Non-nucleoside building blocks for copper-assisted and copper-free click chemistry for the efficient synthesis of RNA conjugates

Article abstract of DOI:10.1021/ol102205j

Novel non-nucleoside alkyne monomers compatible with oligonucleotide synthesis were designed, synthesized, and efficiently incorporated into RNA and RNA analogues during solid-phase synthesis. These modifications allowed site-specific conjugation of ligan

Full text of DOI:10.1021/ol102205j

ORGANIC
LETTERS
2010
Vol. 12, No. 23
5410-5413
Non-Nucleoside Building Blocks for
Copper-Assisted and Copper-Free Click
Chemistry for the Efficient Synthesis of
RNA Conjugates
K. N. Jayaprakash,† Chang Geng Peng,† David Butler,† Jos P. Varghese,‡
Martin A. Maier,† Kallanthottathil G. Rajeev,† and Muthiah Manoharan*,†
Drug DiscoVery, Alnylam Pharmaceuticals, Cambridge, Massachusetts 02142,
United States, and Sanmar Speciality Chemicals Ltd., Chennai, Tamil Nadu, India
mmanoharan@alnylam.com
Received September 14, 2010
ABSTRACT
Novel non-nucleoside alkyne monomers compatible with oligonucleotide synthesis were designed, synthesized, and efficiently incorporated
into RNA and RNA analogues during solid-phase synthesis. These modifications allowed site-specific conjugation of ligands to the RNA
oligonucleotides through copper-assisted (CuAAC) and copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) reactions. The SPAAC
click reactions of cyclooctyne-oligonucleotides with various classes of azido-functionalized ligands in solution phase and on solid phase
were efficient and quantitative and occurred under mild reaction conditions. The SPAAC reaction provides a method for the synthesis of
oligonucleotide-ligand conjugates uncontaminated with copper ions.
Since the discovery of RNA interference (RNAi) as a means
to silence expression of specific genes, small-interfering
RNAs (siRNAs) have proven to be powerful tools for
specifically eliciting degradation of targeted mRNAs of
disease-causing genes. Although siRNAs have the potential
to become a highly effective class of therapeutics,1 efficient
tissue distribution and intracellular delivery in ViVo remain
critical challenges.2 Reported approaches to delivery of
siRNA to particular cell types involve conjugation of lipids
or other small molecules to one of the strands of the siRNA
or formulations with lipid-, polymer-, or peptide-based
delivery systems.3
(1) (a) Manoharan, M. Curr. Opin. Chem. Biol. 2004, 8, 570. (b)
Bumcrot, D.; Manoharan, M.; Koteliansky, V.; Sah, D. W. Y. Nat. Chem.
Biol. 2006, 2, 711.
(2) Manoharan, M. Antisense Nucleic Acid Drug DeV. 2002, 12, 103.
(3) Stanton, M. G.; Colletti, S. L. J. Med. Chem. ASAP; DOI: 10.1021/
jm1003914.
† Alnylam Pharmaceuticals.
‡ Sanmar Speciality Chemicals Ltd.
10.1021/ol102205j  2010 American Chemical Society
Published on Web 11/04/2010

Cu(I)-catalyzed 1,3-dipolar [3 + 2] cycloadditions4 be-
tween azides and alkynes (CuAAC), known generally as
“click reactions”, have been used extensively for conjugation
of various ligands to DNA,5 for DNA ligation,6 and for
fluorophore tagging of oligonucleotides7 and modified nu-
cleosides.8 High chemical stability and selective reactivity
of alkyne and azide precursors and excellent 1,4-regioselec-
tivity4 make CuAAC a promising approach for oligonucleo-
tide-ligand conjugate synthesis.
To achieve CuAAC-mediated RNA conjugation to dif-
ferent ligands, we initially explored a hydroxyprolinol-
derived alkyne derivative. The key alkyne intermediate 3 for
the synthesis of the long-chain aminoalkyl controlled pore
glass (lcaa CPG) support 4 (Scheme 1) and the corresponding
The alkyne-functionalized hydroxyprolinol moiety was
incorporated at the 3′ end of the oligonucleotide using
standard solid-phase synthesis to afford the support-bound
alkyne-RNA 5 (Supporting Information). We evaluated click
reactions of the solid support-bound oligonucleotide 5 with
linoleyl azide10 (6, Scheme 1) under conditions reported for
the synthesis of various solid-supported carbohydrate-DNA
conjugates.11 The goal of linoleyl conjugation was to improve
the tissue distribution and cellular permeation of siRNAs,
similar to what we achieved with cholesterol conjugation
earlier.12 The reaction went to completion upon microwave
irradiation (60 °C, 45 min) of the reaction mixture of alkyne/
azide/CuSO4/tris(benzyltriazolyl-methyl) amine13 (TBTA)/
sodium ascorbate with 1:5.8:0.8:5.6:6.4 molar ratio in
methanol/water/THF (2:2:1 by volume).
The main drawback of the CuAAC reaction is contamina-
tion of the product with copper ions; this is especially a
problem when the reaction is performed in the solution phase
using an unprotected alkyne or azido functionalized oligo-
nucleotide (unpublished results). Contamination with Cu+/
Cu2+ could be problematic due to the potential for increased
nucleic acid hydrolysis14 and potential cytotoxicity due to
heavy metal ion contamination. The development of the
strain-promoted azide-alkyne cycloaddition (SPAAC) reac-
tion, also known as Cu-free click chemistry, has provided a
solution to this issue.15 In addition to this approach, strained
alkene-nitrile oxide16 and alkyne-nitrile oxide17 click
cycloaddition reactions have also been reported for the
modification of DNA in the absence of metal catalysts.
Two research groups have reported the synthesis of
activated cycloalkynes for click chemistry that do not require
Cu(I) catalysis. Bertozzi’s laboratory reported strained cy-
clooctynes 8,15a 9,15b,c 10,15d and 11,15e and Boon’s labora-
tory reported the cyclooctyne 1215g (Figure 1). These
compounds can undergo azide-alkyne cycloaddition in the
absence of copper and enabled the SPAAC15a process. For
Scheme 1.
CuAAC Reaction with Solid-Supported RNAa
a The lowercase letters indicate 2′-O-methyl-ribonucleotides.
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phosporamidite (Scheme S1, Supporting Information) for
incorporation of the alkyne moiety to internal and/or 5′-end
of the oligonucloetide were obtained from trans-4-hydroxy-
prolinol derivative 29 and 5-hexynoic acid (1). The secondary
hydroxyl group of 3 was reacted with succinic anhydride in
the presence of DMAP to afford the hemisuccinate, which
was then loaded onto lcaa CPG support under peptide
coupling conditions to obtain the desired CPG-alkyne support
4 with loading of 90 µmol/g.
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J. M.; Agard, N. J.; Bertozzi, C. R. J. Am. Chem. Soc. 2008, 130, 11486.
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J. Am. Chem. Soc. 2010, 132, 11799. (f) Bertozzi, C. R.; Agard, N. J.;
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Org. Lett., Vol. 12, No. 23, 2010
5411

