Vinyluridine as a Versatile Chemoselective Handle for the Post-transcriptional Chemical Functionalization of RNA

Article abstract of DOI:10.1021/acs.bioconjchem.7b00169

The development of modular and efficient methods to functionalize RNA with biophysical probes is very important in advancing the understanding of the structural and functional relevance of RNA in various cellular events. Herein, we demonstrate a two-step bioorthogonal chemical functionalization approach for the conjugation of multiple probes onto RNA transcripts using a 5-vinyl-modified uridine nucleotide analog (VUTP). VUTP, containing a structurally noninvasive and versatile chemoselective handle, was efficiently incorporated into RNA transcripts by in vitro transcription reactions. Furthermore, we show for the first time the use of a palladium-mediated oxidative Heck reaction in functionalizing RNA with fluorogenic probes by reacting vinyl-labeled RNA transcripts with appropriate boronic acid substrates. The vinyl label also permitted the post-transcriptional functionalization of RNA by a reagent-free inverse electron demand Diels-Alder (IEDDA) reaction in the presence of tetrazine substrates. Collectively, our results demonstrate that the incorporation of VUTP provides newer possibilities for the modular functionalization of RNA with variety of reporters.

Full text of DOI:10.1021/acs.bioconjchem.7b00169

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Article
Vinyluridine as a Versatile Chemoselective Handle for the
Posttranscriptional Chemical Functionalization of RNA
Jerrin Thomas George, and Seergazhi G Srivatsan
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00169 • Publication Date (Web): 13 Apr 2017
Downloaded from http://pubs.acs.org on April 14, 2017
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Vinyluridine as a Versatile Chemoselective Handle for the Posttranscriptional Chemical
Functionalization of RNA
Jerrin Thomas George and Seergazhi G. Srivatsan*
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Department of Chemistry, Indian Institute of Science Education and Research (IISER) Pune,
Dr. Homi Bhabha Road, Pashan, Pune 411008, India.
ABSTRACT: Development of modular and efficient methods to functionalize RNA with
biophysical probes is very important to advance the understanding of the structural and
functional relevance of RNA in various cellular events. Herein we demonstrate a twoꢀstep
bioorthogonal chemical functionalization approach for the conjugation of multiple probes
onto RNA transcripts using a 5ꢀvinylꢀmodified uridine nucleotide analog (VUTP). VUTP,
containing a structurally nonꢀinvasive and versatile chemoselective handle, was efficiently
incorporated into RNA transcripts by in vitro transcription reactions. Further, we show for
the first time the use of a palladiumꢀmediated oxidative Heck reaction in functionalizing
RNA with fluorogenic probes by reacting vinylꢀlabeled RNA transcripts with appropriate
boronic acid substrates.
The vinyl label also permitted the posttranscriptional
functionalization of RNA by a reagentꢀfree inverse electron demand DielsꢀAlder (IEDDA)
reaction in the presence of tetrazine substrates. Collectively, our results demonstrate that the
incorporation of VUTP provides newer possibilities for the modular functionalization of
RNA with variety of reporters.
INTRODUCTION
Functional modification of RNA in vitro and in cells have become necessary for
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understanding the regulation, structure, and function of RNA.
While solidꢀphase
oligonucleotide (ON) synthesis and enzymatic labeling methods using bacteriophage RNA
polymerases are widely used in the generation of labeled RNA suitable for cellꢀfree and inꢀ
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cell analysis, analogous approaches to label endogenous RNA are less prevalent.
Immunostaining of metabolically incorporated nucleoside analogs (e.g., BrU) using
antibodies, structureꢀspecific antibodies (e.g., Gꢀquadruplexes), aptamers (e.g., spinach) and
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dyes have been employed to visualize cellular RNA structure and synthesis.
However,
limited permeability and selectivity of the antibodies and compromised function of aptamerꢀ
tagged RNA have been the downsides of these methods. Alternatively, bioorthogonal
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chemical reactions provide an easy route to label RNA in vitro and cellular milieu for a
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variety of applications.
In this approach, a reactive group is introduced into an RNA ON
by either chemical, enzymatic (RNA polymerases) or chemoꢀenzymatic (transferases) means
and further bioconjugation to label RNA is achieved by performing a chemoselective reaction
with a cognate reactive partner containing a desired biophysical reporter. Several
chemoselective reactions including azideꢀalkyne cycloaddition (AAC), Staudinger ligation,
and IEDDA reactions have been effectively utilized in labeling protein, glycan, lipid and
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DNA.
