Conjugation of an oligonucleotide to Tat, a cell-penetrating peptide, via click chemistry

Article abstract of DOI:10.1016/j.tetlet.2010.07.101

Uptake of diagnostic and therapeutic oligonucleotides that specifically target disease can be enhanced by attachment of a cell-penetrating peptide. Here, we describe the covalent attachment of an oligonucleotide to Tat, a biologically important cell-penetrating peptide, via click chemistry.

Full text of DOI:10.1016/j.tetlet.2010.07.101

Tetrahedron Letters 51 (2010) 5032–5034
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Tetrahedron Letters
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Conjugation of an oligonucleotide to Tat, a cell-penetrating peptide, via
click chemistry
*
Sarah D. Brown, Duncan Graham
Centre for Molecular Nanometrology, WestCHEM, Department of Pure and Applied Chemistry, University of Strathclyde, 295 Cathedral Street, Glasgow G1 1XL, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Uptake of diagnostic and therapeutic oligonucleotides that specifically target disease can be enhanced by
attachment of a cell-penetrating peptide. Here, we describe the covalent attachment of an oligonucleo-
tide to Tat, a biologically important cell-penetrating peptide, via click chemistry.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 12 May 2010
Revised 25 June 2010
Accepted 16 July 2010
Available online 22 July 2010
Keywords:
Oligonucleotide–peptide conjugates
Tat peptide
Click chemistry
Detection and treatment of a disease on a cellular level using oli-
gonucleotides is an elegant strategy with high specificity and low
toxicity.1 Delivery of a nucleic acid sequence into the cell, however,
is made difficult by the efficiency of the cell itself. The plasma mem-
brane is a highly effective barrier with a net negative charge, repel-
ling the phosphate backbone of the oligonucleotides.2 Attachment
of cell-penetrating peptides (CPPs) to oligonucleotides is well docu-
mented and has been found to facilitate transfection and enhance
resistance to the degradation of nucleic sequences.3–9 The conflict-
ing chemistries of peptide and oligonucleotide synthesis make in-
line conjugation challenging. Total solid-phase synthesis overcomes
these problems, however, the method is not very flexible.10 Synthes-
ising the two biomolecules and linking them in solution (fragment
conjugation) avoid these problems but can be labour intensive, can
be time-consuming and can generate poor yields. Tat peptide, de-
rived from HIV-1 Tat protein, is a cell-penetrating peptide of biolog-
ical interest due to its widely reported success in transporting
various cargoes into cells.11–15 Tat peptide, however, is notoriously
difficult to handle, often precipitating from reaction mixtures.16,17
While Gogoi et al. have produced oligonucleotide–peptide conju-
gates (OPCs) using click chemistry,18 we now provide the first report
of an oligonucleotide–Tat peptide conjugate via the copper-cata-
lysedazide–alkynecycloaddition(CuAAC). Inaddition,wehaveused
highly denaturing conditions to ensure that the biomolecules come
together covalently rather than electrostatically. Copper-catalysed
azide–alkyne cycloaddition reactions are chemoselective, fast and
form an irreversible linkage, under ambient conditions.19 The mild
conditions of this reaction have previously been applied to modify
oligonucleotides,20,21 to functionalise nanoparticles with en-
zymes,22 and in fluorescent-labelling of cellular systems.23
A series of modified oligonucleotides as precursors for OPC forma-
tion under click chemistry conditions were synthesised. The alkyne
could be added to either the peptide or the oligonucleotide as could
the azide group, and both the scenarios were examined. To produce
a 50-alkynyl-modified oligonucleotide, 5-hexyn-1-ol was phosphity-
lated and incorporated into the 50-end of the DNA sequence via so-
lid-phase synthesis. Conversely,
a
direct phosphoramidite
derivative of the azido function is not possible due to the reactivity
of this group with phosphines, that is, via the Staudinger reaction.
To overcome this, and to produce an azido-modified oligonucleotide,
succinimidyl azidovalerate was synthesised and reacted with an ami-
no-modified solid support (Scheme 1). The Fmoc protecting group
was removed using piperidine and the free amine was reacted with
the activated ester before being used with standard phosphoramidite
chemistry to yield a 30-azido-modified oligonucleotide.
Table 1
MALDI-TOF mass spectrometric characterisation of modified oligonucleotides, 50-X-
GTT TTC CCA GTC ACG ACG-Y-30 and oligonucleotide–Tat peptide conjugate 1
Calcd m/z
Found m/z
5643.4
5642.2
X =
Y =
5785.3
5782.9
N3
N3
5740.1
7396.0
5739.4
7399.2
X =
1
* Corresponding author. Tel.: +44 141 5484701; fax: +44 141 5520876.
E-mail address: duncan.graham@strath.ac.uk (D. Graham).
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.07.101

