An activated triple bond linker enables 'click' attachment of peptides to oligonucleotides on solid support

Article abstract of DOI:10.1093/nar/gkr603

A general procedure, based on a new activated alkyne linker, for the preparation of peptide-oligonucleotide conjugates (POCs) on solid support has been developed. With this linker, conjugation is effective at room temperature (RT) in millimolar concentrat

Full text of DOI:10.1093/nar/gkr603

Published online 27 July 2011
Nucleic Acids Research, 2011, Vol. 39, No. 20 9047–9059
doi:10.1093/nar/gkr603
An activated triple bond linker enables ‘click’
attachment of peptides to oligonucleotides
on solid support
˚
Malgorzata Wenska*, Margarita Alvira, Peter Steunenberg, Asa Stenberg,
¨
Merita Murtola and Roger Stromberg*
Department of Biosciences and Nutrition, Karolinska Institutet, Novum, SE-141 83 Huddinge, Sweden
Received October 29, 2010; Revised July 5, 2011; Accepted July 6, 2011
nucleic acid uptake into cells is to utilize cell penetrating
peptides (CPPs) either in the form of complexes or in the
form of covalent conjugates, which also protects from di-
gestion by intracellular enzymes (4). A recent successful
example involves a CPP conjugate of a morpholino
phosphorodiamidate oligomer that when given in low
doses leads to highly efficient splice switching and produc-
tion of therapeutically useful levels of dystrophin in
skeletal and heart muscles, which makes this a promising
approach for the treatment of Duchenne muscular dystro-
phy (DMD) (5).
Quite a few approaches have been reported for the
attachment of peptides to oligonucleotides in solution.
This includes a large variety of linkages between the
peptide and oligonucleotide, with the most common
being amide, disulfide, thioether and urea function-
alities and also many other types that have different
advantages or disadvantages (6,7). Two main strategies
have been adopted for the synthesis of peptide–oligo-
nucleotide conjugates (POCs): online solid-phase synthesis
and fragment conjugation methods. In online methods,
the peptide and oligonucleotide fragments are usually
assembled sequentially on the same solid support. This
strategy puts higher demands on compatibility of protect-
ing groups or activating and deblocking agents, and con-
ditions used during synthesis of the peptide are typically
not compatible with the presence of the oligonucleotide
part. Hence, protecting groups and other reaction condi-
tions have to be chosen carefully to minimize subside re-
actions which in most cases requires expertise and a
laboratory equipped with automated synthesizers. A
solid-phase technique for the synthesis of 50-POCs with
a uniform protection strategy for the nucleic acid and
the peptide fragments using acid-labile a-amino protec-
tions has been previously developed in our group (8).
Another procedure, with similar limitations of the need
for an in-house synthesizer and only allowing C-terminal
ABSTRACT
A general procedure, based on a new activated
alkyne linker, for the preparation of peptide–oligo-
nucleotide conjugates (POCs) on solid support has
been developed. With this linker, conjugation is ef-
fective at room temperature (RT) in millimolar con-
centration and submicromolar amounts. This is
made possible since the use of a readily attachable
activated triple bond linker enhances the Cu(I)
catalyzed 1,3-dipolar cycloaddition (‘click’ reaction).
The preferred scheme for conjugate preparation
involves sequential conjugation to oligonucleotides
on solid support of (i) an H-phosphonate-based
aminolinker; (ii) the triple bond donor p-(N-pro-
pynoylamino)toluic acid (PATA); and (iii) azido-
functionalized peptides. The method gives conversion
of oligonucleotide to the POC on solid support, and
only involves a single purification step after com-
plete assembly. The synthesis is flexible and can
be carried out without the need for specific auto-
mated synthesizers since it has been designed to
utilize commercially available oligonucleotide and
peptide derivatives on solid support or in solution.
Methodology for the ready conversion of peptides
into ‘clickable’ azidopeptides with the possibility of
selecting either N-terminus or C-terminus connec-
tion also adds to the flexibility and usability of the
method. Examples of synthesis of POCs include
conjugates of oligonucleotides with peptides known
to be membrane penetrating and nuclear localiza-
tion signals.
