Synthesis and Characterization of Novel Self-Delivering Amino Acid-RNA Conjugates for the ...

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Synthesis and Characterization of Novel Self-Delivering Amino Acid-RNA
Conjugates for the Development of Potent siRNA Prodrugs

Jovanka J. Bogojeski
Department of Chemistry, McGill University
Montreal, QC, Canada
October, 2016

A thesis submitted to McGill University in partial fulfilment of the requirements of the degree of
Doctor of Philosophy

© Jovanka J. Bogojeski, 2016
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For my first teacher, Mira Đorđeska.
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Copyright Statement
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Permission to reprint the images used in Figure 1.4 was obtained directly via email
communication from RSC Publishing.
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Nature Publishing Group and Copyright Clearance Center: 4082650556813.
A license number for the reprint of the images used in Figures 1.13 and 1.16 was obtained from
Nature Publishing Group and Copyright Clearance Center: 4082660445787.
Permission for the reprint of the image used in Figure 1.14 was obtained from ACS Publications
and Copyright Clearance Center.
The image reprinted in Figure 1.15 is free to redistribute under a Creative Commons License:
https://creativecommons.org/licenses/by/4.0/.
Some content in Chapter 2 has been adapted from Johnsson, R.; Lackey, J. G.; Bogojeski, J. J.;
Damha, M. J., New light labile linker for solid phase synthesis of 2′-O-acetalester
oligonucleotides and applications to siRNA prodrug development. Bioorganic & Medicinal
Chemistry Letters 2011, 21 (12), 3721-3725. © 2011 Elsevier Ltd. Elsevier grants authors
publishing in Elsevier journals rights to use their works for scholarly purposes according to the
following URL:
https://www.elsevier.com/__data/assets/pdf_file/0007/55654/AuthorUserRights.pdf. A license
number for the reprint of data in Figure 2.3 was obtained from Elsevier and Copyright Clearance
Center: 4085410520073.

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Abstract
Small interfering RNA (siRNA) has the ability to silence virtually any gene of interest.
The canonical structure, however, is negatively charged and hydrophilic. It is thus repulsed by
the negatively charged phosphodiesters of the cellular membrane and prevented from penetrating
the lipophilic membrane core. The main goal of this thesis is to synthesize siRNA conjugates
containing 2'-sugar modifications that reduce the overall negative charge of the molecule and
cause its unassisted cellular uptake, and to utilize these modifications to synthesize a biolabile
analogue (a promoiety) in a prodrug siRNA (proRNA) construct.
To this end, RNA conjugates were synthesized containing various 2'-O-amino acid acetal
ester (2'-O-AAE) promoieties. The 2'-O-phenylalanine (2'-O-Phe) AAE was most stable in the
aqueous and alcoholic RNA purification and handling conditions to which the 2'-O-AAEs
investigated were exposed. A comparison of hydrolysis rates between the 2'-O-Phe AAE and a
2'-O-hydrocinnamic acetal ester suggested that 2'-O-Phe AAE hydrolysis occurs in aqueous
buffer due to the alpha amino group acting as a general acid catalyst. Anhydrous and aprotic
deprotection and handling solutions were employed, including FMOC and cyanoethyl
elimination with dry triethylamine/acetonitrile, to yield a poly-thymidine oligonucleotide
containing a 2'-O-Phe AAE insert, with minimal evidence of premature cleavage.
In order to validate the incorporation of the 2'-O-Phe AAE modification in a self-
delivering proRNA construct, the analogous 2'-N-acyl-L-phenylalanine modification was
assessed for its ability to cause RNA conjugates to penetrate cellular membranes. Using flow
cytometric fluorescence resonance energy transfer (FCET), naked cyanine-3/5 labeled RNA
conjugates containing 2'-N-acyl-L-phenylalanine modifications were found to penetrate human
glial cells to a much greater extent than the unmodified counterpart. This work suggests that the
analogous 2'-O-Phe AAE modification could also cause increased cellular uptake of RNA
conjugates.
2'-N-acyl-L-phenylalanine modification was incorporated into internal positions of
siRNAs targeting endogenous genes B-cell lymphoma-2 (bcl-2) and downregulated in renal cell
carcinoma (drr), and the siRNAs were transfected into human colonic cells and human glial
cells, respectively. Downregulation was dependent on the placement of these inserts; insertion
of biolabile 2'-O-Phe AAEs at positions particularly vulnerable to modification may improve
potencies. A self-delivering siRNA construct comprised of a phosphorothioate tail and thirteen
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2'-N-acyl-L-phenylalanine inserts achieved potent DRR downregulation in comparison to the
unmodified siRNA containing only a phosphorothioate tail. Delayed onset of silencing by our
highly modified asymmetric siRNA suggests that the construct’s silencing kinetics diverges from
that of canonical siRNA, and thus selective insertion of 2'-O-Phe AAEs may extend the duration
of action of the self-delivering tailed siRNA.
Finally, high-purity RNA was synthesized using a solid support linker and protecting
groups that are orthogonal to the 2'-O-Phe AAE modification in preparation for the synthesis of
proRNA. This work lays the foundation for the rational design of self-delivering and potent
asymmetric proRNA.

