Mercury-Free Automated Synthesis of Guanidinium Backbone Oligonucleotides

Article abstract of DOI:10.1021/jacs.9b09937

A new method for synthesizing deoxynucleic guanidine (DNG) oligonucleotides that uses iodine as a mild and inexpensive coupling reagent is reported. This method eliminates the need for the toxic mercury salts and pungent thiophenol historically used in me

Full text of DOI:10.1021/jacs.9b09937

Article
pubs.acs.org/JACS
Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Mercury-Free Automated Synthesis of Guanidinium Backbone
Oligonucleotides
Kacper Skakuj,† Katherine E. Bujold,† and Chad A. Mirkin*
Department of Chemistry and the International Institute for Nanotechnology, Northwestern University, 2145 Sheridan Road,
Evanston, Illinois 60208, United States
S
* Supporting Information
ABSTRACT: A new method for synthesizing deoxynucleic
guanidine (DNG) oligonucleotides that uses iodine as a mild
and inexpensive coupling reagent is reported. This method
eliminates the need for the toxic mercury salts and pungent
thiophenol historically used in methods aimed at preparing
DNG oligonucleotides. This coupling strategy was readily
translated to a standard MerMade 12 oligonucleotide
synthesizer with coupling yields of 95% and has enabled the
synthesis of a 20-mer DNG oligonucleotide, the longest DNG strand to date, in addition to mixed DNA−DNG sequences with
3−9 DNG inserts. Importantly, DNG oligonucleotides exhibit robust unaided cellular uptake as compared to unmodified
oligonucleotides without apparent cellular toxicity. Taken together, these findings should greatly increase the accessibility of
cationic backbone modifications and assist in the development of oligonucleotide-based drugs.
retain base-pairing fidelity over nonspecific electrostatic
association, in contrast to findings with other positively
charged oligonucleotides; however, duplex formation has not
been studied for DNG sequences longer than eight bases.25−28
As opposed to alternative guanidine-containing backbone
modifications, these oligonucleotides can be synthesized in
the 3′ to 5′ direction using the same solid support and
protection groups afforded by phosphoramidite chemistry,
enabling their synthesis on standard instruments.23 However,
current DNG production protocols require successive
couplings with toxic mercury salts and pungent thiophenol
washes, limiting the use of such structures and their broad scale
adoption on oligonucleotide synthesizers (Scheme 1). For
example, to date the interaction of DNGs with cells has yet to
be explored, and therapeutically relevant lengths have not been
attained.
Herein, we report a single-step coupling for a poly guanidine
synthesis cycle that uses iodine as a mild oxidant to couple
protected thiourea monomers to the 5′-amine of growing
DNG strands anchored on a solid support. This method
removes toxic reagents and increases access to this class of
oligonucleotide modifications. Reaction conditions were
initially identified and optimized through a solution-phase
screen. Promising results from the screen were successfully
translated to an automated oligonucleotide synthesizer and
have enabled the synthesis of the longest DNG strand made to
date. Moreover, for the first time since their development, we
report that DNGs show rapid cellular uptake into mammalian
cells without the need for auxiliary transfection reagents and do
INTRODUCTION
■
The programmable base-pairing of nucleic acids makes them
attractive targets for biological applications such as diagnostic
platforms, intracellular detection agents, drug delivery vehicles,
and therapeutics.1,2 However, these applications are often
limited by the polyanionic backbone of oligonucleotides, which
suffers from high nuclease sensitivity and poor cellular uptake.3
Synthetic backbone modifications have been developed to
address these challenges.4 Among them, phosphorothioate
linkages have been widely adopted due to their ease of
synthesis, increased nuclease stability, and moderate improve-
ment in cellular uptake.5,6 While most backbone modifications
enhance nuclease stability, increasing cellular uptake is more
challenging. Charge alterations are a promising approach to
address this problem, as demonstrated by uncharged peptide
nucleic acid (PNA) linkages7 and through the incorporation of
positively charged groups using phosphoramidate8,9 and amide
backbone modifications.10−15 The variety of advantageous
properties afforded by these modifications, including cell
uptake, have been summarized in recent reviews16,17 and
indicate that the continual development of backbone
modifications expands the possibilities of this class of
molecules. However, their synthesis, often incompatible with
phosphoramidite methods, impedes their wide adoption by
other groups.