INTRODUCTION
conjugation,
synthesis (9).
involves
Fmoc-based
solid
phase
Delivery is a seriously limiting factor for antisense, siRNA
and gene therapy in vivo (1–3). One approach to enhance
*To whom correspondence should be addressed. Tel: +46 8 52481024; Fax: +46 8 52481034; Email: roger.stromberg@ki.se
Correspondence may also be addressed to Malgorzata Wenska. Tel: +46 8 52481025; Fax: +46 8 52481034; Email: malgorzata.wenska@ki.se
ß The Author(s) 2011. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

9048 Nucleic Acids Research, 2011, Vol. 39, No. 20
Solution phase fragment conjugation involves separate
isolation and purification of peptides and oligonucleotides
specifically modified with the functional groups required
for the conjugation reaction. One of the most efficient
liquid-phase conjugation methods is ‘Native ligation’
(10, 11) where the peptides and oligonucleotides are
modified so as to contain cysteine or S-protected thiols
and thioesters at the site of ligation. This methodology
appears to give high yields but requires specific synthesis
of a number of building blocks and reagents, especially if
the thiol function is to be transient. The disulfide linkage is
frequently used in oligonucleotide peptide conjugations
(6) giving high yields, but for some applications there
are concerns regarding the stability of the linkage which
can be cleaved in a reducing environment. Formation of a
thioether linkage is also a common method for making
POCs in solution, typically by one oligomer containing a
thiol and the other a maleimide derivative, which react
giving decent yields of conjugates (6,12). An attractive
solution synthesis approach uses Diels–Alder cycloadd-
ition for linking oligonucleotides with peptides and
results in high yields with slight excess of peptide, while
it will result in isomeric products due to the linkage
formed (13). A reaction that would seem ideal for conju-
gation, due to the compatibility with many other function-
al groups, is the copper (I) catalyzed 1,3-dipolar
cycloaddition between an azide and an alkyne, commonly
referred to as click chemistry (14–17). Recently, synthesis
of a Tat-oligonucleotide conjugate was reported using this
approach in solution. The conjugate was formed in accept-
able yields using a high excess of copper sulfate (ꢀ100)
and peptide analog (ꢀ10), and only an oligonucleotide
with a 30-azide linker gave product and not oligonucleo-
tides with 50-alkyne or 50-azide linkers (18). Another
example of a DNA–peptide conjugate was also prepared
by liquid-phase conjugation, which involves purification
of both peptide and oligonucleotide precursor, but at a
concentration that makes it difficult to perform in
submicromole scale (19).
Solution phase conjugation of highly cationic peptides
can be problematic due to the tendency to form complexes
with the anionic oligonucleotide phosphate backbones.
The reaction mixture can even become highly heteroge-
neous due to precipitation and reduce the conjugation
yields considerably. One way to avoid this problem, and
at the same time gain simplicity and reduce number of
purification steps, is to perform the fragment conjugation
of oligonucleotides and peptides with one of the oligomers
still protected and attached to the solid phase. A method
for solid-phase fragment conjugation (SPFC) in prepar-
ation of POCs by formation of urea linkages has been
developed and results in yields of 3–50% (20,21). Gait
and co-workers have synthesized different types of POCs
by SPFC methods using a variety of functional groups to
link the peptide and oligonucleotide fragments either
directly or using a suitable spacer between the fragments
(22,23). The oligonucleotide is assembled on solid phase
and modified phosphoramidite building blocks are used in
this strategy. The methods above can be used for success-
ful synthesis of POCs. Each method has various limita-
tions in linking position on the oligonucleotide and in
whether the peptide can be linked at the N or C
terminal or both. Other restrictions are that several of
the methods give moderate degrees of conversion and
that in most cases some of the work requires specialists
and access to peptide and/or oligonucleotide synthesizers.
Despite the existence of the above methods, it seemed to
us that there was still a need for versatile and high yielding
method for the synthesis of POCs. It would be preferable
to perform the conjugation with either peptide or oligo-
nucleotide on solid support in order to minimize the
number of purification steps and to make the handling
user friendly. A specific aim was also that it should be
possible to carry out without the need for specific synthe-
sizers, through limited manipulation of commercially
available oligonucleotide and peptide derivatives. This
would be a particular advantage for laboratories that
perform experiments with such conjugates but that do
not have access to automated synthesizers. It would also
be ideal if the general approach would be suitable for con-
jugation of other biomolecules under mild conditions as
well. Oligonucleotides still attached to the solid support
can be ordered from most companies selling oligonucleo-
tides, and peptides still attached to the support can also be
ordered. It would of course not be a disadvantage if the
chemistry involved in the linking gives high versatility,
thus allowing for conjugation to many different kinds of
modified oligonucleotides. It is also advantageous to be
able to choose conjugation to either the N or C terminal
of the peptide as this could play a role in recognition/ac-
cessibility of the functional peptide. Furthermore, it would
be desirable to be able to obtain near quantitative conju-
gation in a submicromole scale, especially for libraries.