tocols (Supporting Information). Both strained alkyne build-
ing blocks 14 and 15 were stable under solid-phase oligo-
nucleotide synthesis and deprotection conditions.
Scheme 3
.
Model Cu-Free Click Reactions with Azido
Derivatives
Figure 1. Strained cyclooctynes enable click reaction with azides
in the absence of copper.15
compound 8, activation of cyclooctyne is due to ring strain,
and the gem-difluoro substitution of compound 9 adjacent
to the triple bond further enhances reactivity. In compounds
11 and 12, activation is accomplished by the orthodibenzo
substitution. Compound 10 combines features of aryl and
gem-difluoro substitutions. We herein describe the syntheses
of conjugates between RNA oligonucleotides and various
ligands that are capable of improving cellular delivery of
siRNAs. Our synthetic adaptations will allow the syntheses
of a variety of RNA conjugates using a copper-free click
protocol.
As shown in Scheme 3, model cycloaddition reactions with
different azido derivatives were evaluated to ensure that the
reactivity of cyclic alkyne 12b was not compromised after
derivatization with a carboxylic acid and to evaluate the
regiochemistry of the products. Compound 12b was reacted
with 1 mol equiv of linoleyl azide 6 or azides derived from
monosaccharides N-acetylgalactosamine (GalNAc) 1618 or
mannose 17.19 These sugars, respectively, target the drugs
to either the asialoglycoprotein receptor, ASGPR, and
mediate delivery to hepatocytes of liver20 or to mannose
binding proteins and enable delivery to dendritic cells and
macrophages.21 The click reaction generated 1,4-/1,5-triazole
derivatives 18a/18b (linoleyl 92%), 19a/19b ([GalNAc]Ac
92%), and 20a/20b ([Mannose]Bz 95%) in high isolated yields
as inseparable mixtures of regioisomers. The rate of reaction
depended upon the nature of the substrate. Lipid derivative
6 took longer to react than the sugar derivatives 16 or 17, a
difference we attribute to the poor solubility of 6 in MeOH
compared to that of the other azides.
Scheme 2
.
Synthesis of Non-Nucleosidic SPAAC Alkyne
Monomers for RNA Synthesis
Next we examined the Cu-free click reaction in solution
between deprotected 5′-alkyne-RNA 21 and [GalNAc]Ac-
azide 16 (Scheme 4). The oligoribonucleotide 21 with the
activated alkyne moiety at the 5′-end underwent cycloaddi-
tion reaction with the azide 16 at room temperature in MeOH/
THF/water (1:1:1.5, v/v). The progress of the reaction was
monitored by HPLC. The cycloaddition reaction was very
efficient; within 45 min all the starting materials were
consumed to afford the product 27. The identity and integrity
of the conjugate 27 were confirmed by LC-MS and HPLC
analysis. We then studied the reaction between 21 and the
As outlined in Scheme 2, the cyclooctyne derivative 1215g
was activated with disuccinimidyl carbonate and further
reacted with methyl 6-aminohexanoate to afford compound
12a in 60% yield. The ester 12a was hydrolyzed to afford
the carboxylic acid-functionalized compound 12b in quan-
titative yield. The carboxylic acid 12b was coupled to
compound 29 to afford compound 13 in 87% isolated yield.
Phosphitylation of compound 13 afforded the phosphora-
midite 14 (79% yield), which was used to introduce the cyclic
alkyne moiety at the 5′-end and at internal positions of
oligonucleotides. Succinylation of compound 13 enabled
loading onto lcaa CPG under peptide coupling conditions to
obtain the support 15 (loading 59.5 µmol/g). The support
15 was then used to introduce the alkyne at the 3′-end of
oligonucleotides by standard oligonucleotide synthesis pro-
(18) Rensen, P. C. N.; van Leeuwen, S. H.; Sliedregt, L. A. J. M.; van
Berkel, T. J. C.; Biessen, E. A. L. J. Med. Chem. 2004, 47, 5798.
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80, 964.
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van Rossenberg, S. M. W.; van Leeuwen, S. H.; van Berkel, T. J. C.;
Biessen, E. A. L. J. Biol. Chem. 2001, 276, 37577.
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Barrett-Bergshoeff, M.; Bijsterbosch, M. K.; Rijken, D. C.; van Berkel,
T. J. C. J. Biol. Chem. 1996, 271, 28024.
5412
Org. Lett., Vol. 12, No. 23, 2010