However, establishing RNA labeling techniques under the conditions used in these
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reactions has always been a challenge due to the inherent instability of the RNA.
Among the various bioorthogonal reactions, AAC reaction has been extensively used
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in labeling RNA postsynthetically.
Typically in this reaction, a copper (I) stabilizing
ligand is used to alleviate the toxic effects of copper ions, which is known to produce species
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harmful to nucleic acids in the redox environment.
In another strategy, the use of toxic
copper has been circumvented by employing strainꢀpromoted AAC (SPAAC) reaction,
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wherein strained alkynes (e.g., cyclooctynes) are used as one of the reactive counterparts.
However, SPAAC reaction efficiency in labeling nucleic acids in cells has been limited by the
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poor permeability and reactivity of bulky cyclooctyne probes.
Other concerns which
have limited the application of AAC reactions are the involvement of alkyne substrates in
side reactions such as homocoupling and thiolꢀyne addition, and low stability of azide
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substrates in the chemical labeling conditions.
Analogous to azide and alkyne groups, the ability of alkene group to participate in a
wide range of chemoselective reactions has been elegantly utilized in devising several
bioconjugation strategies. Bioconjugation methods based on eneꢀthiol and eneꢀtetrazole
reactions have been put to good use in crossꢀlinking and functionalizing biomolecules,
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especially proteins.
However, the use of UV radiation and nonꢀspecific reaction with
cellular thiols has hampered their applications in inꢀcell analysis. Recently, protein labeling
has been achieved by using oxidative Heck reaction between proteinꢀbound alkenes and
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boronic acid reporters.
This reaction is particularly interesting because it can be
effectively performed in aqueous buffer under near physiological conditions (~pH 7, room
temperature and oxygen atmosphere) as compared to other analogous Pdꢀassisted
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biomolecular labeling reactions.
Very recently, baseꢀmodified 2′ꢀdeoxynucleosides,
containing a minimally perturbing vinyl group as a dienophile, have been successfully
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incorporated in vitro into DNA ONs and metabolically into replicating DNA in cells.
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Although the reaction rates are significantly lower as compared to strained dienophiles, vinylꢀ
labeled DNA did enable the labeling as well as visualization of DNA in cellꢀfree and cellular
environments by using IEDDA reaction.
Coupling reactions have been employed to attach heterocyclic aryl moieties via an
alkene to natural nucleobases. Such conjugations have produced microenvironmentꢀsensitive
nucleoside probes, which have been used in studying the conformation of the Gꢀquadruplex
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structure.
Similarly, fluorogenic reporters have been developed to specifically detect
epigenetic and epitranscriptomic nucleobase modifications (e.g., 5ꢀformylcytosine and 5ꢀ
formyluracil) by using a chemoselective reaction between the base and trimethylindole
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derivatives.
Taking advantage of these this knowledge, we rationalized that enzymatic
incorporation of a minimally perturbing vinylꢀmodified ribonucleotide into RNA would
facilitate the generation of microenvironmentꢀsensitive fluorogenic RNA reporters by
posttranscriptional oxidative Heck reaction with appropriate boronic acid derivatives (Figure
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). Also, we envisioned that vinylꢀlabeled RNA transcript would open up possibilities to
investigate its potential to functionalize RNA by reagentless IEDDA reaction. Despite the
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successes with protein and DNA,
the utility of vinyl group as a versatile chemoselective
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handle in developing RNA labeling protocols has not been well explored. In this context,
here we describe the synthesis of 5ꢀvinylꢀmodified UTP analog (VUTP) and its effective
incorporation into RNA ONs by in vitro transcription reactions. Further, we illustrate the
posttranscriptional chemical functionalization of vinylꢀlabeled RNA transcripts with a variety
of biophysical reporters by using oxidative Heck and IEDDA reactions. Notably, this is the
first report on the bioconjugation of an RNA ON by employing Pdꢀmediated oxidative Heck
reaction.
Figure 1. Diagram illustrating the posttranscriptional chemical labeling of RNA by using oxidative
Heck and IEDDA reactions. Incorporation of VUTP into RNA transcripts, followed by oxidative Heck
reaction with boronic acid derivatives and IEDDA reaction with tetrazole derivatives enabled the
synthesis of RNA conjugated to various reporters/tags.