Here, we present a versatile method for the synthesis of
multiple types of POCs that can be performed on solid
support in submicromole scale with commercially avail-
able oligonucleotide and peptide derivatives. Convenient
handling and efficient reaction at low concentration under
mild RT conditions is enabled by use of a new activated
triple bond donor and by performing reaction on solid
support. Convenient and efficient conversion of peptides
into clickable derivatives also contributes to making the
concept user friendly.
MATERIAL AND METHODS
Acetonitrile (MeCN, High Performance Liquid
Chromatography (HPLC) grade, Merck), methanol
(MeOH) and dichloromethane (DCM, Fisher Scientific,
Analytical Grade) were of commercial grade and used as
received. Dimethylformamide (DMF) and pyridine (Pyr)
(both from Merck and analytical grade) were additionally
dried over 4A molecular sieves. Silica gel column chroma-
tography was performed on Merck G60, Thin Layer
Chromatography (TLC) analysis was carried out on
pre-coated Silica Gel 60 F254 (Merck), with detection by
UV light. NMR spectra were recorded on a Bruker
AVANCE DRX-400 instrument (400.13 MHz for 1H,
162.00 MHz for 31P, 100.62 MHz for 13C). Reverse phase
HPLC was carried out on a Jasco HPLC system using the
following columns: Hypersil 5 mm (250 ꢀ 4.6 mm) with

Nucleic Acids Research, 2011, Vol. 39, No. 20 9049
1 ml/min flow rate; Kromasil 100-5-C18, 5 mm
(250 ꢀ 4.6 mm) with 1 ml/min flow rate; and Kromasil
100-5-C18, 5 mm (250 ꢀ 10 mm) at 4 ml/min flow rate.
Buffers for reverse phase chromatography were as
follows: (i) 50 mM triethylammonium acetate (TEAA),
pH 6.5; (ii) 50 mM TEAA, pH 6.5, in 50% CH3CN.
Mass spectra (TOF-MS, ES) were obtained by using a
Micromass LCT electrospray time-of-flight (ES-TOF) in-
strument. Molecular weights of the conjugates were recon-
structed from the m/z values of the multiply charged ions
using the mass deconvolution program (MAXENT) of the
instrument, which gave values with no decimal place.
Oligonucleotide sequences were purchased from: Sigma-
Proligo Sweden, a locked nucleic acid (LNA) sequence
GLGLALMeCLTLTLTL-Am5 (LNA-Am5); Eurogentech
of Seraing, Belgium, dAGAGCCATGAG-C6 (DNA-
ethylene from azidoamine), 53.3 (CH Leu), 60.7
(CH Pro), 169,9 (C=O), 171.1 (C=O), 172.7 (C=O),
172.9 (C=O). ES–MS, calcd m/z (M-H)ꢁ1 394.2281,
found 394.2279 (a similar procedure used for TFA-
SV40-L1-azide and SV40-L1-azide is presented in
Supplementary Data).
Preparation of azide-peptides for N-terminal
conjugation on solid support: C-myc-L2-azide (7)
and TFA-C-myc-L2-azide (8)
The peptide GAAKRVKLD 5 (0.035 mmol) still attached
to solid support and with free N-terminus was placed in a
sealed Eppendorf tube. In another sealed Eppendorf
tube 2-(2-azidoethoxy)ethoxyacetic acid (L2, 3.3 eq.),
2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU, 3.3 eq.) and NMM (6 eq.)
in 350 ml of DMF were shaken for 0.5 h at RT. This
solution was then added to the peptide on solid support.
