, MeOH, 90%; (e) Ac2O, DMAP, pyridine, CHCl3, 96%; (f) TFA, MeOH, 77%
The pKa of the phenolic proton on both CHQ-OAc and CyHQ-OAc were determined by
measuring the UV absorbance spectra of 100 µM substrate in PBS-buffered solutions (1 mL) set
to pH values between 2 and 10 in quartz cuvettes (Figures 7 and 8). A red-shift of the absorption
band correlating to the formation of the phenolate was clearly seen in both compounds. The
absorbance at 369 and 363 nm (for CHQ-OAc and CyHQ-OAc) respectively, which corresponds
to the phenolate species, was plotted against the buffer pH, and the pKa was calculated from the
half-equivalence point of the resulting titration curves (Figures 9 and 10). The pKa of CHQ-OAc
was found to be identical to that of BHQ-OAc at 6.8, whereas the pKa of CyHQ-OAc is 4.9.9
This large difference in pKa’s between the chloro and cyano substituted derivatives, due largely
to the much stronger electron-withdrawing group of the cyano derivative, indicates a possible
difference in distributions of forms of the molecules in aqueous solutions. UV-vis spectrum of
NHQ-OAc was also measured, however no clear isosbestic point was seen for the compound
(Figure 11). This seems to indicate a differing ground state chemistry than the other XHQ-OAc
8
derivatives that requires further investigation.
Figure 7: UV-vis absorption spectra of 100 µmol CHQ-OAc at various pH values in PBS buffer (1 mL)
Figure 8: UV-vis absorption spectra of 100 µmol CyHQ-OAc at various pH values in PBS buffer (1 mL)
9
Figure 9: Titration curve for CHQ-OAc
Figure 10: Titration curve for CyHQ-OAc
10
Figure 11: UV-vis absorption spectra of 100 µmol NHQ-OAc at various pH values in PBS buffer (1 mL)
The Phillips group subsequently measured UV-vis absorbance of BHQ-OAc, CHQ-OAc,
and CyHQ-OAc in MeCN, neutral, and basic solutions.9 Since the pKa is CHQ-OAc is the same
as BHQ-OAc it is not surprising that the absorption spectra of CHQ-OAc is quite similar to that
found for BHQ-OAc. The observed lower pKa affects the UV-spectrum of CyHQ-OAc by
substantially increasing the amount of the anionic form in solution and where it becomes the
other dominant form in the solution coexisting with the neutral species. UV-vis spectroscopy
reveals that the neutral forms of CHQ-OAc and CyHQ-OAc are still the predominant species in
neutral aqueous solutions, with the anionic form increasing significantly in CyHQ-OAc.
Compared to 7-HQ, resonance Raman spectroscopy experiments show that the amount of the
tautomeric form decreases noticeably for BHQ-OAc and CHQ-OAc and is almost nonexistent
for CyHQ-OAc, whereas the anion form substantially increases due to the lower pKa of the
phenol in CyHQ-OAc. Previous work from our collaboration with Dr. Phillips has suggested
11
substitution at the 8-position of 7-HQ affects the formation of the tautomeric form in solution via
steric and/or electronic effects. A number of factors have been postulated as affecting the
tautomeric form including formation of a cyclic hydroxyquinoline-water complex. Another
factor is the inductively electron-withdrawing nature of the substituent at the 8-position can
disfavor formation of a positively charged nitrogen in the quinoline ring. Finally, competitive
hydrogen bonding between the substituent and water molecules could hinder tautomer formation.
The work with CHQ-OAc and CyHQ-OAc in this study along with previous work with BHQ-
OAc seems to support this hypothesis.
Liu et al. have recently derivatized the 8-position of hydroxyquinoline to produce novel
quinoline-based photo-labile groups.8 By substituting the 8-position with pyridine-like
heterocycles up to ten times greater water solubility was achieved than BHQ-OAc as well as 1.5
times faster photolysis. Improved photolysis is likely due to the enlarged conjugation plane of
the pyridinyl-based analogs. With this knowledge, a foundation is laid for detailed time-resolved
spectroscopy experiments to directly examine reaction pathways, intermediates, and mechanisms
involved in the photodeprotection reactions of XHQ-OAc compounds.
12
CHAPTER 2
Development of Bhc-dithiol as a new photochemical protecting group for aldehydes and ketones
Embryonic development is a complex process that requires precise control of gene
expression both spatially and temporally to create complex organisms. Many genes are
expressed in a tissue specific manner further complicating efforts to study development on a
global level. Understanding how genes interact in space and time to modulate cell propagation,
migration, and differentiation is a crucial challenge currently being studied by developmental
biologists. Several methods for controlling gene regulation have been developed to meet this
challenge, the most popular are the Gal4/GR, Cre/Lox, and Tet-ON/Tet-OFF systems.10
The traditional methods developed for controlling gene expression rely on a ligand-
dependent approach (Figure 13). Simply stated, a small molecule is introduced into a system
that contains either a small molecule-dependent transactivator or repressor to induce or silence
gene expression. Introduction of the chemical ligand for embryos that develop ex utero can be
accomplished by addition of the ligand to the culture medium, however for embryos that develop
in utero the ligand can be introduced via intraperitoneal injection, oral gavage, or solubilization
in drinking water.
13
Figure 12: General schematic of traditional ligand-dependent systems for controlling gene expression
The most commonly employed ligand-inducible gene expression system is the
tetracycline mediated TetR/tetO system.11 This system exploits an antibiotic resistance
mechanism found in Escherichia coli. Tetracycline is able to bind to a repressor protein (TetR)
with nanomolar affinity. In the absence of tetracycline, TetR is capable of binding a palindromic
tetracycline operator (tetO) with a dissociation constant of 10-11 M. When tetracycline is
introduced into the system it binds to TetR and the protein undergoes a conformational change
that reduces its affinity for tetO by several orders of magnitude (Figure 13). The resulting
dissociation of TetR from tetO sites promotes tetA transcription and tetracycline clearance from
the organism. Since the TetR protein and tetO operator are not present in eukaryotic genomes, it
was thus possible to use this system to study gene expression in embryos with minimal off-target
perturbation.
14
Two tetracycline-dependent expression systems have been developed for in vivo
studies.12 First, Tet-OFF was developed by fusing the TetR DNA and ligand binding domains
with an activation domain from the herpes simplex virus VP16, thereby creating a tetracycline
controlled transactivator (tTA). Cells expressing rTA and containing a tetO-dependent transgene
constitutively express the targeted gene. Addition of tetracycline or a derivative doxycycline
silences gene transcription. A second gene expression system (Tet-ON) takes advantage of a
tTA varient (rtTA)13 that has been mutagenized to reverse the effects of ligand binding: instead
of dissociating when bound to tetracycline, rtTA only binds tetO sequences when bound with the
antibiotic. Commercial systems of both Tet-ON and Tet-OFF are available from Clontech.