Almost 30 years ago, Bruice and others introduced
deoxyribonucleic guanidines (DNGs), oligonucleotides with
guanidinium moieties in place of the phosphodiester backbone,
as a strategy to increase cellular uptake, inspired by arginine-
rich cell-penetrating peptides.18−21 DNGs are resistant to
nuclease degradation and have been made with all four
nucleobases.22−25 Short DNG sequences were reported to
Received: September 13, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/jacs.9b09937
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Article
a
Scheme 1. Solid-Phase DNG Synthesis Cycle
a
B1 and B2, nucleobases; MMTr, monomethoxy trityl; TEA,
triethylamine; TMP, 2,2,6,6-tetramethylpiperidine; DCA, dichloro-
acetic acid; NMI, N-methylimidazole; DMF, dimethylformamide.
*Indicates steps that are identical to standard phosphoramidite
coupling chemistry.
not affect cell viability. These findings highlight the potential of
DNGs for further development as cationic self-transfecting
gene regulation agents and as probes for making intracellular
measurements.
RESULTS AND DISCUSSION
■
Solution-Phase Screen for Coupling Conditions. We
hypothesized that the mercury salts used during DNG
synthesis could be replaced by less toxic reagents (Scheme
1). Bruice’s coupling chemistry relies on mercury-mediated
sulfur abstraction from the thiourea and addition of an amine
to form a guanidine.29 Therefore, we reviewed alternative
reagents that similarly activate thioureas for a single step
synthesis of carbodiimides and guanidines. Among them are
Mukaiyama’s reagent (2-chloro-1-methylpyridinium iodide,
CMPI),30,31 Sanger’s reagent (1-fluoro-2,4-dinitrobenzene,
DNFB),31,32 hypervalent iodine (e.g., [hydroxy(tosyloxy)-
iodo]benezene, HTIB),33,34 and molecular iodine,35−38 all in
the presence of a nitrogenous base. We began exploring
alternative chemistry by coupling P6 and I3-TIPS under
reported conditions (Figure 1A). The two coupling partners
were synthesized according to published protocols with minor
variations (Schemes S1 and S2). To expedite the screening
process, we carried out these reactions in solution phase with
TIPS-protected 3′-hydoxyl of 5′-deoxyamino thymidine (I3-
TIPS), which would usually be attached to the growing strand
on a solid support. This allowed for rapid reaction screening in
a 96-well format.
Figure 1. Solution-phase screen for coupling reagents and conditions.
(A) Solution-phase reaction between Fmoc protected thiourea P6 and
3′-TIPS protected amine I3-TIPS to give the Fmoc-protected
guanidine product 3. (B) Area under the curve of the UV trace of
the product peak from UPLC analysis. Reactions contained P6, I3-
TIPS, TMP, and indicated coupling reagent in 1:3 dichloromethane
(DCM):acetonitrile (ACN). The structures of some of the coupling
reagents used in the screen are shown. (C) Effects of base identity and
storage in solution with iodine after 30 s, 1 h, and 4 days. Note: Error
bars indicate the standard deviation of three reactions.
resulted in far greater and faster product yields than did CMPI,
DNFB, and HTIB (Figure 1B). We suspected that the active
reagent responsible for the coupling in the iodine and TMP
reaction was an electrophilic iodine source. When iodine and
TMP are mixed in acetonitrile, a TMP-I2 complex crystallizes
from the solution (Figure S1 for crystal structure), which
resembles other electrophilic iodine reagents. To confirm the
hypothesis that electrophilic iodine was responsible for the
sulfur abstraction, we attempted the reaction with two
frequently used sources of electrophilic iodine: N-iodosuccini-
mide (NIS)39 and 1,3-diiodo-5,5-dimiethylhydantoin (DIH).
When either reagent was used, equally high product yields
were observed (Figure 1B). On the basis of these results, we
proceeded to further study a molecular iodine-based
“activator”, due to its low cost and availability.