The tube was sealed and the reaction was agitated on a
vortex for 2 h at RT. The tube was centrifuged, the
solution was carefully removed with a syringe and 200 ml
of DMF was added to the support. The Eppendorf tube
was then agitated on a vortex, centrifuged and the solution
above the solid support was removed. This washing was
repeated 5 times. After this 500 ml of a solution containing
90% trifluoroacetic acid (TFA), 4% triisopropylsilane
(TIS), 4% H2O and 2% 3,5-dioxo-1,8-octanedithiol was
added and the suspension was agitated on a vortex for 4 h.
The tube was centrifuged and the solution was carefully
C6);
Am5 (DNA-Am5), Rasayan Inc., Encinitas, CA, USA,
Cybergene
Sweden,
dAGAGCCATGAG-
20-OMe-CCUCUUACCUCAGUUACA-ODMT
0
[18-merRNA(2 -OMe)]. Nucleobase protecting groups were
benzoyl for cytosine and adenine and isobutyryl for guan-
ine. Solid-supported peptide GAAKRVKLD (C-myc) was
obtained from Eurogentech of Seraing, Belgium. The
amino acid sequence was assembled by standard Fmoc
synthesis on Wang resin and delivered on support with
standard acid-labile protecting groups: t-butyloxycar-
bonyl (Boc) for lysine, t-butyl ester (O-tBu) for aspartic
acid
and
2,2,4,6,7-pentamethyl-dihydrobenzofuran-
5-sulfonyl (Pbf) for arginine. AcPLG (AcMIF1) was
purchased from Bachem. PKKKRKVG (SV40) was
from AnaSpec Inc, and was obtained fully deprotected
but not purified.
collected with
a syringe. Another portion of the
deblocking/support cleaving solution was added and the
suspension was agitated and centrifuged whereupon
the solution was collected with a syringe (this washing
was repeated an additional two times). The combined so-
lutions were collected in round-bottomed flask and TFA
was removed by flushing air into the flask, whereupon
water was added and the solution was washed with cold
diethyl ether (repeated two times). The water layer was
collected and concentrated in vacuo. Methanol was
added and evaporated under reduced pressure whereupon
the residue was dried under vacuum for 0.5 h (peptide 7).
Peptide 7 was purified as detailed in (i). If protection of
basic amino acids (e.g. lysines) is desired, the procedure
indicated in (ii) was followed.
Preparation of azide-peptides for C-terminal conjugation,
e.g. AcMIF1-L1-azide (4)
The acetylated tripeptide AcPLG-COOH (100 mg,
0.3 mmol) was dissolved in 3 ml DMF containing 2 eq.
of N-methylmorpholine (NMM) (67 ml). HBTU was
added (1 eq., 116 mg) and the reaction was stirred at RT
for 30 min. After this time, 2-azidoethylamine was added
(0.95 eq., 25 mg) and the reaction was stirred overnight at
RT. The solvent was then evaporated and the residue was
dried by evaporation of added toluene and dichloro-
methane. The purification was done by silica gel column
chromatography using a linear gradient of methanol in
DCM from 0% to 6%. AcMIF1-L1-azide (4): Yield
(i) The peptide was dissolved in 50 mM triethyl-
ammonium acetate buffer, pH 6.5, containing 15%
acetonitrile and then purified by RP HPLC using a
linear gradient of buffer B in A from 15% to 27%
in 30 min, detection at 220 nm. C-myc-L2-azide (7):
tR = 15.7 min, yield = 60% (HPLC); ES–MS, calcd
m/z (M-H)+1 1128.6, found 1128.8.