The Tet-ON/Tet-OFF systems have been widely used in mice but have not seen as much
use in other metazoans (animals in which the protoplasmic mass, constituting the egg, is
converted into a multitude of cells, which are metamorphosed into the tissues of the body).
Specifically, tetracycline is unable to efficiently traverse the embryonic membrane or vitelline
envelope in frogs, thus requiring microinjection into the embryos for proper localization.14
Efforts to express GFP in zebrafish using the rtTA system failed to plateau after four days
indicating a similar inability of the antibiotic to efficiently cross plasma membranes.15 Zebrafish
development is largely complete after two days, rendering this method ineffective in studying
embryonic patterning.
Another widely used ligand-dependent gene expression system is the dexamethasone
regulated Gal4/GR expression system.16,17 This system works by taking advantage of nuclear
hormone receptors that can act as small molecule inducible transactivators. Glucocorticoid
receptor (GR) is a transcription factor, which is normally sequestered by a cytosolic complex that
contains heat shock protein 90 (Hsp90) bound in an inactive state. Binding of cortisol or other
15
glucocortioids to GR induces a conformational change that causes the transactivator to dissociate
from the complex. GR then translocates to the nucleus, where it binds glucocorticoid response
elements on DNA, and activates transcription of the targeted gene(s). By fusing the GR ligand-
binding domain to any transcriptional activator of interest, it is possible to induce targeted gene
expression using the synthetic GR agonist dexamethasone. Specificity in metazoan biology
however requires a transcription factor Gal4 from Saccharomyces cerevisiae, this transcription
factor was used because it recognizes an upstream activating sequence (UAS) unique to yeast.
This system is limited because of interactions with endogenous GR signaling and the exogenous
transactivator system.18,19
Extending the applicability of using nuclear hormone receptors as small molecule
inducible transactivators, McMahon et al. used DNA-modifying enzymes to create a small
molecule-induced genomic recombination system.20 The group used the bacteriophage P1
recombinase Cre, which catalyzes a site-specific recombination between tandem 34-base pair
loxP sequences.21,22 Simply, DNA sequences that are located between two loxP sites are excised
by Cre and insertion of loxP-flanked DNA cassettes into regulatory elements can effect Cre-
dependant gene expression. The system works by first building an inducible Cre recombinase,
which is done by fusing it to a mutant estrogen receptor ligand-binding domain that responds to a
synthetic agonist 4-hydroxytamoxifen but has no affinity for the endogenous ligand, 17β-
estradiol. Therefore, genetic transcription that has been distrupted by a loxP-flanked insert will
be silenced until 4-hydroxytamoxifen is introduced in the system to bind with the chimeric
protein (Cre-ER) and induces genetic recombination.
Ligand dependent strategies for controlling gene expression have proven useful in
studying gene expression in many diverse model systems. However, they suffer from slow
16
kinetics when compared to events such as the dynamics of embryonic patterning where gene
transcript levels can rise and fall dramatically within minutes. New methods are needed to
control gene expression that work on timescales of seconds to subseconds.
Photoremovable protecting groups are ideal candidates for use in spatio-temporally
controlling ligand release on timescales much faster than traditional systems. These groups are
removed using only light, therefore other sensitive groups in the molecule are untouched. In
recent years, a number of caged systems for regulating gene expression have been developed for
use in biological applications. These include, but are not limited to, caged doxycycline,23,24
toyocamycin,25 estradiol,26 ecdysone,27 anisomycin,28 and progesterone.29
Cambridge et al. developed a caged doxycycline analog to initiate the Tet system for
gene transcription.24 Taking advantage of an essential phenolic β-diketone system needed for the
formation of the doxycycline-magnesium complex, which binds to the rtTA protein with high
affinity, the group synthesized a 1-(4,5-dimethoxy-2-nitrophenyl)ethyl (DMNPE) ether of
doxycycline attached at the phenolic β-diketone system.13 A proof-of-principle experiment
showed that long-wavelength UV irradiation of caged doxycycline induced expression of
enhanced green fluorescent protein (EGFP) similar to that seen with unmodified doxycycline.
To extend this system, photoactivation of caged doxycycline in a three-dimensional tissue was
accomplished using a modified Tet-ON system in transgenic tobacco leafs. A β-glucuronidase
(GUS) reporter gene was silenced via a Tet repressor, however incubation of caged doxcycycline
with leaf tissue followed by irradiation produced a sharp boundary between irradiated and non-
irradiated areas. This was the first example that uses photoactivation based on a ligand-
dependent gene expression paradigm to direct transgene expression with high spatial precision in
compact tissue.
17
Koh et al.26 exploited the estrogen receptor system, that is bound with HSP9030 when the
ligand estradiol is not present and undergoes a conformational shift upon binding of estradiol
resulting in dissocation of HSP90 and subsequent binding of promoters and transcriptional co-
activators,31 with a caged estradiol, which has a hydroxyl group essential for ligand binding
blocked by a photoremovable protecting group. Up to 86% induction of the luceferase reporter
gene was observed upon irradiation. An early example of inhibiting biological processes with a
caged small molecule was the caging of the protein-synthesis inhibitor anisomycin, which blocks
the peptide-bond-forming reaction in eukaryotic ribosomes, by Dore et al.28 Using Bhc-caged
anisomycin, GFP expression was drastically reduced after irradiation in a localized manner.
Aptamers are short, single stranded nucleic acids that can fold into a 3D shape and
subsequently bind to a target molecule. By caging aptamers, spatiotemporal control of a target
molecule can be obtained. Heckel et al. exploited this by caging a known aptamer that targets
the human blood clotting factor α-thrombin,32 as well as the cytoplasmic regulatory protein
cytohesin-1.33 They were able to control the guanine nucleotide exchange factor function (GEF)
of cytohesin in a concentration dependant manner. Recently, Guo et al.8 used BHQ to cage
phosphate backbones of an aptamer targeting α-thrombin. Further exploitation of caging
aptamers with two-photon protecting groups will give spatiotemporal control over aptamer
formation and provide researchers with a more powerful system for inducing expression via
uncaging of a small molecule.
Lauderdale et al. have developed transgenic model systems to carry the GeneSwitch™34
system and used it to study the role the Pax6 gene plays in specifying cell types within the
central nervous system.35, 36 The GeneSwitch™ system consists of two genes, one that codes for
the GeneSwitch™ regulator protein, the other codes for an inducible transgene of interest.