Each of these coupling reagents was incubated with thiourea
P6 in the presence of 2,2,6,6-tetramethylpiperidine (TMP) as
the base, chosen due to its similarity to other bases commonly
used with these reactions (i.e., triethylamine) and its stability
under the reaction conditions (vide infra). Coupling product 3
could be detected in all reactions tested; however, iodine
To preserve the standard operation of the oligonucleotide
synthesizer, we constrained ourselves to storing the iodine and
B
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base in a single bottle, and the thiourea monomer in a separate
bottle, to be delivered simultaneously during the coupling step.
Therefore, it was important to identify a base that could
accomplish the coupling, but also remain stable in solution
with iodine for the duration of the synthesis. For this purpose,
we explored a variety of alkyl and aryl nitrogen bases: 4-
dimethylaminopyridine (DMAP), TMP, TEA, NMI, 1,2,2,6,6-
pentamethylpiperidine (PMP), pyridine (Py), 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU), and 1,4-
diazabicyclo[2.2.2]octane (DABCO). We also measured the
amount of product formed in the solution-phase coupling
reaction with the various iodine−base mixes after they had
remained in the dark at room temperature for 30 s, 1 h, and 4
d.
DMAP, TMP, and NMI all afforded similar amounts of
product. However, DMAP formed a precipitate under these
conditions, while NMI began to afford less product at the
longer time point. Surprisingly, TEA did not perform well in
this screen, although it had been commonly reported as a base
for carbodiimide and guanidine formation from thiour-
eas.32−36,38−40 Similarly, PMP, pyridine, DBU, and DABCO
only afforded the product in low amounts. Most poorly
performing activator solutions changed color over time or
formed a precipitate (Figure S2). These observations suggest
that these formulations may decompose, possibly via a
previously reported iodine-mediated oxidation at the α
position.41 Because TMP did not exhibit any decrease in
product yield over time nor precipitation, it showed promise
for translation to automated synthesis.
the synthesizer could be used to make standard oligonucleo-
tides as well as DNG strands (see the Supporting Information
for automated cycle details).
The initial conditions identified by the solution-phase screen
were used to perform 1−8 DNG dT couplings atop 19- to 11-
mer dT standard DNA strands bearing a 5′-MMTr-protected
amine through coupling with initiator unit I5 (Scheme S3).
Following standard cleavage and deprotection with ammonium
hydroxide and methylamine, the crude reaction afforded
reverse phase chromatography (RP-HPLC) traces that clearly
separated the failure strands lacking an MMTr group from the
full-length strands retarded by the presence of the 5′-MMTr
(Figure 2A). Another late eluting peak was also observed,
which increased in area with more DNG insertions and is
suspected to result from the electrostatic association of
multiple strands. Both the second and the third peaks
contained only the full-length product when analyzed by
MALDI-TOF MS (Figure 2B), confirming successful purifica-
Rapid coupling rates are crucial for translation to an
automated system without prolonged cycle times. We found
the solution-phase rate to increase with the number of
equivalents of base added to the reaction (Figure S3). At
low equivalencies of base, the reaction rate is markedly slower
(amine consumed with a half-life of over 5 min), and the
product degrades via MMTr removal; a large excess of base
increases the reaction rate but can also remove the Fmoc
protecting group. Optimal conditions were identified by
varying the number of base equivalents required to maximize
coupling. More than 2 equiv of base resulted in the highest
product yields; 3 equiv was chosen for the fast rate of reaction,
which reaches completion prior to the first measurement at 1
min. This illustrates the rapid coupling time that this chemistry
can achieve. The extent of the reaction can also be
approximated by the disappearance of the burgundy iodine
color in solution, which occurs in seconds once the
components are mixed together, but does not if iodine, base,
or thiourea are not added (data not shown). Finally, the
solution-phase screen was instrumental in determining solvent
compatibility for this reaction and its impact on coupling
efficiency (Figure S4). Among the five solvents tested (DMF,
tetrahydrofuran, 25%v/v ACN in DCM, 25%v/v DCM in ACN,
and 1-methyl-2-pyrrolidinone (NMP)), 25%v/v DCM in ACN
generated the highest product yields.