(ii) Methanol (5 ml) was added to form a suspension and
the reaction was put on an ice bath. Ethyl
trifluoroacetate (0.3 ml) was added, followed by
0.1 ml of triethylamine. The reaction was left stirring
for 3 days slowly warming up to RT (melting ice
bath). After the reaction was complete, the solvents
are evaporated under reduced pressure and the pep-
tide was dissolved in 50 mM TEAA buffer, pH 6.5,
containing 15% acetonitrile and purified by RP
1
45 mg, 38%. H-NMR (400 MHz, CDCl3), d (ppm): 0.85
(3H, d, CH3 Leu, J = 6.2 Hz), 0.90 (3H, d, CH3 Leu), 1.59
(2H, m, CH Leu+1H from CH2 Leu), 1.71 (1H, m, 1H
from CH2 Leu), 2.00 (3H, m, CH2 Pro), 2.07 (3H, s, CH3,
Ac from Ac-Pro), 2.20 (1H, m, from CH2 Pro), 3.39 (4H,
bs, 2 ꢀ CH2, ethylene from azidoamine), 3.48 (1H, m,
from CH2 Pro), 3.59 (1H, m, from CH2 Pro), 3.88 (2H,
AB-q, CH2 Gly, J = 5.8 Hz, J = 11.0 Hz), 4.17 (1H, m,
aCH Leu), 4.42 (1H, m, aCH Pro), 7.21 (2H, bs,
2 ꢀ NH, Leu+Gly) 13C-NMR (100.62 MHz, CDCl3),
d (ppm): 21.7 (CH3, Ac from Ac-Pro), 22.7 (CH3 Leu),
23.1 (CH3 Leu), 25.2 (CH Leu+CH2 Pro), 28.5 (CH2
Pro), 39.1 (CH2, ethylene from azidoamine), 40.6
(CH2 Leu), 43.4 (CH Gly), 48.7 (CH2 Pro), 50.5 (CH2,

9050 Nucleic Acids Research, 2011, Vol. 39, No. 20
HPLC using a linear gradient of buffer B in A from
30% to 100% in 30 min, detection at 220 nm.
TFA-C-myc-L2-azide (8): tR = 16.8 min, yield =
44% (HPLC); ES–MS, calcd m/z (M-H)ꢁ11318.6,
found 1318.6.
by sodium ascorbate solution [6 eq., 0.6 mmol, 5 ml from
a stock solution of 24 mg/ml in tBuOH/H2O (1:1)], where-
upon the tube was gently shaken and CuSO4 was added
(2.4 eq., 0.24 mmol, 5 ml from a stock solution of 12 mg/ml
in H2O). The Eppendorf tubes were sealed, connected
together with Parafilm and horizontally shaken on the
vortex over night at RT. After this time, the tube was
centrifuged, the solution was carefully removed from the
solid support with a syringe and the residue was washed
two times with 200 ml of tBuOH/H2O (1:1) and two times
with DMF. To each mixture was 500 ml of 28% NH3 (aq)
added and the Eppendorf tube was sealed, agitated on a
vortex for 1 min and left at 55ꢂC over night. The tube was
centrifuged and the solution was carefully collected with a
syringe. Another portion of NH3 solution was added and
the suspension agitated, centrifuged and the solution was
collected. To the combined solutions, water was added
(5 ml) and the solution with the POC was concentrated
in vacuo. The reaction was analyzed and purified by
RP-HPLC using a linear gradient of buffer B in A from
0% to 50% in 20 min (UV detection at 260 nm) at 50ꢂC.
LNA-AcMIF1 (entry 1, Table 1): tR = 15.2 min, ES–
MS, calcd (M) 3067, found 3066; DNA-Am5-AcMIF1
(entry 6, Table 1): tR = 16.4 min ES–MS, calcd (M)
4138, found 4138; LNA-Am5-C-myc (entries 2 and 3,
Table 1): tR = 16.2 min; ES–MS, calcd (M) 3802, found
3803; DNA-Am5-C-myc (entries 7 and 8, Table 1):
tR = 15.1 min; ES–MS, calcd (M) 4870, found 4870;
DNA-C6-C-myc (entry 9, Table 1): tR = 16.3 min; ES–
MS, calcd (M) 4882, found 4882; LNA-Am5-L2-azide
(oligonucleotide with azide linker used in conjugate syn-
thesis for entry 5, Table 1): tR = 15.0 min; ES–MS, calcd
(M) 2659, found 2659; LNA-TFA-SV40 (non-isolated,
entry 5, Table 1): ES–MS, calcd (M) 3680, found 3680.
