Mechanochemical Synthesis of Short DNA Fragments

Article abstract of DOI:10.1002/chem.202001193

We demonstrate the first mechanochemical synthesis of DNA fragments by ball milling, enabling the synthesis of oligomers of controllable sequence and length using multi-step, one-pot reactions, without bulk solvent or the need to isolate intermediates. Me

Full text of DOI:10.1002/chem.202001193

A Journal of
Accepted Article
Title: Mechanochemical synthesis of short DNA fragments
Authors: James D. Thorpe, Daniel O'Reilly, Tomislav Friscic, and
Masad J. Damha
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10.1002/chem.202001193
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RESEARCH ARTICLE
Mechanochemical synthesis of short DNA fragments
James D. Thorpe,[+] Daniel O’Reilly,[+] Tomislav Friščić* and Masad J. Damha*
[*]
J. D. Thorpe, Dr. D. O’Reilly, Prof. T. Friščić, Prof. M. J. Damha
Department of Chemistry
McGill University
801 Sherbrooke Street West, Montreal, QC, H3A 0B8, Canada
E-mail: masad.damha@mcgill.ca
[+]
These authors contributed equally
Supporting information for this article is given via a link at the end of the document.
Abstract: We demonstrate the first mechanochemical synthesis of
DNA fragments by ball milling, enabling the synthesis of oligomers of
controllable sequence and length using multi-step, one-pot reactions,
without bulk solvent or the need to isolate intermediates.
Mechanochemistry allowed for coupling of phosphoramidite
monomers to the 5′-hydroxyl group of nucleosides, iodine/water
oxidation of the resulting phosphite triester linkage, and removal of
the 5′-dimethoxytrityl (DMTr) protecting group in situ in good yields
(up to 60% over three steps) to produce DNA dimers in a one-pot
manner. H-phosphonate chemistry under milling conditions enabled
coupling and protection of the H-phosphonate linkage, as well as
removal of the 5′-DMTr protecting group in situ, enabling a one-pot
process with good yields (up to 65% over three steps, or ca. 87% per
step). Sulfurization of the internucleotide linkage was possible using
elemental sulfur (S8) or sulfur transfer reagents, yielding the target
DNA phosphorothioate dimers in good yield (up to 80% over two
steps). This work opens the door to creation of solvent-free synthesis
methodologies for DNA and RNA therapeutics.
materials, through grinding, milling or shearing without the need
for bulk solvents or elevated temperatures.[9] Performed either by
neat milling or promoted by a small amount of liquid phase (liquid-
assisted grinding, LAG), mechanochemistry can provide access
to chemical reactions that are not only rapid, scalable and void of
bulk solvents, but also yield products, catalytic transformations
and reaction selectivities that are difficult or not at all observable
in solution.[10] The amount of liquid in LAG synthesis is expressed
by η, the ratio of liquid volume to the weight of reactants, and lies
between 0-2 μL/mg.[11] While mechanochemistry was shown to
be applicable for the synthesis of nucleosides, nucleotides and
related materials by several groups, including those of Vyle and
of Roy, the area remains under-developed.[12] For instance,
recent studies have focused on protecting group chemistry,[13]
phosphitylations,[14]
pyrophosphate
formation,[15]
and
dinucleoside 5′,5′-polyphosphates.[16] Encouraged by this prior
work, and the growing need for rapid, simple and efficient
approach to synthetic ONs, we envisioned the use of
mechanochemistry as a general platform to synthesize ON
structures in a solvent-less environment. Herein, we report the
first strategy for the ball millling synthesis of 3′-5′-linked di- and
trinucleotides based on both phosphoramidite and H-
phosphonate chemistry.
Introduction
Oligonucleotides (ONs) have become an exciting new class of
therapeutics with multiple already approved for treating a wide
range of genetic diseases.[1] With increasing numbers of ON
therapeutics nearing the market, the manufacturing process for
ONs is assuming increased importance, with consideration of
building block (monomer, dimer, trimers, etc.) availability, solvent
use (waste management cost), product yield, purity, and
scalability. The demand for synthetic ONs has reached an all-time
high and will only continue to grow.[2]
Solid-phase ON synthesis is well established and takes
advantage of well-developed solution-based chemistry using
phosphoramidite building blocks on insoluble polymer supports[3]
such as controlled pore glass (CPG) (Figure S1).[4] Many different
studies have focused on the use of soluble ionic tags,[5] soluble
polymer supports,[6] and block coupling[7] to address some of the
challenges of solid-phase synthesis.[2, 8] Whereas this approach
to ON targets is based on surface immobilization of growing
chains, the chemistry itself is done through solution processes
that are among the most solvent-demanding synthesis
procedures, demanding an enormous amount of acetonitrile as
solvent per kilogram of target, making the assembly of these
valuable targets a major environmental challenge.
