Protected pyrimidine nucleosides for cell-specific metabolic labeling of RNA

Article abstract of DOI:10.1016/j.tetlet.2018.09.040

RNA molecules can perform a myriad of functions, from the regulation of gene expression to providing the genetic blueprint for protein synthesis. Characterizing RNA expression dynamics, in a cell-specific manner, still remains a great challenge in biology. Herein we present a new set of protected alkynyl nucleosides for cell-specific metabolic labeling of RNA. We anticipate these analogs will find wide spread utility toward the goal of understanding RNA expression in complex cellular and tissue environments, even within living animals.

Full text of DOI:10.1016/j.tetlet.2018.09.040

Accepted Manuscript
Protected pyrimidine nucleosides for cell-specific metabolic labeling of RNA
Samantha Beasley, Kim Nguyen, Michael Fazio, Robert C. Spitale
PII:
S0040-4039(18)31130-4
https://doi.org/10.1016/j.tetlet.2018.09.040
TETL 50279
DOI:
Reference:
Tetrahedron Letters
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9 July 2018
13 September 2018
14 September 2018
Please cite this article as: Beasley, S., Nguyen, K., Fazio, M., Spitale, R.C., Protected pyrimidine nucleosides for
cell-specific metabolic labeling of RNA, Tetrahedron Letters (2018), doi: https://doi.org/10.1016/j.tetlet.
2018.09.040
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Protected Pyrimidine Nucleosides for Cell-
Specific Metabolic Labeling of RNA
Leave this area blank for abstract info.
Samantha Beasley, Kim Nguyen, Michael Fazio, and Robert C. Spitale
Tetrahedron Letters
Protected pyrimidine nucleosides for cell-specific metabolic labeling of RNA
Samantha Beasleya, Kim Nguyena, Michael Fazioa, and Robert C. Spitalea,b,*
aDepartment of Pharmaceutical Sciences, University of California, Irvine. Irvine, California 92697
bDepartment of Chemistry, University of California, Irvine. Irvine, California 92697
*Corresponding author. e-mail address: rspitale@uci.edu
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
RNA molecules can perform a myriad of functions, from the regulation of gene
expression to providing the genetic blueprint for protein synthesis. Characterizing
RNA expression dynamics, in a cell-specific manner, still remains a great challenge in
biology. Herein we present a new set of protected alkynyl nucleosides for cell-specific
metabolic labeling of RNA. We anticipate these analogs will find wide spread utility
toward the goal of understanding RNA expression in complex cellular and tissue
environments, even within living animals.
Available online
Keywords:
RNA
Metabolic Labeling
Alkynyl Nucleosides
.