S. D. Brown, D. Graham / Tetrahedron Letters 51 (2010) 5032–5034
5033
O
Fmoc HN
*
O
H2N
*
ODMTr
ODMTr
N
N3
O
O
(i)
O
O
H
N
N3
*
ODMTr
O
O
DNA Synthesiser
H
N
(ii)
N3
*
O
Oligonucleotide
O
OH
*
Scheme 1. Reagents: (i) 20% piperidine in MeCN; (ii) concentrated NH4OH. indicates a chiral centre.
In a similar approach, a 50-azido-modified oligonucleotide was
produced using a two-step process: 50-monomethoxytrityl (MMT)
aminomodifier phosphoramidite was used to modify the 50-end of
the oligonucleotide with a protected amine group. Removal of the
MMTprotectinggroupallowed thefreeamine toreactwithsuccinim-
idyl azidovalerate to generate a 50-azido-modified oligonucleotide.
The modified sequences were cleaved, purified and characterized
by MALDI-TOF mass spectrometry (Table 1).
Cu(I) and accelerate the reaction (Scheme 2).25 An aliquot of form-
amide was added to ensure the covalent attachment of the biomol-
ecules and prevent them coming together electrostatically.17 Each
solution was agitated at room temperature overnight.
Ion-exchange HPLC analysis of the reaction between the 30-azi-
do-modified oligonucleotide and the alkyne-modified peptide
showed the formation of a new peak with a shorter retention time
than that of the unconjugated oligonucleotide (Fig. 1).
Azide-modified Tat peptide was synthesised by a reaction of the
N-terminus of the peptide with succinimidyl azidovalerate. Propiol-
ic acid was coupled to the N-terminus of Tat peptide to form an
amide bond which gave the alkyne-modified peptide. Conjugation
of the 50-alkyne-modified oligonucleotide with the azido-modified
Tat peptide derivative (YGRKKRRQRRR) and the 50- and 30-azido-
modified oligonucleotides with the alkyne-modified Tat peptide
was carried out using the reaction conditions as recommended by
Kolca˘lka et al.24 This included tris(benzyltriazolylmethyl)amine
(TBTA), an additional ligand which has been shown to stabilise
The OPC is overall less negatively charged in comparison to the
unconjugated oligonucleotide as the ionic charges are negated due
to the positively charged peptide. The peak appearing at approxi-
mately 11 min, thought to be the OPC product, was collected, dia-
lysed to remove the remaining formamide and further purified
using ZipTip™ C18 pipette tips. Formation of the OPC was con-
firmed by MALDI-TOF mass spectrometry in positive mode (Table
1). Based on peak ratios, the conjugate was formed in 56% yield.
All conjugation reactions described were carried out under ar-
gon to prevent the breakdown of the copper catalyst. It was subse-
O
H
N
RRRQRRKKRGY
N3
*
O
Oligonucleotide
N
H
O
OH
(i)
O
N
*
N
N
H
O
Oligonucleotide
N
OH
RRRQRRKKRGY
N
H
O
Scheme 2. Reagents: (i) TBTA, sodium ascorbate, CuSO4, formamide, phosphate buffer, 56%.

5034
S. D. Brown, D. Graham / Tetrahedron Letters 51 (2010) 5032–5034
first report of the preparation of an OPC via CuAAC using Tat. The
reaction proceeds under aerobic conditions, and at room tempera-
ture, in water.19,28 These are attractive properties in the develop-
ment of biological tools for diagnostics and therapeutics.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2010.07.101.
References and notes
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Figure 1. Ion-exchange HPLC traces at 260 nm of 30-azido-modified oligonucleo-
tide–Tat OPC (solid line) and a control (dashed line): (i) unreacted catalyst mixture,
(ii) OPC, (iii) unreacted oligonucleotide. The control contained TBTA, CuSO4, sodium
ascorbate, 30-azido-modified oligonucleotide, formamide, phosphate buffer, but not
the Tat peptide.
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quently found, however, that this made no difference to the out-
come of the reactions. The arginine side chain is known to stabilise
Cu(I) which may prevent the anticipated oligonucleotide degrada-
tion negating the need for an inert atmosphere.26
No OPC peak was observed for the synthesis of the 50-azido-
modified or 50-alkyne-modified oligonucleotide–Tat peptide conju-
gates. It is not fully understood as to why the reaction between 50-
azido-modified oligonucleotide and alkyne-modified Tat peptide
did not proceed, however, the successful formation of oligonucleo-
tide–Tat peptide conjugates may require an activated alkyne which
was present during the formation of OPC 1.27 The amino acid side
chains of the peptide can have a significant effect on the reaction
outcome and this underlines the difficulty in using biologically rel-
evant peptides such as Tat.
In conclusion, a series of modified oligonucleotides as precur-
sors for CuAAC synthesis of OPCs were generated, however, OPC
formation was only observed upon reaction with a 30-azido-modi-
fied oligonucleotide and an alkyne-modified Tat peptide. This is the
27. Li, Z.; Seo, T. S.; Ju, J. Terahedron Lett. 2004, 45, 3143.
28. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int. Ed.
2002, 41, 2596.