Figure 2. Synthesis conditions and yield quantification of DNA−
DNG chimeras. (A) Representative RP-HPLC trace of a 22-mer with
three DNG linkages after cleavage and deprotection. (B) MALDI-
TOF spectra of HPLC peaks containing the full-length peak (∼22
min) and the assembly peak (∼32 min). (C) Coupling yields of three
DNG additions atop a DNA strand on a MerMade 12 automated
synthesizer. (a) Coupling yields calculated per-coupling based on
HPLC UV trace AUC of product and failure peaks. (b) Alternative
wash contained ACN instead of DMF. (c) Iodine extracted with
saturated NaHCO3 and dried with MgSO4 prior to making the
activator solution. (d) Coupling yield measured by trityl absorbance
of deprotection solution following each coupling cycle for 8 cycles
atop an 11-mer DNA strand.
Automated Synthesis on Solid Support. We next
translated the iodine coupling reaction to a MerMade 12
(BioAutomation) synthesizer. The synthesizer was minimally
modified to accommodate both the iodine/TMP and the
standard activators used for phosphoramidite chemistry, while
the thiourea nucleoside monomer was placed into an empty
amidite position (Figure S5). An additional wash step of neat
DMF (vide infra) was incorporated into the cycle by following
the manufacturer’s instructions. Following these modifications,
C
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tion. Coupling yield was estimated using the integrated peak
areas of the UV absorption traces from batches of CPGs
deprotected immediately prior to and following the addition of
the three DNG bases. This method provided a simple way of
comparing coupling conditions (Figure S6).
mass (Figure 3A). To demonstrate the ability of this method
to synthesize DNG strands of lengths relevant to therapeutic
We found that the activator retained a constant coupling
yield when stored on the synthesizer for up to 24 h but showed
a significantly decreased coupling yield after 48 h (Figure 2C,
entries 1−3). This suggests that the iodine−TMP solution
should only be used for 1 day before being replaced, which is
long enough to complete even extensive oligonucleotide
syntheses.
During the optimization process, we noticed that the drain
line that transfers reagents from the column to the waste
system would periodically become clogged, which did not
occur during the standard phosphoramidite syntheses. We
suspected that a precipitate, at times visible during the
coupling step, was the cause of the clogs. The introduction
of an additional DMF wash step to dissolve any precipitates
prevented the clog from occurring since and did not impact the
coupling efficiency (Figure 2C, entries 1 and 4).
In addition to testing the number of base equivalents in
solution phase, we also tested this on a solid support. Three
equivalents of base with respect to both thiourea and iodine
resulted in the highest yield and did not remove the Fmoc
protecting groups from the growing strand (Figure S7), while 2
or 4 equiv caused a decrease in coupling efficiency (Figure 2C,
entries 6−8). Finally, increasing the amount of thiourea from 1
to 1.5 and 3 equiv with respect to iodine, while keeping the
base constant at 3 equiv, caused a decreased yield (Figure 2C,
entries 4, 5, and 7). This finding suggests that the excess
unactivated thiourea may consume the activated thiourea
monomer in a side reaction. Similar reactivity has been
reported; however, we did not attempt to isolate these side
products.42 The coupling yield of these optimized conditions
was measured using the established trityl cation absorption
protocol, and showed an average coupling efficiency of 95%
over eight coupling cycles (Figure 2C, entry 5; Figure S8).43
Once a high yielding coupling was attained, a primarily
positively charged strand was synthesized. Controlled pore
glass (CPG) beads bearing a disulfide moiety, to provide a
thiol handle for subsequent conjugation reactions, were
coupled with initiator phosphoramidite I5 through a single
phosphate linkage. Afterward, a 9-mer strand of DNG dT
nucleosides was added to this nascent strand (sequence: 5′-
(Tg)9(Tp)-OC3H6SSC3H6OH-3′, p is phosphate and g is
guanidinium linkages). MALDI-TOF MS of the crude material
showed the presence of the product peak along with a number
of failure strands. The full-length strand was purified using a
serendipitously discovered extraction procedure by washing
the solid support with water and acetonitrile, which removed
the failure strands, and then a 20% aqueous solution of acetic
acid, which extracted the full-length product. HPLC
purification of the strand proved elusive due to a strong and
likely electrostatic attraction between the DNG strand and
surfaces, and a general purification strategy is under develop-
ment (Figures S9 and S10). The 10-mer DNG strand was
characterized by SDS-PAGE and stained with SimplyBlue, a
negatively charged protein stain that confirms the structure’s
overall positive charge (Figure S11B). One main band was
observed, attesting to the purity of the material. The purified
10-mer product showed a single primary mass peak when
analyzed via MALDI-TOF MS, which matches the calculated
Figure 3. Synthesis, cellular uptake, and toxicity of DNG (Tg)10
strands. (A) MALDI-TOF spectra of the crude and purified DNG
strands, shot with DHAP matrix; (B) confocal fluorescence images of
C166 cells after treatment with fluorophore-labeled DNA or DNG
(red) with stained nuclei (blue); (C) uptake of fluorophore-labeled
DNA and DNG strands into C166 cells as measured by flow
cytometry; and (D) toxicity of DNA and DNG oligonucleotides
toward C166 cells measured by Alamar Blue assay.