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RESULTS AND DISCUSSION
Synthesis and enzymatic incorporation of 5-vinyluridine triphosphate (VUTP) 3. VUTP
was synthesized in simple steps starting from 5ꢀiodouridine 1 (Scheme 1). 5ꢀVinyluridine
(VU) 2 was synthesized by Stille crossꢀcoupling reaction using vinyltributylstannane and a
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palladium catalyst, tris(dibenzylideneacetone)dipalladium(0).
VU was further
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phosphorylated in the presence of POCl and bisꢀtributylammonium pyrophosphate to afford
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VUTP. The ability of VUTP to serve as a UTP analog for RNA polymerase was studied by
performing in vitro transcription reactions in the presence of a small series of DNA templates
S1–S4 (Figure 2). The templates contained one or two dA residues in the coding region to
guide the insertion of monophosphate of VUTP into RNA during polymerization reaction.
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The 5′ꢀend of each template ended with a dT residue to introduce a radiolabeled αꢀ P
adenosine at the 3′ꢀend of the fullꢀlength transcript. Transcription was performed using GTP,
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CTP, UTP/3, αꢀ P ATP in the presence of T7 RNA polymerase. The transcription products
were then electrophoresed on an analytical polyacrylamide gel and phosphor imaged.
Scheme 1. Synthesis of VUTP 3. (a) Pd
2 3 3
(dba) ,P(Fur) , vinyltributylstannane, DMF, 60 °C, 3 h, 53 %.
(b) (i) POCl , proton sponge, trimethylphosphate, ~4 °C, 30 min, (ii) bisꢀtributylammonium
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pyrophosphate, tributylamine, ~4 °C, 30 min, 21 %.
Figure 2. Incorporation of vinyl group into RNA ONs by in vitro transcription reaction using DNA
templates S1–S4 and VUTP 3. “r” preceding the sequence in parenthesis represents RNA ON
transcripts.
The incorporation of VUTP 3 in the presence of template S1 proceeded with very
good efficiency at +7 position yielding a 10 mer fullꢀlength RNA transcript 4 (Figure 3, lane
2). Slower migration of the transcription product 4 in lane 2 indicated hinted the
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incorporation of 3 into the transcript (compare lane 1 and 2). While a control reaction in the
absence of UTP and 3 did not yield fullꢀlength RNA transcript, at equimolar concentrations,
both natural UTP and 3 got incorporated into RNA (Figure 3, lane 3 and 4, respectively).
Templates S2–S4 incorporated modified UTP near the promoter region and at more than one
site with good to excellent efficiency (Figure 3, lane 6, 8 and 10).
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Figure 3. Phosphor image of transcripts obtained by transcription of templates S1–S4 in the presence
of UTP and or 3. % Incorporation of 3 is reported relative to a control transcription with UTP. For
complete gel picture see Figure S1.
Transcript 4 was scaled up by performing largeꢀscale reactions with S1 in the
presence of cold NTPs. Typically, a reaction in the presence of 75 pmol of the template gave
nearly 15 nmol of the modified transcript after gel electrophoretic purification, which was
further used for posttranscriptional chemical labeling experiments. The presence of VU in
transcript 4 was confirmed by mass analysis and enzymatic digestion assay (Figure S2, S3
and Table S1).
Oxidative Heck reaction on vinyl-labeled RNA transcript. Electronically unbiased
terminal alkene (e.g., allylic) substrates have been reported to undergo oxidative Heck
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reaction with boronic acids in the presence of waterꢀsoluble PdꢀEDTA complex. But the
feasibility of such a reaction on RNA has not been explored. In this regard, we sought to
investigate the possibility of performing oxidative Heck reaction on vinylꢀmodified RNA
with suitable boronic acid and ester substrates. For this purpose we chose a range of
heterobicycles, namely benzothiopheneꢀ2ꢀ (5), benzofuranꢀ2ꢀ (6), indoleꢀ5ꢀ (7),
dibenzothiopheneꢀ4ꢀboronic acid (8) and a previously reported pinacol boronic ester,
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benzothiopheneꢀ2ꢀvinyl boronic ester (9), which upon conjugation to uracil ring could
impart fluorescence to otherwise nonꢀemissive nucleobase (Figure 4).
Fluorescent
nucleosides have been developed by attaching heterocycles to pyrimidine and purine bases,
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respectively, and such analogs incorporated into ONs have been used as probes in various
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nucleic acid studies.
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Figure 4. Oxidative Heck reaction between vinylꢀmodified RNA ON 4 and boronic acid substrates 5–
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or boronic ester 9 in the presence of PdꢀEDTA complex.
For performing oxidative Heck reaction on RNA, transcript 4 was incubated with
boronic acid 5 in the presence of PdꢀEDTA complex at 37 °C. Aliquots of reaction mixture at
various time intervals were resolved by PAGE under denaturing conditions. Almost complete
conversion was observed at 18 h, and rewardingly, we observed an intense fluorescent band
corresponding to the coupled product along with a minor band (Figure 5 and Figure S4).
Reactions with other substrates (6–8) also proceeded well (Figure S5). Further, products
from largeꢀscale reactions between transcript 4 and boronic acid 5–8 or boronic ester 9 were
analyzed by RPꢀHPLC at 260 nm and 338 nm (conjugation of heterocycles to pyrimidine
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bases results in a strong absorption band near 338 nm).
Mass analysis of HPLC fractions
corresponding to both 260 nm and 338 nm absorption bands revealed the formation of the
oxidative Heck products 5a–9a (Figure S6, S7 and Table S2). Reactions performed at a 5
nmole scale of the RNA ON 4 provided 0.5 to 1.1 nmole of the coupled products (Table S3).
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Figure 5. (A) UVꢀtransilluminator image (364 nm) of the gel of a reaction between RNA 4 and 5. See
Figure S4 for the UVꢀshadow image of the gel at 254 nm. (B) Fluorescence of RNA transcript before
and after the reaction between RNA 4 and boronic acid 5 under UV lamp (364 nm). (C) Fluorescence
spectra (0.2 ꢁM) of RNA transcript 4, boronic acid 5 and RNA product 5a (HPLC fraction
corresponding to 5a′). Samples were excited at 289 nm, 310 nm and 340 nm, respectively, with an
excitation and emission slit width of 8 nm and 9 nm. (D) Fluorescence spectra (0.2 ꢁM) of RNA
transcript 4, boronic ester 9 and RNA product 9a (HPLC fraction corresponding to 9a′). Samples were
excited at 289 nm, 310 nm and 372 nm, respectively, with an excitation and emission slit width of 7
nm and 8 nm. See Figure S6 for HPLC profiles.
An oxidative Heck reaction between an electronically unbiased terminal alkene (e.g.,
allylic system) and arylboronic acid substrates, typically gives linear and branched crossꢀ
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coupled products. However, reactions with electronically biased aryl vinyl systems (e.g.,
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styrene) give predominantly linear transꢀcoupled product.
Formation of branched
regioisomer product has also been reported using Pd in the presence of certain bulky ligands
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(e. g., 2,9ꢀdimethylꢀ1,10ꢀphenanthroline).
Although it is not a major concern in most
bioconjugation strategies, we sought to determine the formation of different isomers in the
oxidative Heck reaction of vinylꢀlabeled RNA transcript. First, we carried out a reaction
between free vinylꢀmodified uridine 2 and boronic acid 5 under similar conditions used for
RNA ligation. Interestingly, at the ribonucleoside level a very low conversion was observed
even after prolonged incubation time. Varying the Pdꢀligand complex and oxidant did not
give a better yield (data not shown). However, a fluorescent product was isolated in
analytical quantities, and further characterization by NMR revealed the formation of a linear
transꢀisomer product 2a (Figure S8). Further, the coupled product gave a unique longer
wavelength absorption band at ~338 nm as compared to native nucleoside/nucleic acid,
which absorbs at 260 nm (Figure S8B). Next, the major HPLC fraction corresponding to the
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RNA products (designated as 5a′) of a reaction between vinylꢀlabeled RNA ON 4 and
boronic acid 5 was subjected to enzymatic digestion in the presence of calf intestinal alkaline
phosphatase, RNase A, RNase T1 and snake venom phosphodiesterase. Enzymatic digestion
of 5a′ gave individual ribonucleosides as well as Heckꢀcoupled ribonucleosides, whose
identity was determined by comparing the HPLC chromatogram of the digested sample and
oxidative Heck reaction product 2a obtained from a reaction between uridine 2 and boronic
acid 5 (Figure S9). The chromatogram of the digest revealed the presence of native
ribonucleosides (rC, rG and rA) and two peaks corresponding to coupled products absorbing
at 338 nm. One of the peaks clearly matched with the transꢀisomer product 2a, whereas the
chemical structure of the peak X could not be determined. However, both 2a and X gave the
same mass indicating that that a mixture of isomers (X may be cis isomer) are formed in
these reactions. Formation of a mixture of regioꢀ and stereoisomers isomers, which is an
inherent “limitation” of this reaction, has also been observed in protein ligation
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experiments.