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Résumé
Les petits ARN interférents (siARN) ont la possibilité d’inhiber l’expression de
pratiquement n’importe quel gène d’intérêt. Un siARN étant hydrophile et chargé négativement,
il est soumis à une répulsion électrostatique avec les phosphodiesters de la membrane cellulaire
chargés négativement et, ainsi, n’est pas à même de traverser la membrane lipophile. L’objectif
principal de cette thèse est de synthétiser des conjugués de siARNs comportant des modifications
chimiques sur la position 2ʹ du sucre réduisant sa charge globale négative et susceptibles
d’entraîner une pénétration cellulaire sans assistance afin, à terme, de fabriquer des analogues
biolabiles (progroupes) dans une approche de synthèse de prodrogues de siARN (proARN).
Pour ce faire, des brins d’ARN contenant des progroupes d’acétalesters d’acides aminés
en position 2ʹ ont été synthétisés (2ʹ-O-AAE). L’acétalester de la phénylalanine en position 2ʹ
(2ʹ-O-Phe AAE) était la plus stable des modifications étudiées, en milieu aqueux et alcoolique
nécessaires à la purification d’ARN et face aux conditions expérimentales de manipulation. En
comparant les taux d’hydrolyse des AAE 2ʹ-O-Phe et d’acide hydrocinnamique, nous avons
estimé que l’hydrolyse des 2ʹ-O-Phe AAE en milieu aqueux s’effectue à l’aide de la fonction -
amine jouant le rôle d’un catalyseur acide. Sous des conditions de déprotection et de
manipulation anhydres et aprotiques, et ce pendant la déprotection des groupements Fmoc et
cyanoéthyles requérant l’utilisation de triéthylamine/acétonitrile secs, nous avons pu isoler un
polythymidylate contenant une modification 2ʹ-O-Phe AAE en minimisant son hydrolyse
prématurée.
Afin de valider l’intérêt de l’incorporation d’une modification 2ʹ-O-Phe AAE dans un
siARN capable de s’auto-administrer dans une cellule, nous avons testé la capacité d’une
modification analogue, la 2ʹ-N-acyl-L-phénylalanine, à induire la pénétration cellulaire d’un
conjugué d’ARN. En utilisant la technique de transfert d’énergie entre molécules fluorescentes
suivi par cytométrie en flux (FCET « flow cytometric fluorescence resonance energy transfer »),
il a été possible de voir que des conjugués « nus » d’ARN, possédant un marqueur fluorescent
cyanine3 ou 5 ainsi que des modifications 2ʹ-N-acyl-L-phénylalanine étaient capables de pénétrer
des cellules gliales humaines en plus grande proportion que les versions d’ARN non-modifié.
Cette étude suggère que la modification analogue 2ʹ-O-Phe AAE aurait un effet similaire sur la
pénétration cellulaire de conjugués d’ARN.
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La modification 2ʹ-N-acyl-L-phénylalanine fut ensuite intégrée à des siARNs ciblant les
gènes des lymphomes à cellule B (B-cell lymphoma 2, bcl-2) de cellules humaines du côlon et les
oncogènes de carcinomes à cellules rénales (downregulated in renal cell, drr) dans des cellules
gliales humaines. La régulation négative s’est révélée dépendante du positionnement de ces
insertions et la présence de groupements biolabiles 2ʹ-O-Phe AAE à des positions
particulièrement sensibles aux modifications pourrait améliorer l’efficacité des siARNs. Un
siARNs modifié avec treize incorporations de 2ʹ-N-acyl-L-phénylalanine ainsi que des liens
phosphorothioate sur la partie terminale de la chaîne oligonucléotidique (queue) s’est montré
capable de régulation négative de drr en l’absence d’agent de transfection, contrairement à des
siARNs ne contenant que des liens phosphorothioate en queue de chaîne. L’action retardée de
silençage de l’expression génétique induite par nos siARN largement modifiés suggère que la
cinétique du silençage diffère de celle des siARNs non-modifiés, et laisse ainsi entrevoir la
possibilité que l’insertion sélective de 2ʹ-O-Phe AAEs puisse accroître la durée d’action de
siARNs auto-délivrants.
Enfin, la synthèse d’ARN de grande pureté s’est effectuée à l’aide d’un lien sur support
solide et de groupements protecteurs orthogonaux à la modification 2ʹ-O-Phe AAE, et ce en
préparation de la synthèse de prodrogues de siARN. Le travail de cette thèse représente ainsi un
dévelopement important vers la conception méthodique et la préparation de prodrogues auto-
délivrantes et efficaces de siARN.