Results and Discussion
Phosphoramidite Chemistry. The first aim of the current study
was to recreate the phosphoramidite synthesis cycle in a ball
millling environment, with dinucleotides as targets. Thus, 1a-d (2
equivalents) were coupled with 3′-O-tert-butyldimethylsilyl
thymidine (2),[17] in the presence of 5-(ethylthio)-1H-tetrazole
(ETT, 3 equivalents) by milling for 30 minutes in a steel or a
Teflon® assembly, at a frequency of 30 Hz (Scheme 1, the
mechanochemical conditions indicated by the symbol proposed
by Rightmire and Hanusa).[18] The 31P NMR spectrum of the crude
product displayed two sets of signals at 140 ppm[19] corresponding
to the expected diastereomeric phosphite triesters (3a-d), and
another set of signals at ca. 8 ppm, assigned to the H-
phosphonate by-product resulting from the hydrolysis of excess
1a-d (Figure S3). Formation of the H-phosphonate by-product
was reduced by drying the reaction vessels as well as reagents
under high vacuum prior to performing the reaction. After
purification, 3a-d were produced in good yields (63-80%) and
were identical to standards prepared in solution (see
Supplemental Information).
Mechanochemistry has recently emerged as a versatile approach
for the synthesis of a wide range of molecular targets and
1
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10.1002/chem.202001193
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RESEARCH ARTICLE
a carbonyl chloride or chlorophosphate activator.[22] We first
attempted to mechanochemically couple protected H-
phosphonate 6a with 7 using pivaloyl chloride (PivCl) as an
activator in the presence of stoichiometric amounts of pyridine
(Scheme 3).
Scheme 1. Mechanochemical synthesis of DNA dimers via phosphoramidite
chemistry.
The next step in the cycle is the oxidation reaction required to
Scheme 3. One-pot, two-step mechanochemical synthesis of fully protected
DNA dimers via H-phosphonate chemistry
convert the reactive phosphite triester P(III) species to the more
19a]
stable phosphate triester P(V) species.[4c,
While this is
accomplished in standard solid-phase synthesis through I2 in
aqueous pyridine/THF solution, we found that the transformation
was readily achieved by one-pot mechanochemistry from 1a-d
and 2, by adding solid iodine, water and pyridine (LAG, η  0.1
μL/mg) into the milled reaction mixture and milling for an
additional 20 minutes (Scheme 1). As a result, compounds 4a, 4b
and 4d were produced with variable yields (32-80%). In the case
of dGpT 4c, the dimer could not be isolated, partly due to
premature loss of the 5′-O-dimethoxytrityl (DMTr) group during
the milling and purification steps. Switching the oxidant from I2 to
meta-chloroperbenzoic acid (mCPBA),[20] however, provided 4c in
36% yield, again observing partial removal of the DMTr group
from this dimer during its isolation.
Despite screening a wide range of conditions, including reactant
ratio, time, and milling frequency, no formation of H-phosphonate
diesters was observed according to 31P NMR (Figure S2a and
b).[22a] However, using adamantoyl chloride (AdaCl) as an
activator, upon 15 minutes milling of equimolar amounts of 6a and
7 in the presence of 5 equivalents of AdaCl and 10 equivalents of
pyridine (η = 0.39 μL/mg), we observed complete consumption of
6a and formation of two peaks around 7-9 ppm in the 31P NMR
spectrum, corresponding to the two diastereomers of the H-
phosphonate diester (Figure S2c). However, the product was
difficult to isolate due to low stability of H-phosphonate diesters
under basic conditions.[23]
To deal with the challenge of premature detritylation of 4c, we
attempted to perform coupling, oxidation and detritylation steps in
a one-pot sequence without isolation of intermediates (Scheme
2), effectively recreating the synthesis cycle currently used on
automated synthesizers.
For this reason, we turned to a modified H-phosphonate
approach, using thiophosphoric esters as protecting groups for
24]
the linkage.[22d,
From our initial studies, we knew that H-
phosphonate diesters could be prepared by ball milling in 15
minutes. Gratifyingly, upon subsequent addition of the sulfur-
transfer reagent N-(phenylthio)phthalimide (PTP) and pyridine (η
= 0.34 μL/mg) to the crude reaction mixture, the protected dimers
8a-d (Scheme 3) were obtained in 15 minutes under the same
milling conditions. This was evidenced by appearance of two new
peaks around 24 ppm in the 31P-NMR spectrum (Figure S2d),
corresponding to the thiophosphoric ester, and the disappearance
of the peaks of the H-phosphonate diester. Despite conducting
these reactions under basic conditions, the previously mentioned
hydrolysis was avoided by using dry pyridine and drying reaction
vessels and reagents under high vacuum prior to performing the
reactions. These compounds (8a-d) were much more stable, and
easily isolable by simple column chromatography in modest yields
(27-65%; or, average yield of 52-80% per step; entries 1-4, Table
S1). Similarly, to the phosphoramidite chemistry, we observed
premature detritylation and problems in isolation for dGpT (8c)
(entry 3, Table S1).
Screening of various activators for the coupling reaction revealed
that the coupling and sulfurization steps could be carried out in
one-pot simultaneously, using diphenyl phosphoryl chloride
(DPC) as the activator in place of AdaCl.[22d] Additionally, by
varying the amount of base used it was also possible to remove
the 5′-DMTr protecting group during the same reaction, yielding a
strategy to synthesize partially protected dimers 9a-d in one step
(Scheme 4). We were similarly able to isolate these compounds
by column chromatography in 44-65% yields, corresponding to
76-87% per step (entries 5-8, Table S1).