Information). The two product peaks shown in the HPLC
profile correspond to compounds with the same molecular
weight as shown by LC-MS analysis. We presume that these
are the two regioisomers expected to form from the click
reaction. The regioisomers obtained from the azides 16, 17,
25, and 26 were inseparable under the HPLC conditions
evaluated. All click products had the expected molecular
weights upon LC/MS analysis.
Scheme 4. RNA Conjugation by Cu(I)-Free Click Reactions
We also examined the click reactions of alkynes with
oligonucleotides derivatized at the 3′ terminus (22) and at an
internal position (23). All the reactions were clean and quantita-
tive. The click reactions between solid-support-bound 21 and
azides proceeded to completion after stirring overnight at room
temperature (Supporting Information). The heterogeneity of the
reaction presumably contributed to the slow reactions between
support-bound alkyne-oligoribonucleotide and the azides tested.
Several fluorophore-tagged azides were also incorporated into
the RNA in a similar fashion (data not shown).
Thus, using this strategy one can choose where to place a
ligand and also at what stage of the RNA synthesis to
incorporate it. For example, when screening a particular
biological target using a library of ligands, it may be
preferable to obtain purified RNA precursors to be utilized
for multiple, parallel click reactions in solution, whereas for
large-scale synthesis, conjugation of the ligand to the
oligonucleotide on solid support may confer a considerable
economic advantage as purification steps can be avoided.
To summarize, we have demonstrated that a Cu-free strained
alkyne-azide click reaction is applicable to the general synthesis
of oligonucleotide-ligand conjugates. All common steps of
phosphoramidite chemistry, including the oxidation with iodine,
are compatible with the activated, strained cyclooctyne deriva-
tive. Copper-free click reactions of RNA oligonucleotides
functionalized at 5′ or 3′ termini or at an internal position were
efficient in the solution phase as well as on solid support. The
various azides evaluated reacted quantitatively and efficiently
as determined by HPLC analysis. These procedures will serve
as the foundation for the development of methods for the
introduction of all kinds of functional molecules into RNA,
DNA, and chimeric oligonucleotides without the generation of
toxic byproducts or reactive intermediates. As pointed out, the
only remaining limitation with this approach appears to be the
mixed regiochemistry of the products. We are in the process
of identifying methods to separate the isomers and evaluating
their uptake, delivery, and gene silencing improvements of
siRNAs derived from these RNA conjugates and will report
the results in due course.
trivalent GalNAc3-azide 25 to test the efficiency of the
coupling with a large hydrophilic azide. Figure 2 depicts the
Figure 2. Progress of the click reaction of deprotected 5′-alkyne-
RNA 21 with [GalNAc]Ac-azide 16 and GalNAc3 azide 25
monitored by HPLC.
time course of the SPAAC reaction of the sugar azides 16
and 25 with alkyne-RNA 21 in solution as monitored by
HPLC. Both reactions were complete within 3 h at ambient
temperature.
Reactions of alkyne-oligoribonucleotide 21 with other
azido derivatives (6, 17, and 2622) were also quantitative
(Table S3, Supporting Information). For the hydrophobic
azide 17, additional THF was required to improve solubility.
Reaction of the hydrophobic linoleyl azide 6 with the alkyne
21 was slow in comparison to the sugar azides; HPLC
analysis showed only 38% conversion in the first 3 h with
completion of reaction in 18 h (Figure S1d, Supporting
Acknowledgment. We thank Professor Michael E. Jung
(UCLA) and Dr. Bhuvana Sridhar (Sanmar Speciality
Chemicals Ltd.) for valuable discussions and Ms. Kathy Mills
and Dr. Bo Pang (Alnylam) for MS analyses.
Supporting Information Available: Experimental pro-
cedures and compound characterization; HPLC profiles; and
MS characterizations of oligonucleotides and conjugates.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(22) Li, J.; Zacharek, S.; Chen, X.; Wang, J.; Zhang, W.; Janczuk, A.;
Wang, P. G. Bioorg. Med. Chem. 1999, 7, 1549.
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