18
Expression of the GeneSwitch™ regulator protein is driven by a constitutive promoter. The
GeneSwitch™ regulator protein is a chimeric transcription factor that is allosterically activated
by synthetic antiprogestin drugs. The chimeric protein contains three important functional
domains: (1) a mutant form of the ligand binding domain of the human progesterone receptor,
(2) the DNA binding domain from the yeast GAL4 protein, and (3) the transcriptional activation
domain from the VP16 protein of herpes simplex virus or from the p65 subunit of human NF-kB.
Mifepristone activates the GeneSwitch™ regulator protein by binding to the ligand binding
domain causing a conformational shift of the chimeric protein. This causes heat shock proteins
(Hsp90, Hsp70) and accessory proteins (immunophilins, Hop, p23) to dissociate and activate the
monomer for dimerization. This dimer subsequently binds to the inducible transgene, which has
a promoter that consists of a minimal promoter sequence linked to multiple copies of the 17 bp
palindromic GAL4 DNA binding site, and activates expression. Each 17 bp binding site is
capable of binding a homodimer of the GeneSwitch™ regulator protein. The GeneSwitch™
regulator protein is not activated by progesterone or other natural steroids. By using a caged
version of mifepristone, spatial and temporal control of gene expression would enable deeper
understanding of processes involved during development. Using Bhc, two-photon excitation
would also enable less phototoxicity and three-dimensional spatial selectivity not accessible
using photochemical protecting groups that are only sensitive to one-photon excitation.
Carbonyl groups are often found in small molecules of interest and have biological
activities.37,38 Photochemical release of caged carbonyl compounds is of interest in answering
basic biological questions and in biomedical applications. Despite decades of research into
developing caging groups for carbonyl moieties, only a few have been found to be practically
useful.39-45 Long irradiation times, low absorption, and insufficient quantum efficiencies has
19
plagued development of useful systems. More recently, work done by Wang et al.46 and Popik et
al.47 have expanded the toolset available for protecting carbonyls with photoremovable
protecting groups with higher photorelease yields and improved solubilities. However, these
protecting groups have little to no sensitivity to two-photon excitation, which would allow for
lower phototoxicity and improved spatial resolution in biological studies. A recent example of a
caged Bhc-diol-progesterone is the first example of a photoactivated carbonyl compound in a
biological system, which also used two-photon excitation.29
Dore et al. envisioned using Bhc-diol to cage mifepristone, a synthetic steroid that can be
used to induce gene expression via the GeneSwitch™ system (Dore et al. unpublished results).
This molecule has an enone moiety that is essential for function. By caging the ketone, control
of gene expression via photorelease was achieved. Bhc-diol-mifepristone was synthesized and
tested for its ability to induce GFP expression. Significantly less expression was observed in
cells with Bhc-diol-mifepristone when compared to cells incubated with mifepristone. (Dore et
al. unpublished results) Nevertheless, some GFP expression was observed indicating possible
dark hydrolysis of the ketal that releases mifepristone. Alkene rearrangement is also known to
occur when enones are protected as ketals.48,49 We hypothesize that a Bhc-dithiol-mifepristone
analog would be more resistant to hydrolysis50 and that formation of thioketals from enones
would proceed without alkene rearrangement (Figure 13).51,52
20
Figure 13: Activation of gene under GeneSwitch™ control using Bhc-mifepristone
Thioketals are much less susceptible to hydrolysis than their oxygen analogs.50 With this
knowledge we attempted to synthesize a Bhc-dithiol derivative to test as a potentially more
robust photoremovable protecting group than Bhc-diol for protecting ketones and aldehydes.
(Figure 14) Thiolates are also better leaving groups than alkoxides, thus we expect the
photochemical release of the carbonyl to be as good or better than Bhc-diol. This synthesis
started from a previously known Bhc-alkene 37 where the phenol was first MOM-protected
yielding 38 and then the alkene was dihydroxylated giving 39 using Upjohn conditions.53 Next,
mesylation of both hydroxl groups provided courmarin 40 with two excellent leaving groups to
react with a sulfur nucleophile. To our disappointment we found that common sulfur
nucleophiles such as thioacetic acid, sodium sulfhydrate, and various sulfur carbonates did not
yield the expected disubstituted product (Figure 15). In most cases, substitution on the primary
position was achieved, but reduction occurred at the secondary position. Varying reaction
21
conditions such as solvent, concentrations of reagents, or temperature did not change the reaction
outcome. Other reaction pathways were also attempted such as converting the alkene into an
episulfide to open with a sulfur nucleophile, however these attempts failed to generate desirable
outcomes. Finally, we found that reacting MOM protected Bhc-dimesylate with sodium
dimethyldithiocarbamate in methanol gave MOM-Bhc-bis-dimethyldithiocarbamate 41.54,55 This
gave the compound with sulfurs in the desired position with only the cleavage of the
dimethyldithiocarbamate group left to obtain target 42.
Figure 14: Synthesis route for Bhc-dithiol (a) Ac2O, DMAP, pyridine, CHCl3, 75%; (b) PPh3, CH3CN, 85 C, 82%; (c) 37% CH2O, 15% Na2CO3, H2O, 72%; (d) MOMCl, Et3N, THF, 83%; (e) OsO4, NMO, H2O, acetone, 80%; (f) MsCl, Et3N, THF, 88%; (g) NaNS2C3H6, MeOH, 71%
22
Figure 15: Attempts to synthesize Bhc-dithiol using various sulfur nucleophiles
Dimethyldithiocarbamate protecting groups are cleaved to the free thiols using refluxing
basic conditions or LAH reduction.56,57 These conditions proved too harsh, opening the lactone
portion of the coumarin ring or reducing it. Reactions using simple amines to achieve cleavage,
such as cyclohexylamine and hydrazine, yielded no reaction at room temperature and ring
degradation when heated (Figure 16). In spite of varying reaction conditions including
temperature, solvent, various amines and bases as a means of cleavage, as well as using strong
acid to attempt cleavage (since the coumarin ring is stable to acidic conditions) a suitable method
for cleaving the dimethyldithiocarbamate protecting group without affecting the coumarin ring
23
evaded us. Despite the failure to reduce the compound to the final vicinal dithiol product using
the Bhc photochemical protecting group, this method may provide a new route in synthesizing
vicinal dithiols provided the final cleavage can be accomplished.
Figure 16: Various attempts at cleaving dimethyldithiocarbamate protecting group
24
CHAPTER 3
Caging antisense reagents to control gene expression
Antisense reagents have long been used in controlling gene expression in a variety of
model systems. These reagents work via microinjection into cells where they then
complementarily bind to a target RNA sequence that has been transcribed from a gene of
interest. By capturing mRNA sequences before they are spliced or translated, gene expression is
silenced selectively. Common antisense reagents include small interfering RNA (siRNA),
negatively charged peptide nucleic acids (ncPNAs), and morpholinos (MOs) (Figure 17).58
Morpholinos and ncPNAs were developed with modified phosphate backbones to be resistant to
endogenous nucleases that can degrade siRNAs. Whereas these methods have proven effective
they are not without limitations. Microinjection of the reagent must be done before a certain
time point in cell differentiation to be capable of observing a targeted phenotype among all cells.
This prevents researchers from studying genes that have pleiotropic functions since the effects of
antisense reagents are immediate and global; therefore, a method was needed to spatio-
temporally control the release of active antisense reagents within cells. By linking inhibitory
sequences to the antisense reagents via a photocleavable linker that can be released upon
irradiation with light, chemists have devised a “caging” method to spatio-temporally control
gene expression.
25
Figure 17: Structures of RNA and common antisense reagents
Caging small interfering RNA (siRNA) has been accomplished as a means to block gene
expression (Figure 18). Friedman et al. developed a GFP-targeting siRNA caged with 1-(1-
diazoethyl)-4,5-dimethoxy-2-nitrobenzene on approximately 5-15% of the phosphate
backbone.59 Experiments with this caged-siRNA showed a range of basal and light-induced gene
silencing that was inversely correlated to the number of caging groups per siRNA. However,
relative changes in expression levels remained two-fold or less from basal levels, not useful for
most biological studies. McMaster et al.60 was able to overcome this limitation by placing a
single protecting group at the 5' phosphate position. While this modification did not completely
block siRNA function (5-10% residual activity), uncaging of the nitrobenzyl-based protecting
26
group restored gene silencing to near unmodified siRNA levels. This method of gene silencing
via caging at the 5' position may prove to be beneficial in probing embryonic development.
Figure 18: Schematic of caging antisense reagents and subsequent uncaging with light irradiation.
Dieters et al.61 have developed caged antisense reagents using a DNA phosphorothioate
(PS DNA) backbone based system. These oligomers are more stable than their oxygen
substituted analogs. By substituting three or four NPOM (6-nitropiperonyloxymethyl)-caged
thymidine residues into PS DNA 19-mers, they were able to silence expression of a luciferase
gene by up to 70%.
Caged negatively charged peptide nucleic acids (ncPNA) control gene expression via
caging RNA transcripts that bind ncPNA’s can bind to complementary unbound DNA and RNA
sequences with high affinity (Figure 18). One advantage is that they are resistant to cleavage by
endogenous proteases and nucleases. A basic strategy for constructing caged ncPNA’s involves
attaching a complementary 2'-O-methyl RNA to the ncPNA through a nitrobenzyl-based linker.
Subsequent photolysis cleaves the linker, which forms an unstable ncPNA/RNA complex that
falls apart releasing ncPNA. Dmochowski et al used this strategy to develop caged ncPNAs
targeting both bozozok (boz) and chordin (chd) genes with known roles in early development.62
27
After global irradiation, loss of gene expression was seen in both cases.
Caging morpholinos is another antisense technique that has been developed to control
gene expression and study embryonic development. Morpholinos are synthetic oligomers
containing a modified non-ionic phosphorodiamidate background that is resistant to enzymatic
cleavage. The morpholino rings serve as an analog to the ribose rings found in both RNA and
DNA. Developed recently by Mayer et al63, Photomorphs™ are morpholinos that are bound to a
complementary sequence with a nitrophenol photocleavable linker that is built into the backbone
of the complementary sequence. Irradiation led to cleavage releasing free morpholino and
subsequent knockdown of GFP, ntl, and cdh1 (E-cadherin) genes. While this technology
provides a facile way to cage a wide range of morpholinos, long irradiation times (~30 mins) can
lead to significant toxicity and undesired off target phenotypes.
Chen et al. envisioned caging morpholinos (cMOs) as a way to spatiotemporally control
morpholino activity.64 By caging morpholinos with a photoreactive protecting group, researchers
would be able to spatially and temporally control gene expression using UV light to catalyze
photolysis and subsequent release of the reactive morpholino. To test this hypothesis the group
tethered a complementary inhibitor to a 25-base targeting sequence of a specific gene using a
dimethoxynitrobenzyl (DMNB)-based photocleavable linker (Figure 19). This linkage resulted
in forming a hairpin structure. This hairpin shaped complex suppresses binding to the target
RNA sequence until irradiation with 360-nm light, when the linker is cleaved and the inhibitor
sequence dissociates from the 25-base targeting sequence. This active sequence may then bind
to its complementary RNA target sequence and suppress splicing or translation.
28
Figure 19: Schematic of DMNB cMO and light activation for gene silencing
Chen et al. developed a caged morpholino targeting a T-box transcription factor called no
tail-a (ntla) that is required for the differentiation of axial mesodermal cells into a transient,
chordate-specific organ called the notochord.64 They found that ntla is required not only for
specification of the mesoderm cells into a notochord, but also for maturation of notochord
progenitors into highly vacuolated tissues.64 Uncaging morpholinos in a subset of the cells in the
experiment selectively redirected the irradiated cells to form medial floor plate cells. These
experiments demonstrated the spatio-temporal control of photocaged morpholinos.
Another aim of the Chen lab was to determine what factors affect the stability of cMOs in
vitro. To this end, a series of cMOs was built with varying lengths as well as both blunt and
staggered ends. It was found that “blunt” hairpin cMOs have higher caging efficiencies than
their “staggered” counterparts due to greater stabilization of the intramolecular MO/inhibitor
duplex. Binding free energy calculations were done to determine the stability of the in vitro ntla
MO/inhibitor. Values between ∆G = -12 and -14 kcal/mol yielded optimum balance between
caged and uncaged activities. A number of factors can affect activity differences between
morpholinos, such as divergent photolysis pathways, differences in inhibitor dissociation rates,
etc. in vivo. It is currently unclear how all these factors might benefit or inhibit activity
difference in vivo. Using this knowledge, the Chen lab was able to develop cMOs for three
29
additional genes and show effective uncaging and phenotype formation.
Some downsides of the technique are that long periods of exposure to UV light have
phototoxicity issues and this type of irradiation only offers 2D spatial control. Two-photon
excitation uses wavelengths above 700 nm and provides greater special resolution than one-
photon excitation (Figure 20). Two-photon excitation uses lower energy IR light at
approximately double the wavelength used in one-photon photolysis. Because cells and tissues
are relatively transparent to IR light less cell damage is incurred as irradiation times are
dramatically reduced and deeper penetration into tissues can be achieved. The use of a two-
photon sensitive caging group would therefore provide an improved method for uncaging cMOs,
since the DMNB photolinker is insufficient to be used for two-photon applications because of its
low two-photon cross section. With this in mind, Chen et al. prepared a BHQ-based bifunctional
two-photon derivative (46) to test for its biological application (Figure 21).65 While the BHQ-
caged morpholino was shown to photoactivate and inhibit translation as intended, long
irradiation times and insufficient uncaging led the group to seek a better photoremovable
protecting group for use in future studies.
Figure 20: Three-dimensional spatial selectivity of single vs. two-photon excitation
30
Figure 21: Structure of BHQ-based caged morpholino
As a follow-up to this work, we envisioned using a different two-photon photoprotecting
group as a linker for use in caging MOs. A cyano-based derivative (CyHQ) of BHQ was chosen
for study because of its larger one-photon cross section and higher molar absorptivity than
BHQ.6 These features of CyHQ will provide researchers with a photolinker with better uncaging
abilities with the BHQ analog and faster irradiation times resulting in less cell damage. The δu
(GM) for CyHQ (0.17) is also over the 0.1 GM limit suggested for biological application.66 With
this in mind, a synthetic strategy similar to the BHQ analog used in the Chen lab has been
followed.
The synthesis of the CyHQ linker began by protecting the phenol of 47 with a
benzenesulfonyl group in 60% yield (Figure 22). Next, the 2-methyl group was oxidized with
selenium dioxide to give the aldehyde 48 in 88% yield. The aldehyde was allylated with Barbier
conditions to give the terminal alkene product 49 in 78% yield. Sonication was needed to clean
the indium powder before it would react with allyl bromide to give the metal-halogen complex,
which was then added dropwise to a mixture of the substrate in THF. The alkene was first
dihydroxylated and then oxidatively cleaved to give the aldehyde 50 albeit in moderate yield
(45%). Presumably, over-oxidation led to byproducts such as the α-hydroxyketone.7 The
oxidized product was subjected to indirect reductive amination using 2 M methylamine in THF
under nitrogen giving the amino alcohol product 51. The amine first reacts with the carbonyl
31
group to form a hemiaminal species, which subsequently loses one molecule of water in a
reversible manner via elimination to form the imine. Sodium triacetoxyborohydride was then
used to reduce the imine to obtain the product. Taking advantage of the nucleophilicity of the
nitrogen atom, methyl adipoyl chloride was added dropwise to a mixture of the substrate in
dichloromethane. DIPEA was slowly added neutralize the solution (as a product of the reaction
is HCl) and push the reaction to completion and give 52 in 78% yield. Next, activation of the
secondary hydroxyl group with carbonyl diimidazole in dichloromethane yielded the activated
imidazole group. Subsequently, in basic conditions 6-amino-N-(prop-2-ynyl)hexanamide was
coupled to give the propargyl intermediate 53 in 28% yield. Saponification of the methyl ester
was performed which also deprotected the benzenesulfonyl group. Succinimide coupling on the
carboxylic acid has been attempted with EDCI, N-hydroxysuccinimide, and DMF without
success so far. Current work is ongoing in synthesizing the final succinimide coupled product 54
which will be reacted with a 5'-amine inhibitor sequence and then with a morpholino sequence
via click chemistry to form the caged morpholino 57 to be tested in various applications (Figure
23).
32
Figure 22: Synthesis strategy of photolabile CyHQ linker (a) benzenesulfonyl chloride, DIPEA, CH2Cl2, 60%; (b) SeO2, dioxane, 80 °C, 88%; (c) In powder, allyl bromide, THF (sonication), 78%; (d) K2OsO4·2H2O, lutidine, dioxane, H2O; then NaIO4, 43%; (e) methylamine (aq), NaBH(OAc)3, MeOH, H2O, 40%; (f) methyl adipoyl chloride, DIPEA, CH2Cl2, 78%; (g) carbonyl diimidazole, CH2Cl2, 78%; then 6-amino-N-(prop-2-ynyl)hexanamide, DIPEA, CH2Cl2, 78%; (h) 0.2 N NaOH (aq.), THF, MeOH; (i) N-hydroxysuccinimide, EDCI, DMF
33
Figure 23: Proposed synthesis of CyHQ-caged morpholino
34
CHAPTER 4
Experimental Section
All reagents and solvents were purchased from commercial sources and used without
further purification with the following exceptions. THF was dried by passing it through
activated alumina under nitrogen pressure (Solv-tek, Berryville, VA). Pyridine and acetonitrile
were refluxed with calcium hydride under nitrogen and then distilled. Benzenesulfonyl chloride
was distilled before use. 1HNMR and 13C NMR spectra were recorded on a Varian Mercury Plus
400 MHz spectrometer using tetramethylsilane (TMS) as an internal standard. Chemical shift
data for the proton resonances were reported in parts per million (δ) relative to internal standard
TMS (δ 0.00). Chromatographic solvent proportions are expressed on a volume/volume basis.
UV spectra were recorded on a Cary 300 Bio UV-Visible spectrophotometer (Varian). Mass
spectrometry was performed on a Sciex API-1 Plus quadrupole mass spectrometer with an
electrospray ionization source. Standard PBS buffer solutions were made and titrated to
appropriate pH values. Thin layer and column chromatography were performed on precoated
silica gel 60 F254 plates (Sorbent Technologies) and 230-400 mesh silica gel 60 (Sorbent
Technologies), respectively.
35
Procedure for titration curves for XHQ-OAc derivatives
The pKa of the phenolic proton of CHQ-OAc and CyHQ-OAc was determined by measuring the
UV absorbance spectra of 100 µM substrate in PBS-buffered solutions (1 mL) set to pH values
between 2 and 10 in quartz cuvettes. Solutions of phosphate buffered saline (PBS) were titrated
to the desired pH by using 1 M HCl or 1 M NaOH. The absorbance at 369 and 363 nm (for
CHQ-OAc and CyHQ-OAc, respectively), which corresponds to the phenolate form of the
compound, was plotted against the buffer pH, and the pKa was calculated from the half-
equivalence point of the resulting titration curves.
36
(8-chloro-7-hydroxyquinolin-2-yl)methyl acetate (18)
Quinoline 17 was dissolved in methanol and conc. TFA was added dropwise to the solution. The
solvent was then removed in vacuo. The reaction was monitored by TLC. Upon completion, the
mixture was neutralized with 1 M NaOH. The solvent was removed in vacuo and the residue
was subjected to silica gel column chromatography (hexanes/ethyl acetate 2:1) to yield 18 as a
white solid (70%).