and detection applications, we also made a 20-mer DNG
(Figure S11), the first example of a DNG strand of this length.
Cell-Penetrating Capabilities. The improved coupling
method and automated synthesis made the production of
DNG strands at lengths not achieved to date practical and
allowed us to finally test the 30-year hypothesis that the
incorporation of guanidinium linkages as the backbone of
oligonucleotides will affect their uptake into cells. We used
AlexaFluor 647 to fluorophore-label the 10-mer DNG strand
and a standard phosphate-backbone 10-mer DNA strand.
Mouse endothelial C166 cells were separately treated with
both labeled strands at a 600 nM concentration for 2.5 h to
assess their uptake at concentrations relevant for oligonucleo-
tide drug delivery. Confocal microscopy and flow cytometry
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were used to characterize the extent of uptake into cells
(Figures 3B,C and S12−S15). Minimal fluorescence is visible
in the DNA treatment group, attesting to the inefficient uptake
of free oligonucleotides into cells. However, the DNG treated
cells show intense fluorescence, both throughout the cytoplasm
and in punctate areas arrayed inside the cell. Flow cytometry
analysis further highlighted the high cellular uptake of DNGs,
which is 38-fold higher than their DNA equivalent. To our
delight, no toxicity was observed between 78 nM and 5 μM
concentrations using an Alamar Blue cell viability assay
(Figures 3D and S16).
necessarily represent the official views of the National
Institutes of Health. This work made use of the IMSERC at
Northwestern University, which has received support from the
Soft and Hybrid Nanotechnology Experimental (SHyNE)
Resource (NSF ECCS-1542205), the State of Illinois, and the
International Institute for Nanotechnology (IIN). K.E.B. is
supported by a Banting Fellowship from the Government of
Canada. We would like to thank Professor Karl A. Scheidt for
helpful and illuminating discussions.
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CONCLUSIONS
■
Herein, we have introduced a practical method for the
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.9b09937.
■
S
Detailed protocols for the synthesis of monomers I5, P6,
and I3-TIPS; NMR spectra and HRMS data of
monomers; crystal structure of iodine-TMP activator
precipitate; further reaction characterization data;
images of activator solutions over time; details of the
automated synthesis cycle and synthesizer setup; flow
cytometry gating strategy and results; confocal micros-
copy images and Z-stack analysis; and cell viability
results (PDF)
AUTHOR INFORMATION
Corresponding Author
*chadnano@northwestern.edu
■
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ORCID
Kacper Skakuj: 0000-0003-1563-0382
Katherine E. Bujold: 0000-0002-7292-6617
Chad A. Mirkin: 0000-0002-6634-7627
̈
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Author Contributions
†K.S. and K.E.B. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We would like to acknowledge support from the Vannevar
Bush Faculty Fellowship program sponsored by the Basic
Research Office of the Assistant Secretary of Defense for
Research and Engineering and funded by the Office of Naval
Research through grant N00014-15-1-0043; the Prostate
Cancer Foundation and the Movember Foundation under
award 17CHAL08; and the National Cancer Institute of the
National Institutes of Health under award U54CA199091. The
content is solely the responsibility of the authors and does not
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DOI: 10.1021/jacs.9b09937
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX