An immobilized and reusable Cu(i) catalyst for metal ion-free conjugation of ligands to fully deprotected oligonucleotides through click reaction

Article abstract of DOI:10.1039/c2cc36811k

Chelation of Cu(i) ions to an immobilized hydrophilic tris(triazolylmethyl) amine chelator on a solid support allowed synthesis of RNA oligonucleotide conjugates from completely deprotected alkyne-oligonucleotides. No oligonucleotide strand degradation or metal ion contamination was observed. Furthermore, use of the immobilized copper(i) ion overcame regioselectivity issues associated with strain-promoted copper-free azide-alkyne cycloaddition. This journal is The Royal Society of Chemistry.

Full text of DOI:10.1039/c2cc36811k

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An immobilized and reusable Cu(I) catalyst for metal
ion-free conjugation of ligands to fully deprotected
oligonucleotides through click reaction†
Cite this: DOI: 10.1039/c2cc36811k
Received 18th September 2012,
Accepted 5th November 2012
Laxman Eltepu, Muthusamy Jayaraman, Kallanthottathil G. Rajeev and
Muthiah Manoharan*
DOI: 10.1039/c2cc36811k
www.rsc.org/chemcomm
Chelation of Cu(I) ions to an immobilized hydrophilic tris(triazolyl-
methyl)amine chelator on a solid support allowed synthesis of RNA
oligonucleotide conjugates from completely deprotected alkyne-
oligonucleotides. No oligonucleotide strand degradation or metal ion
contamination was observed. Furthermore, use of the immobilized
copper(I) ion overcame regioselectivity issues associated with strain-
promoted copper-free azide–alkyne cycloaddition.
Fig. 1 Immobilized Cu(I) ion on a solid support.
Both copper-assisted and copper-free strain-promoted click reac-
tions have been used for bioconjugation.1 We recently reported use
of both approaches for conjugation of various ligands to oligo-
nucleotide strands of small interfering RNA (siRNA) duplexes with
the goal of improving cellular uptake of these therapeutic
agents.2,3 The drawbacks of copper-assisted azide–alkyne cyclo-
addition of a ligand to a therapeutic siRNA are the difficulties
associated with complete removal of copper ions from the product
and strand degradation during conjugation.2,4 These limitations
create chemistry, manufacturing, and control (CMC) issues in drug
development. The disadvantages of the copper-free strain
promoted method are that synthesis of the activated alkyne
requires multiple steps and, more importantly, results in non-
regioselective bicyclic triazole (both 1,4- and 1,5-) adducts.3
We therefore explored alternative strategies of copper-assisted
regioselective 1,4-cycloaddition of an azide and a simple alkyne for
oligonucleotide conjugation. Heterogeneous and immobilized
copper-assisted click reactions effectively minimize heavy metal
contamination of the product,5 and the support can be reused.5e,6
Chan et al. reported stabilization of Cu(I) ions by chelation to
tris(benzyltriazolylmethyl)amine (TBTA); chelation protects the
metal ion from oxidation and disproportionation while enhancing
its catalytic activity.7 Immobilization of TBTA onto TantaGel resin
allows parallel synthesis and direct screening of cycloaddition
products.6 We rationalized that immobilizing a hydrophilic analog
of TBTA, such as tris(triazolylmethyl)amine, chelated with Cu(I)
ions onto a solid support would allow use of unstrained alkynes
and azides in click conjugation of highly hydrophilic oligo-
nucleotides to ligands without copper ion contamination of the
product.