1H NMR (400 MHz, CD3OD) δ 8.18 (d, J = 8.0 Hz, 1H), 7.67 (d, J = 8.6 Hz, 1H), 7.37 (d, J =
8.0 Hz, 1H), 7.23 (d, J = 8.6 Hz, 1H), 5.35 (s, 2H), 2.16 (s, 3H).
Literature values7
1H NMR (400 MHz, CDCl3) δ 8.13 (1H, d, J = 8.5 Hz), 7.68 (1H, d, J = 8.5 Hz) 7.40 (1H, d, J
= 8.5 Hz) 7.34 (1H, d, J = 9.0 Hz) 5.46 (2H, s), 2.23 (3H, s)
37
38
8-cyano-7-hydroxyquinolin-2-yl)methyl acetate (24)
Quinoline 23 was dissolved in methanol and conc. TFA was added dropwise to the
solution. The solvent was then removed in vacuo. The reaction was monitored by TLC. Upon
completion, the mixture was neutralized with 1 M NaOH. The solvent was removed in vacuo
and the residue was subjected to silica gel column chromatography (hexanes/ethyl acetate 2:1) to
yield 34 as an off-white solid (64%).
1H NMR (400 MHz, acetone-d6) δ 8.36 (d, J = 8.4 Hz, 1H), 8.11 (d, J = 9.0 Hz, 1H), 7.51 (d, J =
8.4 Hz, 1H), 7.43 (d, J = 9.0 Hz, 1H), 5.39 (s, 2H), 2.20 (s, 3H)
Literature values7
1H NMR (400 MHz, CDCl3) δ 8.14 (1H, d, J = 8.4 Hz), 7.92 (1H, d, J = 8.8Hz), 7.42 (1H, d, J
= 8.8 Hz), 7.262 (1H, d, J = 8.4 Hz), 5.45 (2H, s), 2.26 (3H, s)
39
NHO
CN
OAc
40
(7-hydroxy-8-nitroquinolin-2-yl)methyl acetate (30)
Quinoline 29 was dissolved in methanol and conc. TFA was added dropwise to the solution. The
solvent was then removed in vacuo. The reaction was monitored by TLC. Upon completion, the
mixture was neutralized with 1 M NaOH. The solvent was removed in vacuo and the residue
was subjected to silica gel column chromatography (hexanes/ethyl acetate 7:3) to yield 30 as a
light brown solid (77%).
1H NMR (400 MHz, acetone-d6) δ 8.39 (d, J = 8.5 Hz, 1H), 8.04 (d, J = 9.1 Hz, 1H), 7.53 (d, J =
8.5 Hz, 1H), 7.47 (d, J = 9.1 Hz, 1H), 5.31 (s, 2H), 2.16 (s, 3H)
Literature values7
1H NMR (400 MHz, DMSO-d6) δ 8.42 (1H, d, J = 8.4 Hz), 8.04 (1H, d, J = 9.2 Hz), 7.48 (1H, d,
J = 8.4 Hz), 7.41 (1H, d, J = 8.8 Hz), 5.27 (2H, s), 2.15 (3H, s)
41
42
1-(6-bromo-7-(methoxymethoxy)-2-oxo-2H-chromen-4-yl)ethane-1,2-diyl
dimethanesulfonate (40)
Under a nitrogen atmosphere, 39 (466 mg, 1.35 mmol) was stirred with TEA (0.94 mL,
6.75 mmol) into anhydrous THF. Mesyl chloride (387 mg, 3.38 mmol) was added dropwise over
10 minutes and the reaction was stirred for 4 h at room temperature. The solvent was removed in
vacuo and the resulting gel was dissolved into ethyl acetate. Resulting solution was washed
with water, brine and dried over anhydrous NaSO4. Resulting crude product was purified by
silica gel column chromatography (hexanes/ethyl acetate 9:1) to 40 as a clear gel (555 mg, 1.11
mmol, 82%).
1H NMR (400 MHz, acetone) δ 8.22 (s, 1H), 7.22 (s, 1H), 6.63 (s, 1H), 6.39 (m, J = 6.6, 2.2 Hz,
1H), 5.46 (s, 2H), 4.80 (dd, J = 12.3, 2.7 Hz, 1H), 4.67 (dd, J = 12.3, 6.8 Hz, 1H), 3.50 (s, 3H),
3.36 (s, 3H), 3.24 (s, 3H).
MS-ESI (m/z): [M + H]+ calcd for C15H18BrO10S2 500.95 and 502.95 ; found, 500.80 and
502.80.
43
O
Br
OMOMO
MsO
OMs
44
1-(6-bromo-7-(methoxymethoxy)-2-oxo-2H-chromen-4-yl)ethane-1,2-diyl
bis(dimethylcarbamodithioate) (41)
Coumarin 40 was dissolved into methanol and sodium dimethyldithiocarbamate was
added. The reaction was stirred at room temperature for 18 hrs. Solvent was removed and
resulting gel was dissolved into CHCl3. Solvent was washed with water, brine, and dried over
anhydrous Na2SO4. Crude product was purified by silica gel column chromatography
(hexanes/ethyl acetate 7:3) to give 41 as an orange gel.
1H NMR (400 MHz, CDCl3) δ 8.28 (s, 1H), 7.13 (s, 1H), 6.56 (s, 1H), 5.80 (t, J = 7.6 Hz, 1H),
5,31 (s, 2H), 4.07 (dd, J = 13.8, 7.9 Hz, 1H), 3.91 (dd, J = 13.8, 7.2 Hz, 1H), 3.56 (s, 3H), 3.52
(s, 3H), 3.50 (s, 3H), 3.39 (s, 3H), 3.31 (s, 3H)
MS-ESI (m/z): [M + H]+ calcd for C19H24BrN2O4S4 550.98 and 552.98 ; found, 552.60 and
552.80.
45
46
8-cyano-2-methylquinolin-7-yl benzenesulfonate (58)
A mixture of 47 (2.5 g, 13.6 mmol) and N,N-diisopropylethylamine (4.74 mL, 27.2
mmol) were dissolved in anhydrous CH2Cl2 (10 mL), and the solution was cooled to 0 °C.
Benzenesulfonyl chloride (3.0 g, 17 mmol) in anhydrous CH2Cl2 (5 mL) was added over 10 min,
and the reaction mixture was stirred for 14 h at room temperature under nitrogen. The solvent
was removed in vacuo, and the residue was dissolved in EtOAc and washed twice with saturated
aq NaHCO3 and then dried over anhydrous Na2SO4. Solvent was removed in vacuo, and the
residue was purified silica gel column chromatography (Hexanes/EtOAc 1:1) to yield 58 as a
white solid (2.65g, 8.17 mmol, 60%).
1H NMR (500 MHz CDCl3) δ 8.10 (d, J = 8.5 Hz, 1H), 8.04 (d, J = 8.4 Hz, 1H), 8.02 (t, J = 8.5
Hz, 2H), 7.72 (t, J = 7.5 Hz, 1H), 7.68 (d, J = 9.0 Hz, 1H), 7.58 (t, J = 7.9 Hz, 2H), 7.41 (d, J =
8.5 Hz, 1H), 2.77 (s, 3H)
13C NMR (126 MHz, CDCl3) δ 163.2, 152.6, 147.7, 136.1, 135.2, 133.5, 129.6, 128.8, 124.8,
123.9, 121.1, 112.9, 106.0, 25.7
HRMS-ESI (m/z): [M + H]+ calcd for C17H13N2O3S 325.0647; found, 325.0639.