(N-Propynoylamino)-p-toluic acid (PATA, 11)
Propynoic acid (1 mmol, 61 ml) was added to stirred cold
(ice bath) solution of dicyclohexylcarbodiimide (DCC, 1.2
eq., 247 mg) in dry acetonitrile (7 ml) under argon. After
10 min, 4-(aminomethyl)benzoic acid (1 eq., 151 mg)
dissolved in 1.5 ml of acetonitrile was added dropwise
and the resulting reaction mixture was stirred for 1 h at
0ꢂC, allowed to warm up to RT and then reacted for an
additional 1 h at this temperature. The solution was
evaporated and the compound purified by column chro-
matography using a linear gradient of methanol in
dichloromethane (0–10%). After collecting fractions con-
taining the product, the solvent was evaporated under
reduced pressure and the compound precipitated by the
addition of dichloromethane. Pure 11 was collected by
filtration, dried under vacuum and can be kept at ꢁ20ꢂC
for several weeks. Yield of 102 mg, 50%. 1H-NMR
(400 MHz, DMSO d6), d (ppm): 4.20 (1H, s, CH), 4.34
(2H, d, CH2, J = 6.1 Hz), 7.32 (2H, d, 2 ꢀ CH Ph,
J = 8.1 Hz), 7.90 (2H, d, 2 ꢀ CH Ph, J = 8.1 Hz), 9.32
(1H, t, NH, J = 6.1 Hz), 13C-NMR (100.62 MHz,
DMSO d6), d (ppm): 42.1 (CH2), 76.3 (CH), 78.1 (CH),
127.1 (2 ꢀ CH Ph), 129.4 (2 ꢀ CH Ph), 142.9 (C=O),
151.8 (C=O) ES–MS, calcd m/z (M-H)ꢁ1 202.0582,
found 202.0570.
Synthesis of POCs by conjugation of peptide-azides to
solid-supported, aminolinker-carrying commercial
oligonucleotides
2-(N-(4-monomethoxytrityl)aminoethoxy)ethyl
H-phosphonate triethylammonium salt (13)
Commercially available solid-supported 50-aminolinker
oligonucleotides were placed in sealed 500 ml Eppendorf
tubes (1.5–3 mg support, 0.1 mmol in each tube). In
another Eppendorf tube, HBTU (479 eq., 18 mg) and ac-
tive linker PATA (500 eq., 10 mg) were dissolved in 560 ml
of dry DMF containing 55 ml (4950 eq.) of NMM. The
tube was sealed and placed horizontally on a vortex and
shaken for 0.5 h, after which time a one-quarter of the
solution was taken by syringe and added to the solid-
supported oligonucleotide. The Eppendorf tubes were
sealed, connected together by means of Parafilm and
placed horizontally on a vortex, and then shaken for 2 h.
After this time, the mixture was centrifuged, the solution
was carefully removed from the solid support with a
syringe and 200 ml of DMF was added to each tube. The
tube was closed, centrifuged and washed with an addition-
al portion of DMF; this process is repeated to have, in
total, 10 washings with DMF and finally 2 with tBuOH/
H2O (1:1). The protected peptide-azide TFA-C-myc-
L2-azide (1.6 mmol, 2.1 mg) or Ac-MIF1-L2-azide
(1.6 mmol, 0.63 mg) dissolved in 140 ml of tBuOH/H2O
(1:1) and 35 ml of this peptide solution (4 eq., 0.4 mmol)
was added to the supported oligonucleotide. To each
reaction was added 5 ml of tBuOH/H2O (1:1) followed
To 2-(2-aminoethoxy)ethanol (2 mmol, 200 ml) dissolved
in 20 ml of dry pyridine, triethylamine (2 mmol, 288 ml)
was added, followed by monomethoxytrityl chloride
(2.2 mmol, 679 mg). The reaction was stirred under
argon atmosphere for 2 h (RT). After confirmation (by
TLC with ninhydrine staining) of consumption of the
starting material, diphenyl H-phosphonate was added to
the reaction mixture (4 mmol, 780 ml). The reaction was
left stirring for an additional 1 h. After confirmation that
all monomethoxytritylated linker was consumed (TLC
and MS), 5 ml of water was added to the reaction that
was then allowed to stir overnight at RT. The reaction
was then evaporated to dryness under reduced pressure,
dissolved in dichloromethane and washed with a saturated
aqueous solution of NaHCO3 (two times). The organic
phase was collected, dried over MgSO4, concentrated
and purified by column chromatography on silica using
a gradient of methanol in dichloromethane (0–5%, with
0.3% triethylamine). Fractions containing 13 were col-
lected and concentrated whereupon added dichloro-
methane was evaporated (three times) giving a pale
1
orange oil. Yield 1 g, 92%. H-NMR (400 MHz, CDCl3),
d (ppm): 1.18 (9H, t, J = 7.3 Hz, CH3), 2.24 (2H, t, CH2,

Nucleic Acids Research, 2011, Vol. 39, No. 20 9051
J = 5.4 Hz), 2.89 (6H, q, CH2, J = 7.3 Hz), 3.47 (2H, t,
CH2, J = 5.0 Hz), 3.51 (2H, t, CH2, J = 5.2 Hz), 3.67
(3H, s, OCH3), 3.87 (2H, m), 6.01 (0.5H, s, 1/2PH,
J = 614 Hz), 6.72 (4H, m, PhH), 7.07-7.38 (10H, m,
PhH), 7.55 (0.5H, s, 1/2PH, J = 614 Hz), 31P-NMR
CH2 (TEAH+), J = 7.3 Hz), 3.30 (2H, q, CH2,
J = 5.0 Hz), 3.54 (2H, t, CH2, J = 4.9 Hz), 3.66 (2H, t,
CH2, J = 4.8 Hz), 4.04 (2H, m, CH2), 6.14 (0.5H, s, 1/
2PH, J = 624 Hz), 7.33 (1H, m, PhH), 7.41-7.47 (4H, m,
PhH), 7.55-7.60 (4H, m, PhH), 7.70 (0.5H, s, 1/2PH,
J = 624 Hz); 31P-NMR (161.9 MHz, CDCl3) d (ppm):
4.30, 8.13 (1P, dt, J = 620 Hz, J = 8.3 Hz); 13C-NMR
(100.62 MHz, CDCl3), d (ppm): 27.9 (CH3), 29.1 (CH3),
40.6 (CH2), 62.8 (CH2), 70.0 (CH2), 70.9 (CH2), 80.4 (C),
124.8, 127.0, 127.1, 128.7, 139.5, 140.9, 145.7, (C, CH, Ph),
155.4 (C=O). ES–MS, calcd m/z (M-H)ꢁ1 405.3870,
found 405.9834.
(161.9 MHz, CDCl3)
d (ppm): 4.01, 7.86 (1P, dt,
J = 614 Hz, J = 7.7 Hz), 13C-NMR (100.62 MHz,
CDCl3), d (ppm): 7.6 (CH3), 38.3 (CH2), 44.1 (CH2),
54.1 (OCH3), 61.6 (CH2), 69.1 (CH2), 69.5 (C), 70.1
(CH2), 112.1 (CH, Ph), 125.1 (CH, Ph), 126.8 (CH, Ph),
127.1 (CH, Ph), 127.5 (CH, Ph), 137.1 (CH, Ph), 145.2
(CH, Ph), 157.1 (CH, Ph), ES–MS, calcd m/z (M-H)ꢁ1
440.1785, found 439.9181.
General Method for synthesis of POCs on solid support
by subsequent coupling of aminolinker H-phosphonate and
PATA followed by Cu(I) catalyzed cycloaddition with
peptide-azides
2-(N-(2-(4-Biphenylyl)-prop-2-yl-
oxycarbonyl)aminoethoxy)ethyl H-phosphonate
triethylammonium salt (16)
2-(Biphenyl)prop-2-yl 4-methoxycarbonylphenyl carbon-
ate (Bpoc reagent) (2 mmol, 780 mg) was dissolved in
10 ml of DMF containing 10% of triethylamine and
placed in at 55ꢂC in a silicon oil bath. A 1.1 equivalent
of 2-(2-aminoethoxy)-ethanol (2.2 mmol, 220 ml) was
added to the solution and the mixture was stirred for
3.5 h, after which the solvent was removed under
reduced pressure. The resulting oil was diluted with H2O
(20 ml), 5% NaHCO3 (2 ml) and extracted three times with
DCM (3 ꢀ 20 ml). The aqueous phase was cooled in an ice
bath and extracted once more with DCM (20 ml). The
combined organic layers were dried over Na2SO4,
filtered and concentrated in vacuo, to a pale yellow oil.
The crude product was purified by column chromatog-
raphy on silica using a mixture of DCM/MeOH/NEt3
(98:2:10). Fractions containing pure compound were col-
lected and the solvent was evaporated under reduced
pressure to give the desired product (15) in 87% yield.
1H-NMR (400 MHz, CDCl3), d (ppm): 1.80 (6H, s,
CH3), 3.32 (2H, m, CH2), 3.53 (2H, t, CH2, J = 4.5 Hz),
3.57 (2H, t, CH2, J = 4.5 Hz), 3.74 (2H, t, CH2,
J = 4.6 Hz), 7.32 (1H, m, PhH), 7.40-7.45 (4H, m, PhH),
7.54-7.59 (4H, m, PhH); ES–MS, calcd m/z (M-H
+N a+)ꢁ1 366.4074, found 366.4141.