2
Tetrahedron
1. Introduction
refractory to incorporation into nascent RNA due to the
placement of the PGA-specific protecting group (Fig. 1, B).
Cytidine and uridine are more reasonable choices for metabolic
labeling as they are evenly distributed throughout the genome,
and as we have seen before bulky substituents can prevent
processing by endogenous kinase enzymes, such as
uridine/cytidine kinase 1, thus preventing production of the
triphosphate.8 Furthermore, functionalizing the 5-position with
an alkyne is not predicted to alter the sugar orientation or the
anti-position of the nucleobase to disrupt RNA structure.
Cells exist in complex environments in Nature. Understanding
the molecular signatures of individual cell types has emerged as a
key challenge in biology and medicine. However, precise
chemical tools to do so are still overall lacking.1 RNA molecules,
once regarded as transient messengers between the genome and
proteome, have now emerged as key players in regulating cell-
fate decisions, development, cancer and neurological disorders.2-4
Cell-specific profiling of RNA molecules, in the form of gene
expression analysis, is extremely challenging. Such analyses rely
on cell-sorting techniques, which have been demonstrated to be
both notoriously dirty and often detrimental to the stability of
RNA molecules.5, 6 Such obstacles manifest themselves in high
false-positive rates in data analysis that can often mislead
biologists aiming to understand changes in gene expression or the
function of RNA.7 Cell-specific metabolic labeling has emerged
as a powerful technique in understanding the proteome and
metabolome, yet, with few exceptions, analogous methods for
RNA are still lacking. We recently reported the utility of
protected nucleoside and nucleobase analogs for cell-specific
metabolic labeling of RNA (Fig. 1, A).8, 9 In such a case an
exogenous enzyme can be expressed, within a cell-type of
interest, to reveal RNA metabolic intermediates containing bio-
orthogonal chemical handles. This approach allows the imaging
and isolation of nascent and cell-type specific RNA. Such an
approach is extremely powerful as it opens the door for cell-
specific RNA profiling with the potential to reveal novel
functions and gene expression signatures.
We previously demonstrated the enzyme uracil
phosphoribosyl transferase (UPRT) can be used to incorporate 5-
ethynyluracil into nascent RNA.8 Although useful, UPRT is
expressed in many lower organisms and as such the host
pyrimidine salvage pathway compromises its utility inside these
animals.10 As a viable alternative, we matched a N6-
phenylacetyladenosine analog with a 2’-azido handle (2’-AzA),
with an enzyme, penicillin G amidase (PGA).8 PGA is a
bacterial-specific enzyme that cleaves an amide bond.11 In our
work, cellular RNA is endowed with azido functionality only
when cells express PGA. While useful, we reasoned extending
the toolset of the PGA enzymatic removal paradigm could further
reveal additional nucleosides that can be used for metabolic
labeling of RNA.
Figure 1. Protected analogs for cell-specific metabolic labeling
of RNA. (A) Schematic of cell-specific metabolic labeling of
RNA. (B) Structures of analogs synthesized herein and their
proposed reactions with PGA.
PAC-5EC was synthesized in five steps starting from
commercially available 5-iodocytidine (5-IC; ESI). 5-IC was
subjected to a Sonogashira coupling to install the trimethylsilyl
alkyne handle at the 5-position, followed by protection of the
sugar alcohols with tert-butyldimethylsilyl groups. To synthesize
the PAC portion, 2-phenylacetic acid was treated with DCC to
yield 2-phenylacetic anhydride. The exocyclic amine of the silyl-
protected cytidine was then reacted with 2-phenylacetic
anhydride to install the phenylacetyl group, followed by
tetrabutylammonium fluoride deprotection of the silyl groups to
yield the final product, PAC-5EC, in an overall yield of 19%.
PACQM-5EU was synthesized in 13 steps starting from
commercially available uridine (ESI). Iodination of uridine
yielded 5-iodouridine, which was then subjected to a Sonogashira
coupling to install the trimethylsilyl alkyne handle at the 5
position. To synthesize the PAC portion, para-
aminobenzylalcohol was coupled to phenylacetic acid using 2-
ethoxy-1-ethoxycarbonyl-1,2-dihyrdoquinoline to generate N-(4-
(hydroxymethyl)phenyl)-2-phenylacetamide. The benzyl alcohol
was then brominated with PBr3, followed by Finkelstein reaction
to install the iodine. The iodo-intermediate was then coupled with
the TMS-protected uridine using potassium carbonate, followed
by tetrabutylammonium fluoride deprotection of the TMS group
to yield the final product, PACQM-5EU, in an overall yield of
11%.
We reasoned that the azido moiety of 2’-AzA could be prone
to reduction inside the cell, and more complicated environments
in vivo, which would negate its ability to be utilized for affinity
and purification experiments. Azide reduction has been observed
with many other azido-containing analogs, and modified
nucleosides, and can have a gross effect on their activity in cells
and animals.12 We also reasoned that adenosine analogs would be
highly biased towards RNAs that have long polyA tails, a
hallmark of messenger RNAs and long non-coding RNAs. Our
own work with 2’-Az-A has demonstrated that it is largely
incorporated into polyA tails.13 This is a well-documented
problem when understanding RNA expression levels through
RNA sequencing – the current state of the art method of
characterizing RNA expression.14 Together, these matters
underscore the need to expand the scope of novel analogs for
cell-specific metabolic labeling of RNA.
2. Results and Discussion
We sought to utilize a different nucleobase structure for
metabolic labeling of RNA. Penicillin G amidase is our current
enzyme of choice and as such we decided to design and
synthesize an alkynyl cytidine and uridine, which would be