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Acknowledgements
I want to first express my appreciation to Dr. Masad Damha for his guidance, enthusiasm,
and trust in me. I will always be in debt to him for this unique and rich experience. I am grateful
for the many opportunities to convene with diverse and talented individuals and to expand my
knowledge, which were made possible to me through the Damha lab. Indeed, a large part of this
thesis would not have been possible without the graciousness of Dr. Kevin Petrecca in making
his laboratory space at the Montreal Neurological Institute available to me. I thank Dr. Phuong
Le for showing me the ropes there and for testing ‘one last compound’ several times over. Elena
Moroz in the lab of Dr. Jean-Christophe Leroux, as well as Dr. Johans Fakhoury of the Dr.
Hanadi Sleiman lab, kindly performed additional tests with high quality. I was also very
fortunate to have worked with Dr. Ken Yamada on expanding our catalogue of testable
compounds. His attention to detail improved the quality of my work a great deal. I am also
grateful for the lending hand I received from Erika Steels in making more material needed for the
dimer studies carried out in Chapter 2. Dr. Alexander Wahba and Nadim Saadeh were also
invaluable in providing mass spectrometry data to support my research over the years.
Many past and present members of the Damha lab provided assistance in editing parts of
this thesis. I thank Dr. Jory Liétard and Dr. Adam Katolik for taking time out of their post-
doctoral research to share their thoughts, and Brandon Payliss for making suggestions that were
very helpful to clarifying my introductory comments. Current members Danielle Vlaho and Dan
O’Reilly were gracious in offering their advice at a time that was convenient to me. I will miss
Danielle’s enthusiasm and Dan’s wise advice. I must also thank other current members that will
also be missed – Maryam Habibian’s funny stories, Hala Abou Assi’s calming morning talks,
and Elise Malek-Adamian’s generous smiles and laughs. I am also thankful for the time I was
able to spend with past members Dr. Jeremy Lackey (and his enthusiasm and guidance on
proRNA), Fereshteh Azizi, Pascal Gallant, Dr. Robert Donga, Dr. Richard Johnsson, Dr. Dilip
Dixit, Dr. Mathew Hassler, Dr. Glen Deleavey, Dr. Maryam Yahyaee-Anazahaee, and Dr. Saúl
Martinez-Montero who all made the lab a fun and enlightening experience when I joined. I
thank Chantal Marotte for having all the administrative answers and being available at any time.
There is so much I could say about the influence of my family, but I will sum it up by
acknowledging the fact that they taught me to believe in myself and to pursue anything my heart
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desires. Without my first teacher, Mom, I am nothing. With my best friend and sister, Nancy, I
always have the feeling of a supporter in my corner. Thank you to my brother-in-law Radoslav
for being a good friend and to my sweet nephews Miloš and Milan for lighting up my world.

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