Here we demonstrate the chelation of Cu(I) ions to an immo-
bilized hydrophilic tris(triazolylmethyl)amine on a solid support
(Fig. 1). The coupling of a carboxylate moiety of the hydrophilic
tris(triazolylmethyl)amine to an amino-functionalized solid
support followed by chelation with Cu(I) ions using tetrakis-
(acetonitrile)copper(I) hexafluorophosphate [Cu(CH3CN)4PF6]
afforded the desired support (7). The utility of the immobilized
Cu(I) catalyst for quantitative azide–alkyne cycloaddition of
completely deprotected alkyne-oligonucleotides to azides
was evaluated under azide–alkyne cycloaddition conditions
reported previously.8
The Cu (I) ion-chelated solid support 7 was synthesized from
commercially available 3-[2-[2-[2-(2-azidoethoxy)ethoxy]-ethoxy]-
ethoxy]-propanoic acid (1) as shown in Scheme 1. Azido
carboxylic acid 1 was refluxed with benzyl bromide in acetone
in the presence of solid potassium carbonate to afford benzyl
ester 2 as colourless oil in quantitative yield. The click reaction
of tripropargylamine 3 with 3 molar equivalents of the azide 2
in the presence of sodium ascorbate (10 mol%) and CuSO4ꢀ
5H2O (1 mol%) in a 1 : 1 mixture of tert-BuOH and H2O (v/v)
yielded the desired tris(triazolylmethyl)amine derivative 4.
The reaction was performed both at ambient temperature
and under microwave-assisted conditions. The ambient
temperature reaction was complete after overnight stirring,
whereas the microwave-assisted reaction at 80 1C was complete
Drug Discovery, Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA 02142,
USA. E-mail: mmanoharan@alnylam.com; Fax: +1 617-551-8101;
Tel: +1 617-551-8319
† Electronic supplementary information (ESI) available: Experimental
procedures and HPLC and MS characterization of oligonucleotide conjugates.
See DOI: 10.1039/c2cc36811k
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Scheme 2 Immobilized Cu(I) ion-mediated azide–alkyne cycloaddition.
effectively solubilizes hydrophobic azides like 9 and 10. Use of 10
mol% of the immobilized Cu(I) ion was sufficient to drive the
reaction to completion. With a 1 : 1 molar ratio of the azide to
Scheme 1 Synthesis of Cu(I) ion chelated polystyrene resin 7.
after 1 h in almost quantitative yield. The product after pur- alkyne the desired product was obtained in excellent purity as
ification was pale green in colour, presumably due to partial determined by TLC and NMR. No detritylation of the nucleoside
chelation of copper ions (from CuSO4ꢀ5H2O) to the product. was observed with the heterogeneous immobilized CuAAC reaction,
Catalytic hydrogenation of 4 over 10% Pd–C at atmospheric whereas dimethoxytrityl (DMTr) deprotection is an issue with the
pressure in methanol afforded the tricarboxylic acid 5 as an oil conventional CuAAC reaction due to the acidic nature of the
in quantitative yield. The tricarboxylic acid 5 was activated with
0.5 molar equivalents of 2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetra-
reagents employed.
Recyclability of the catalyst was investigated using the alkyne 8
methyluronium hexafluoro-phosphate (HBTU) in the presence and azides 9 and 10. The immobilized catalyst 7 was filtered,
of diisopropylethylamine in DMF and charged with an amine washed, and reused repeatedly to determine the catalytic efficiency
functionalized polystyrene resin (amine content 230 mmol gꢁ1). of the chelated Cu(I) ion. The performance of the immobilized
After gentle agitation at ambient temperature overnight, the catalyst remained the same after five cycles, and the recyclability of
chelator-functionalized resin 6 was suspended in DMF and the catalyst was solvent independent. There was no detectable
mixed with Cu(CH3CN)4PF6 followed by gentle shaking to leaching of the copper ion from the chelator to the reaction media
obtain Cu(I)-chelated resin 7. The Cu(I) ion content of the resin
was estimated by inductively coupled plasma optical emission
under any reaction conditions tested. We also evaluated the stability
of the catalyst at ambient temperature and confirmed similar
spectrometry (ICP-OES); the observed loading of the metal ion activity over a period of several months.
was between 160 and 220 mmol gꢁ1 with variation from batch to
We next evaluated the usefulness of the approach for azide
batch. Coupling of the chelator to the resin was roughly equimolar cycloaddition to fully deprotected oligonucleotides containing
with amine present on the support. Loading of compound 5 under an alkyne.2 Due to the highly anionic nature of fully depro-
similar conditions to long-chain amino alkyl controlled pore glass tected oligonucleotides, copper ion contamination in the pro-
(lcaa CPG) afforded the chelator-immobilized CPG. The Cu(I) ion duct and RNA strand degradation are limitations of the copper-
content of this resin was proportionate to the amine content
(loading capacity) of the resin used and the number of molar
equivalents of compound 5 added.
assisted click conjugation approach with fully deprotected
oligonucleotides.2,4 The chelated copper complex 7 was used
to catalyse click reactions between a fully deprotected alkyne-
We first evaluated the heterogeneous copper-assisted azide– modified oligonucleotide 13 and azido functionalized ligands
alkyne 1,4-cycloaddition (CuAAC) reaction in different solvent sys- 14,9 15,2 and 162 as shown in Scheme 3. DMF was chosen as the
tems including DCM, DMF, DCM : DMF (1 : 2), THF, toluene, and solvent to overcome solubility limitations of azides and unpro-
acetone using 50-O-[bis(4-methoxyphenyl)phenylmethyl]-5-methyl-30- tected oligonucleotides. As observed in the reactions of small
O-2-propyn-1-yl-uridine 8 and alkyl azides2 9 and 10 as reactants molecules, 10 mol% of the immobilized catalyst 7 was suffi-
(Scheme 2). Of the solvents evaluated, acetone, DCM : DMF (1 : 2),
and DMF were optimal based on reaction time and yield at ambient
cient to drive the reaction to completion overnight at ambient
temperature. The catalyst was recovered by filtration and the
temperature. The mixture of DCM and DMF was selected as it integrity of the conjugates (18–20) was confirmed by LC-MS
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overcame regioselectivity issues associated with strain-
promoted copper-free azide–alkyne cycloaddition and the metal
ion contamination observed with the conventional CuAAC
reaction of fully deprotected alkyne-oligonucleotides with
azides. Moreover, the recyclability of the catalyst makes this
an environmentally friendly and economical strategy5a,5e,6 for
ligand conjugation to oligonucleotides. To expand the scope of
this reagent, we are evaluating the utility of this catalyst in
synthesizing other click chemistry products including targeted
nanoparticles for delivery of siRNAs.
We are grateful to our colleagues Drs T. Yamada, C. G. Peng,
K. N. Jayaprakash, and S. Matsuda for synthesizing intermediates
used in this study.
Notes and references
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Scheme 3 Chelated Cu(I)-assisted azide–alkyne cycloaddition of fully deprotected
alkyne oligonucleotides to azides.
analysis. Importantly, LC-MS did not show detectable levels of
copper ion in the product or strand cleavage. In our earlier work
using the conventional click reaction, strand cleavage was
observed and copper ion was detected in the product even after
stringent purification.2
We then investigated conjugation of oligonucleotide 13 to a
lipopeptide. The azido lipopeptide 17 is based on the HIV Tat
48–60 cell permeation peptide (CPP)10 and was synthesized
under standard Fmoc solid-phase peptide synthesis conditions.
After coupling of the last amino acid to the solid support,
16-azidohexadecanoic acid11 was coupled to the N-terminus to
obtain the desired peptide. The lipophilic alkyl chain
functioned as a tether and is considered as an additional
pharmacokinetic modulator of the siRNA.12 The reaction
between the alkyne-oligonucleotide 13 and the azido peptide
17 was quantitative with 2 molar equivalents of the azide to the
alkyne, and there was no copper ion contamination in the
product as confirmed by LC-MS analysis. The catalyst retained
activity after repeated use. There was no distinguishable
difference in the catalytic activity of the immobilized Cu(I) ion
on polystyrene compared to the CPG solid support (data not
shown).
In summary, we have demonstrated efficient chelation of
Cu(I) ions to derivatized hydrophilic tris(triazolylmethyl)amine
on a solid support. The immobilized reagent effectively cata-
lysed the CuAAC reaction under heterogeneous conditions and
resulted in quantitative and regioselective conjugation of an
azido ligand to a completely deprotected alkyne oligonucleotide
with no detectable levels of copper ions in the product or strand
degradation. Use of the immobilized and chelated Cu(I) ion
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This journal is The Royal Society of Chemistry 2012
Chem. Commun.