Scheme 2. One-pot, three-step mechanochemical synthesis of DNA dimers via
phosphoramidite chemistry.
Indeed, coupling of 1a with 2 (30 min, 30 Hz), followed by
oxidation with I2 crystals and wet pyridine (20 min, 30 Hz, η = 0.07
μL/mg), and detritylation with solid trichloroacetic acid (TCA) and
methanol (MeOH, 10 min, 30 Hz, η = 0.06 μL/mg) produced 5a in
60% yield (over three steps; Scheme 2). The presence of a small
amount of MeOH as a LAG additive was necessary, since TCA
alone failed to cleave the DMTr group. We hypothesize that LAG
promotes 5′-O protonation, as well as releases and quenches the
trityl cation, driving the detritylation reaction to completion. When
this three-step procedure was applied to the in-situ synthesis of
dGpT, 5c was synthesized in the same manner in 44% yield
(three steps from 1c; Scheme 2).
H-Phosphonate Chemistry. While phosphoramidite chemistry is
the method of choice for typical solid-phase oligonucleotide
synthesis, H-phosphonate chemistry has also been shown to be
an effective strategy.[21] Nucleotide H-phosphonate couplings are
rapid and are most often performed in pyridine in the presence of
These two coupling strategies give us solventless routes to
synthetically useful DNA dimer building blocks simply based on
the choice of activator, equivalents of base, and the order of
2
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10.1002/chem.202001193
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RESEARCH ARTICLE
addition of reagents. It was also possible to prepare trimer 10 by
coupling 9a with monomer 6a (Scheme 4). Trimer 10 was isolated Conclusion
by column chromatography in 64% yield, corresponding to an
average yield of 86.2% per step.
In summary, we have demonstrated the ability to prepare
synthetically useful DNA building blocks of controlled length and
sequence via ball milling mechanochemistry, in the absence of
bulk solvents. These dimers and trimers can be applied to
traditional solid-phase synthesis of oligonucleotides in order to
reduce the total number of synthesis cycles. This proof-of-
principle study not only demonstrates that complex
oligonucleotide structures can be assembled in a controlled way
under ball milling conditions, but also that such structures readily
form by multi-step reactions in one-pot fashion. Whereas the
presented work has focused on the synthesis of dimers and
trimers, the herein presented strategies are generally applicable
and we are confident that the synthesis of longer sequences via
block coupling under mechanochemical conditions, combined
with ionic-tags to selectively precipitate intermediate products
should also be possible, eliminating the use of chromatography
for purifications of products.[5] Furthermore, we see no reason why
the herein presented reaction sequences should not be
compatible with industrial-scale mills and/or continuous
mechanochemical manufacture via twin screw extrusion,[26] as
has recently been done in the context of small organic molecule
targets and metal-organic frameworks.[27] Finally, we note that the
ability to obtain functional oligonucleotide structures without a
bulk solvent might suggest the potential role of solid-state
transformations in the early stages of chemical evolution, as
recently highlighted by Trapp and co-workers,[28] and Hernández
and co-workers.[29]
Scheme 4. One-pot, single-step mechanochemical synthesis of partially-
protected DNA dimers and trimer via H-phosphonate chemistry
Phosphorothioate formation. One of the most widely-used
phosphate modifications in oligonucleotide therapeutics is the
phosphorothioate (PS) linkage.[25] PS linkages alleviate a major
challenge associated with using oligonucleotides in vivo by
providing resistance against a variety of extra- and intracellular
nucleases.
To produce the desired PS backbone, two strategies were
pursued. First, 1a-d were coupled to 2 (30 min, 30 Hz), and the
resulting phosphite triester intermediates were treated in situ with
yellow sulfur powder (20 min, 30 Hz) to give 11a-d (55-80% over
two steps; Scheme 5A). Compound 11a was identical to a sample
prepared by reacting phosphite triester 3a directly with S8 at 30
Hz for 5 min. In this case, the PS-TpT dimer was obtained as a
mixture of PS-TpT (triester, 11a) and PS-TpT (diester) in 8:2 ratio,
respectively; the diester most likely results from the premature
removal of the beta-cyanoethyl protecting group (31P-NMR,
Figures S30 and S31).
Acknowledgements
This research was supported by funding from the McGill
Sustainability Systems Initiative (MSSI) through the
ideas/innovation fund and the National Science and Engineering
Council of Canada (NSERC) to M. J. Damha and T. Friščić.
Keywords: Mechanochemistry • DNA • Phosphoramidite • H-
Phosphonate • Green Chemistry
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Entry for the Table of Contents
Insert graphic for Table of Contents here. ((Please ensure your graphic is in one of following formats))
Shake to make DNA. Utilizing mechanochemistry to make DNA fragments using both phosphoramidite and H-phosphonate
chemistries under minimal solvent conditions.
Institute and/or researcher Twitter usernames:@DANorDNA, @F_ANAtico, @Tomislav Friscic, @JamesDThorpe
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