47
48
49
50
8-cyano-2-formylquinolin-7-yl benzenesulfonate (48)
A mixture of SeO2 (1.14 g, 10.2 mmol) and 1,4-dioxane (10 mL) was heated to 80 °C.
Quinoline 58 (2.2 g, 6.8 mmol) in 1,4-dioxane (5 mL) was added. After stirring at 80 °C for 24 h,
the reaction was cooled and vacuum filtered. The filtrate was collected and concentrated, leaving
a yellow solid. Purification by silica gel column chromatography (hexanes/ethyl acetate 4:1)
gave 48 as a white solid (2.03 g, 6 mmol, 88%).
1H NMR (500 MHz, CDCl3) δ 10.25 (s, 1H), 8.44 (d, J = 8.4 Hz, 1H), 8.20 (d, J = 9.1 Hz, 1H),
8.16 (d, J = 8.4 Hz, 1H), 8.04 (dd, J = 8.4, 1.0 Hz, 2H), 7.94 (d, J = 9.1 Hz, 1H), 7.77 (t, 1H),
7.63 (t, 2H)
13C NMR (126 MHz, CDCl3) δ 192.7, 154.3, 153.7, 147.4, 138.0, 135.5, 134.4, 133.7, 129.8,
128.9, 128.2, 124.8, 119.0, 112.0, 107.4
HRMS-ESI (m/z): [M + H]+ calcd for C17H11N2O4S 339.0440; found, 339.0431.
51
52
53
54
8-cyano-2-(1-hydroxybut-3-enyl)quinolin-7-yl benzenesulfonate (49)
In powder (340 mg, 2.96 mmol) and allyl bromide (385µl, 4.45 mmol) and THF (10 ml)
were sonicated until no more powder was present. Quinoline 48 (500 mg, 1.48 mmol) was then
added and stirred for 1 h. THF was removed in vacuo, and the residue was dissolved into EtOAc,
which was dried over anhydrous Na2SO4. Solvent was removed in vacuo, and the residue was
purified by silica gel column chromatography (hexanes/ethyl acetate 2:1) to yield 49 as a white
solid (440 mg, 1.16 mmol, 78%)
1H NMR (500 MHz, CDCl3) δ 8.24 (t, J = 7.2 Hz, 1H), 8.10 (dd, J = 9.1, 4.6 Hz, 1H), 8.05-8.00
(m, 2H), 7.78 (d, J = 8.1 Hz, 1H), 7.72 (t, 1H), 7.64-7.58 (t, 2H), 7.57 (d, J = 8.5 Hz, 1H), 5.86
(ddt, J = 17.2, 10.2, 7.0 Hz, 1H), 5.17-5.07 (m, 2H), 5.05-4.99 (m, 1H), 2.81-2.70 (m, 1H), 2.62-
2.51 (m, 1H)
13C NMR (126 MHz, CDCl3) δ 165.6, 153.0, 146.3, 137.0, 135.3, 134.5, 133.6, 133.5, 129.6,
129.6, 128.8, 125.7, 122.1, 120.5, 118.5, 112.3, 106.2, 72.6, 55.3, 42.4
HRMS-ESI (m/z): [M + H]+ calcd for C20H17N2O4S 381.0909; found, 381.0908.
55
56
57
58
8-cyano-2-(1-hydroxy-3-oxopropyl)quinolin-7-yl benzenesulfonate (50)
To a solution of 49 (500 mg, 1.31 mmol) in dioxane/water (3:1, 8 mL) were added 2,6-
lutidine (0.382 mL, 3.3 mmol), K2OsO4•2H2O (6 mg, 0.016 mmol), and after 15 mins NaIO4
(1.12 g, 5.24 mmol). The reaction was stirred at 25°C and monitored by TLC. After the reaction
was complete, water (10 mL) and CH2Cl2 (20 mL) were added. The organic layer was separated,
and the aqueous layer was extracted by CH2Cl2 (10 mL) three times. The combined organic
phase was washed with brine and dried over anhydrous Na2SO4. The solvent was removed, and
the product was purified with silica gel column chromatography to afford 50 (215 mg, 0.56
mmol, 43%) as a colorless oil.
1H NMR (500 MHz, CDCl3) δ 9.95 (s, 1H), 8.28 (d, J = 8.6 Hz, 1H), 8.10 (t, J = 7.3 Hz, 1H),
8.05-8.01 (m, 2H), 7.76 (m, J = 8.8, 6.0 Hz, 3H), 7.61 (m, J = 8.0 Hz, 2H), 5.43 (dd, J = 7.4, 4.2
Hz, 1H), 3.24 (dd, J = 17.4, 3.9 Hz, 1H), 3.10 (dd, J = 17.0, 6.9 Hz, 1H)
13C NMR (126 MHz, CDCl3) δ 201.5, 164.7, 153.1, 146.5, 137.4, 135.3, 134.6, 133.6, 129.7,
128.8, 125.7, 122.3, 120.4, 112.4, 69.6, 50.2
HRMS-ESI (m/z): [M + H]+ calcd for C19H15N2O5S 383.0702; found, 383.0695.
59
60
61
62
8-cyano-2-(1-hydroxy-3-(methylamino)propyl)quinolin-7-yl benzenesulfonate (51)
To a solution of anhydrous THF (15 ml) was added 50 (0.24 g, 0.63 mmol) and 2 M
methyl amine (1.89 mL, 40 mmol). The reaction was stirred under nitrogen for 3 h at room
temperature. NaBH(OAc)3 (267 mg, 1.26 mmol) was added slowly to the solution and stirred for
an additional 30 minutes. After completion, the THF was removed in vacuo and the product was
dissolved in ethyl acetate and washed with water, brine, and dried over anhydrous Na2SO4. The
product was purified with silica gel column chromatography (chloroform/acetone 1:1) to afford
51 (100 mg, 0.25 mmol, 40%) as a yellowish oil.
1H NMR (500 MHz, CDCl3) δ 8.27 (d, J = 8.5 Hz, 1H), 8.11 (d, J = 9.0, 1H), 8.03 (d, J = 8.0
Hz, 2H), 7.77 (dd, J = 14.9, 8.3 Hz, 2H), 7.61 (t, J = 8.4, 3H), 5.19 (dd, J = 9.2, 3.4 Hz, 1H),
3.96 (dd, J = 10.8, 4.4 Hz, 2H), 2.28-2.19 (m, 1H), 1.96 (dd, J = 20.8, 14.2 Hz, 2H), 1.60, (m,
4H)
13C NMR (126 Mhz, CDCl3) δ 165.8, 153.0, 137.3, 135.3, 134.6, 133.6, 129.7, 128.8, 125.7,
124.0, 122.2, 120.1, 112.29, 72.7, 60.9, 39.5
HRMS-ESI (m/z): [M + H]+ calcd for C20H20N3O4S 398.1175; found, 398.1184.
63
64
65
66
Methyl 6-((3-(8-cyano-7-((phenylsulfonyl)oxy)quinolin-2-yl)-3-
hydroxypropyl)(methyl)amino)-6-oxohexanoate (52)
A solution of 51 (75 mg, 0.19 mmol) and N,N-diisopropylethylamine (42 µl, 0.24 mmol)
were dissolved in anhydrous CH2Cl2 (5 mL), and the solution was cooled to 0 °C. Methyl
adipoyl chloride (49 mg, 0.25 mmol) was added over 5 min, and the reaction mixture was stirred
for 6 h at room temperature under nitrogen. Solvent was removed in vacuo, and residue was
dissolved in EtOAc and washed twice with saturated aq NaHCO3 and then dried over anhydrous
Na2SO4. Solvent was removed in vacuo, and the residue was purified by silica gel column
chromatography (chloroform/acetone 3:1) to yield 52 (80 mg, 0.15 mmol, 78%) as a yellow oil.
1H NMR (500 MHz, CDCl3) δ 8.26-8.22 (m, 1H), 8.08 (t, J = 6.3, 1H), 8.02 (dd, J = 8.5, 1.1 Hz,
2H), 7.93-7.88 (m, 1H), 7.74 (t, J = 10.8, 4.3 Hz, 1H) 7.70 (d, J = 9.0 Hz, 1H), 7.60 (dd, J = 8.2,
7.7 Hz, 2H), 5.36 (s, 1H), 4.82 (dd, J = 8.7, 3.4 Hz, 1H), 3.91 (ddd, J = 14.2, 9.4, 4.8 Hz, 1H),
3.65 (s, 3H), 3.33 (ddt, J = 24.7, 14.0, 5.3 Hz, 1H), 3.00 (s, 3H), 2.37-2.29 (m, 4H), 2.03 (tt, J =
9.9, 7.0 Hz, 1H), 1.94-1.78 (m, 2H), 1.65-1.53 (m, 5H)
13C NMR (126 MHz, CDCl3) δ 174.0, 167.3, 152.8, 146.6, 136.8, 135.2, 133.6, 129.6, 128.8,
125.5, 121.6, 120.6, 112.7, 70.6, 55.6, 44.5, 35.9, 34.1, 33.8, 33.8, 33.0, 33.0, 24.6, 24.6, 24.3
HRMS-ESI (m/z): [M + H]+ calcd for C27H29N3O7S 540.1804; found, 540.1798.
67
68
69
70
Methyl 6-((3-(8-cyano-7-((phenylsulfonyl)oxy)quinolin-2-yl)-3-
hydroxypropyl)(methyl)amino)-6-oxohexanoate (59)
Quinoline 52 (80 mg, 0.15 mmol) was dissolved in anhydrous CH2Cl2 (1 mL) and added
to carbonyl diimidazole (73 mg, 0.45 mmol) in anhydrous CH2Cl2 (1.5 mL). The reaction
mixture was stirred for 4 h at room temperature under nitrogen, diluted with CH2Cl2, washed two
times with water, and dried over anhydrous MgSO4. Solvent was removed in vacuo to yield
activated imidazole carbamate crude as a gum. This crude material was purified by silica gel
column chromatography (CHCl3/acetone 1:1) to yield 59 (68 mg, 0.11 mmol, 74%) as a yellow
gum.
1H NMR (400 MHz, CDCl3) δ 8.35 (d, 1H), 8.25 (s, 1H), 8.12 (d, J = 9.1 Hz, 1H), 8.02 (d, J =
7.5 Hz, 2H), 7.77 (dd, J = 16.4, 9.3 Hz, 2H), 7.68 (d, J = 8.6, 1H), 7.61 (t, J = 7.9, 2H), 7.53 (s,
1H), 7.12 (s, 1H), 6.17 (t, J = 6.4, 1H), 3.65 (s, 3H), 3.56 (dq, J = 20.4, 6.9 Hz, 1H), 3.02 (s, 3H)
2.62-2.40 (m, 2H), 3.29-2.14 (m, 4H), 1.76-1.46 (m, 5H)
HRMS-ESI (m/z): [M + H]+ calcd for C31H31N5O8S 634.1972; found, 634.1986.
71
72
73
Methyl 14-(8-cyano-7-((phenylsulfonyl)oxy)quinolin-2-yl)-17-methyl-5,12,18-trioxo-13-oxa-
4,11,17-triazatricos-1-yn-23-oate (60)
Quinoline 59 (66 mg, 0.1 mmol) was dissolved in anhydrous CH2Cl2 (1.5 mL) and N,N-
diisopropylethylamine (33 µL, 0.190 mmol). To this mixture was added 6-oxo-6-(prop-2-
ynylamino)hexan-1-aminium hydrochloride salt (36 mg, 0.177 mmol) in anhydrous CH2Cl2 (1.4
mL). The reaction mixture was stirred overnight at room temperature under nitrogen. Solvent
was then removed in vacuo. Resulting gum was dissolved into CHCl3 and washed with 1 N HCl,
then with 5% saturated aq NaHCO3, and brine. The organic layer was then dried over anhydrous
Na2SO4. Solvent was removed in vacuo and the residue was purified via silica gel
chromatography (chloroform/acetone 4:1) to yield 60 (21 mg, 0.029 mmol, 28%) as a yellow
gum.
1H NMR (400 MHz, CD3OD) δ 8.07 (t, J = 8.2 Hz, 2H), 7.74 (t, J = 7.6 Hz, 2H), 7.65-7.58 (m,
1H), 7.55 (t, J = 7.4 Hz, 1H), 7.22 (t, J = 7.9 Hz, 1H), 7.01 (d, J = 8.8 Hz, 1H), 6.92 (d, J = 7.2
Hz, 1H), 5.78 (s, 1H), 3.91 (t, J = 4.1 Hz, 3H), 3.63 (d, J = 5.1 Hz, 2H), 3.60-3.51 (m, 1H), 3.20
(s, 3H), 3.15-3.07 (m, 1H), 2.82 (t, J = 7.0 Hz, 1H), 2.44-2.07 (m, 10H), 1.73-1.17 (m, 12H).
HRMS-ESI (m/z): [M + H]+ calcd for C32H43N5O9S 734.2860; found, 734.2859.
74
75
76
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