Although, at the present, this reaction may not be viable to introduce a
specific type of conformationꢀsensitive probe into RNA, development in chelateꢀcontrol
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strategies could vastly improve the selective of the reaction.
We next studied the fluorogenic nature of the RNA products by steadyꢀstate
fluorescence spectroscopy. Benzothiopheneꢀcoupled RNA product 5a (HPLC fraction
corresponding to 5a′) showed a ~40ꢀfold enhancement in fluorescence intensity as compared
to the boronic acid substrate 5 (Figure 4, 5C and Figure S6). Interestingly, benzothiopheneꢀ
alkeneꢀcoupled RNA product 9a (HPLC fraction corresponding to 9a′) showed a remarkable
~
170ꢀfold enhancement in fluorescence intensity as compared to substrate 9. Further, the
emission profile of 9a showed a significant bathochromic shift compared to RNA 5a as a
direct result of increased conjugation between the uracil and benzothiophene rings (Figure 4,
5D and Figure S6). Reactions with benzofuran (6), indole (7), dibenzothiophene (8) boronic
acid substrates also produced fluorescent RNA products (Figure S10). While benzofuranꢀ
and dibenzothiopheneꢀconjugated RNA products were reasonably fluorescent, indoleꢀ
conjugated RNA was found to be weakly fluorescent. Taken together, this approach of
functionalizing vinylꢀlabeled RNA transcripts by fluorogenic Heckꢀtype coupling reaction
with appropriate boronic acid/boronic ester substrates could provide direct access to RNA
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functionalized with fluorescent reporters.
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IEDDA reaction on vinyl-labeled RNA transcripts. Analogous to AAC chemistry, IEDDA
reaction between an electronꢀrich dienophile and electronꢀdeficient tetrazine is gaining
particular attention as a useful biomacromolecular labeling strategy because this reaction is
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reagent free, reasonably fast, highly selective and biocompatible.
Bioconjugation
strategies based on IEDDA reaction commonly use ONs labeled with reactive dienophiles
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such as norbornene, transꢀcyclooctene and cyclopropene groups.
While reaction rates of
strained dienophiles with tetrazine derivatives are generally fast, such bulky dienophile
substituents on nucleoside are particularly not suitable for labeling cellular nucleic acids as
they may not serve as good substrates for endogenous polymerases. Albeit low reactivity as
compared to strained dienophiles, vinylated 2′ꢀdeoxynucleosides have been used as chemical
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reporters to label and visualize DNA in cells by IEDDA reaction. Encouraged by these
results, we decided to study the reactivity of vinyl label of transcript 4 towards diene
counterpart in IEDDA reaction. For this purpose, we chose two tetrazine cores, which had
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reportedly shown a good reactivity against an electronꢀrich strained alkene.
The
reactivity of transcript 4 was tested by using a commercially available tetrazine 10, and
biotinylated (11) and Cy5ꢀconjugated (12) tetrazines, which were synthesized by following an
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analogous literature procedure (Figure 6 and Scheme S1).
Figure 6. (A) Tetrazine substrates 10–12 used in this study. (B) IEDDA reaction between RNA ON 4
and tetrazine substrates. 10a–12a are RNA products obtained from a reaction between RNA ON 4 and
tetrazines 10–12, respectively.
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Initially, the IEDDA reaction was performed using the free nucleoside 2 and a
tetrazine substrate 10. The reaction proceeded well and almost complete conversion was
observed after 21 h (Scheme S2 and Figure S11). The reaction resulted in a mixture of ligated
products (2b and 2c), which were isolated and characterized. 2a was found to be oxidized
pyridazine product, whereas 2c corresponded to the dihydropyridazine product, which
underwent slow oxidation to form 2b. Formation of a mixture of products and slow oxidation
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event has been documented earlier in DNA conjugation experiments.