With PAC-5EC and PACQM-5EU in hand, we next focused
on testing whether these analogs would display selectivity for
metabolic labeling inside cells (Fig. 2). Briefly, HEK293T cells
were grown in culture either with or without PGA transfected
with plasmid. To the cells, 200uM of PAC-5EC (5EC as positive
control) or PACQM-5EU (5EU as positive control) was added.
Figure 3. Imaging of sub-RNA population-specific incorporation
in HEK cells. (A) Imaging demonstrates that PAC-5EC is
incorporated into cellular RNA in a PGA-dependent manner. (B)
Imaging demonstrates differential RNA incorporation for PAC-
5EC and PAC-2’-AzA mediated by PGA.
To test our analogs further, we aimed to demonstrate that
multiple analogs could be differentiated from each other using
orthogonal chemistries, and we reasoned our work herein with
PAC-5EC and PGA could be used to satisfy this goal. In our
previous work we demonstrated that PAC-2’-AzA is
Figure 2. Incorporation of protected analogs into total RNA in
HEK cells expressing PGA. (A) Schematic of experiments to test
incorporation. (B) Demonstration of PGA-dependent
incorporation of PAC-5EC. (C) Demonstration of PGA-
dependent incorporation of PAC-QM-5EU.
incorporated into cellular RNA only in the presence of PGA, but
that was mostly incorporated into polyA tails, by polyA
polymerase. As such, PAC-2’-AzA staining is almost exclusively
cytoplasmic. We compared PAC-2’-AzA and PAC-5EC staining
within the same cells and demonstrated their localization patterns
are indeed different, further demonstrating that the two analogs
have different distributions in RNA. Together, these data further
suggest that PAC-5EC (or PACQM-5EU) are likely better
analogs for analyzing incorporation into all types of RNAs.
Total RNA was isolated using TriZol. RNA was then appended
with bitoin using Cu mediated Alkyne-Azide cycloaddition
(CuAAC) and biotin-azide. Dot blot analysis demonstrated that
PAC-5EC and PACQM-5EU can both be robustly deprotected
within 1 h in PGA-expressing cells. In contrast, when PAC-5EC
and PACQM-5EU were incubated in cells not expressing
transfected PGA there was nearly undetectable background
incorporation (Fig. 2, B and C). These results demonstrated that
PAC-5EC and PACQM-5EU are selectively incorporated into
cells expressing PGA.
In conclusion, we have reported the design, synthesis, and
utilization of protected alkynyl analogs for cell-specific
metabolic labeling of RNA. We have demonstrated that PAC-
protected analogs are incorporated into cellular RNA only in the
presence of PGA. Furthermore, we have utilized imaging to
further demonstrate their utility in imaging RNA, and also that
the pyrimidine nucleoside analogs herein are incorporated into a
different pool of RNA from our previously reported PAC-2’-
AzA. Importantly, we envision these analogs will be used widely
for understanding changes in RNA expression in a cell-specific
manner, and could also find utility for such efforts in vivo.
A major advantage to using analogs that contain alkyne
functionality is the ability to image them and their incorporation
into cellular RNA; this is in contrast to utilizing 4-thiouridine,
which cannot be imaged. We wanted to test whether our novel
analogs could be imaged for RNA incorporation in a PGA-
dependent manner. As shown in Figure 3, we observed punctate
staining within the nucleolus only in PGA transfected cells with
the addition of PAC-5EC to the media. A similar labeled pattern
was observed with PACQM-5EU (Fig. S1, ESI). This staining is
consistent with what has been observed with other analogs and is
known to represent rRNA synthesis within the nucleolus.
Staining in the nucleolus nicely demonstrates that our analogs are
being robustly incorporated into RNA.
Acknowledgments
We thank members of the Spitale lab for their careful reading of the
manuscript. This work is supported by the NIH (1DP2GM11916 and
1R21MH113062 to RCS). RCS is a Pew Biomedical Scholar.
Samantha Beasley is supported through an NIH Training Grant
(5T32CA009054)
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Supplementary Material
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A chemical-genetic strategy to enable cell-
specific metabolic labeling of RNA.
Two small molecule-enzyme pairs that
enable cell-specific RNA labeling.
Cell-specific labeling can be imaged with
orthogonal “click” chemistry pairs.