Next, IEDDA reactions were performed between RNA ON 4 and tetrazine substrates
in TrisꢀHCl buffer (pH 7.5) at 37 °C, and aliquots of reaction mixture at various time intervals
were analyzed by analytical PAGE under denaturing conditions. A reaction with tetrazine 10
was found to be almost complete in 12 h, whereas with substrates 11 and 12 the reaction was
partially complete even after 15 h (Figure S12). Reactions at elevated temperatures (45 °C
and 55 °C) did not result in noticeable improvement in reaction efficiency (data not shown).
This is in agreement with reports of less reactivity of the tetrazine core of 11 and 12 as
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compared to tetrazine core of 10.
Largeꢀscale reactions were then performed to isolate and characterize the RNA
products. Reaction with substrates 10–12 produced major and minor (could not be isolated)
bands (Figure S13). Mass analysis of major bands confirmed the formation of pyridazine
72
products 10a–12a (Figure S14 and Table S2) similar to previous report. UVꢀvis profile of
purified products was distinguishingly different from the profile of transcript 4, and further
fluorescence analysis also confirmed the bioconjugation by IEDDA reaction (Figure S15).
While HPLC analysis of the major band obtained from a reaction with symmetrical tetrazine
10 gave a single peak corresponding to the pyridazine 10a, reactions with asymmetrical
tetrazines 11 and 12 afforded mixture of pyridazine isomers (Figure S16). The formation of
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mixture of isomeric products is consistent with literature reports.
High density labeling of VUTP into a longer RNA transcript was studied by using a
DNA template that would generate a 59 mer RNA containing 9 modifications. T7 RNA
polymerase effectively incorporated the vinyl analog into the 59 mer RNA 13 with efficiency
comparable to that of natural UTP (Figure 7A). Transcript 13 was subsequently reacted with
sulfoꢀCy5 labeled tetrazine 12 and the reaction product was resolved on a 2% agarose gel.
The image captured using UVꢀtransilluminator (364 nm) clearly revealed the fluorescence
labeling of the longer RNA transcript (Figure 7B).
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Figure 7. (A) Agarose gel picture of 59 mer control (lane 2) and VUꢀmodified RNA transcript 13
(lane 3). Lane 1: 50 bp DNA ladder. (B) Agarose gel picture of IEDDA reaction between RNA 13 and
Cy5ꢀtetrazine 12. UVꢀtransilluminator image at (i) 254 nm and (ii) 364 nm. Lane 1: 50 bp DNA
ladder.
CONCLUSIONS
We have successfully incorporated the vinyl functionality into RNA by in vitro transcription
reaction using VUTP 3. The results demonstrate that VU incorporated into RNA transcripts
can serve as a useful handle for chemoselective functionalization of RNA in a modular
fashion by oxidative Heck and IEDDA reactions. In particular, the generation of fluorogenic
RNA by oxidative Heck reaction with boronic acid/ester is advantageous. It suggests that a
screening reaction with appropriate boronic acid/ester substrates could provide direct access
to RNA emitting at different wavelengths. Even though conjugation by utilizing oxidative
Heck reaction resulted in a mixture of isomers, this study represents a promising initial step
on further research on oxidative Heck reaction for the design of madeꢀtoꢀorder
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biologically compatible regioselective and stereoselective ligands for palladium catalysis.
Such studies would pave way for selective incorporation of desired biophysical probes, for
example, microenvironment sensing probes onto RNA. Furthermore, metabolic labeling of
VU followed by posttranscriptional functionalization by these methods could potentially
enable the imaging of RNA in cells. Taken together, the studies presented here are expected
to complement other bioorthogonal RNA labeling strategies by providing alternative access
to RNA labeled with biophysical probes and tags.
ASSOCIATED CONTENT
Supporting Information
The supporting Information is available free of charge in the ACS Publication website.
Experimental procedure, characterization data, Figure S1–S16 and Table S1–S4 (PDF).
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AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: srivatsan@iiserpune.ac.in
Notes
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The authors declare no competing financial interest.
ACKNOWLEDGMENT
S.G.S. thanks DST, India (EMR/2014/000419) and Wellcome TrustꢀDBT India Alliance
(IA/S/16/1/502360) for the research grants. J.T.G. is grateful to IISER, Pune for graduate
research fellowship. We thank Manish Walunj for providing one of the boronic acid
substrates.
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