One millimole of 15 (343 mg) was dissolved in 5 ml of
2 M H3PO3 solution in dry pyridine (10 mmol). The
reaction mixture was stirred and pivaloyl chloride (PvCl;
675 ml, 5.5 mmol) was added slowly. The reaction was
monitored, for disappearance of the starting alcohol and
the formation of a baseline material, by TLC on silica gel
plates using 10:1 DCM/MeOH as solvent. When the
reaction was complete (after overnight at RT), 1 ml of
1 M triethylammonium bicarbonate (TEAB, pH 7.8) was
added and the mixture was partitioned between 40 ml of
DCM and 25 ml of 2 M TEAB (pH 7.8). The organic layer
was dried over Na2SO4, filtered and concentrated in vacuo.
The crude product was purified by column chromatog-
raphy on silica using a mixture of DCM/MeOH/NEt3
(9:1:0.1). Fractions containing the desired product were
combined and concentrated under reduced pressure to
yield pure 16 as the triethylammonium salt in 88% yield.
1H-NMR (400 MHz, CDCl3), d (ppm):1.27 (9H, t, CH3
(TEAH+), J = 7.3 Hz,), 1.80 (6H, s, CH3), 2.99 (6H, q,
The oligonucleotide [52 mg (2 mmol) of controlled pore
glass (CPG) supported 18-mer RNA(20-OMe)] was
placed in a syringe with a frit and then detritylated with
2% dichloroacetic acid (DCA) in DCM (two times 1 min)
and subsequently washed with DCM (five times), MeCN
(2 times) and pyridine. The protected (Bpoc or MMT)-
aminolinker H-phosphonate was dissolved in anhydrous
pyridine (62 mM, coupling solution) and added to the sup-
ported oligonucleotide. 1.1 eq. of pivaloyl chloride (PvCl)
in acetonitrile was then added so that the final proportion
of pyr/MeCN is 10:1 and the reaction mixture was slowly
agitated at RT for 20 min. After this time, the coupling
solution was removed and the support washed with
pyridine. Two percent of I2 in pyridine/water (98:2) was
added to the support which was left shaking for 15 min.
The support was then washed extensively with pyridine
(five times), MeCN (five times) and DCM (five times).
The 50-amino protecting group (MMT or Bpoc) was
then removed by treatment with 2% DCA in DCM (two
times for 2 min) and the support was washed with DCM
(five times) and MeCN (two times). In a separate tube,
compound 11 (PATA linker, 60 mg, 150 eq.) was dissolved
in 2 ml of anhydrous DMF and mixed with HBTU (150
eq., 112 mg). NMM (400 ml, 1800 eq.) was added and the
tube was agitated slowly on a vortex for 30 min at RT.
This preactivation solution was then transferred to the
support in the syringe which was then gently agitated on
a vortex for 2 h at RT. The resulting solid supported
alkyne-modified oligonucleotide was then washed with
DMF (25 ml), MeCN (20 ml) and DCM (3 ml), dried
and split in 0.2 mmol fractions to perform click reactions.
Thus, 0.2 mmol of the supported 50-alkyneoligonucleotide
(5 mg resin) was placed in a small glass vial with a screw
cap. The 4 eq. of peptide-azide (0.316 mg of Ac-MIF1-
L2-azide or 1.1 mg of TFA-C-myc-L2-azide) was dis-
solved in 80 ml of tBuOH/H2O (1:1) and added into the
vial. The 1 eq. of CuI in 10 ml of DMSO (from a stock
solution of 4.3 mg/ml in DMSO) was added to the mixture
followed by diisopropyl ethyl amine (DIPEA) solution [8
eq., 1.6 mmol, 10 ml from a stock solution of 28 ml in 1 ml of
tBuOH/H2O (1:1)] and sodium ascorbate [3 eq., 0.6 mmol,
10 ml from a stock solution of 12 mg/ml in tBuOH/H2O
(1:1)] whereupon the solution was gently agitated on a

9052 Nucleic Acids Research, 2011, Vol. 39, No. 20
vortex at RT over night. The solution was then removed
from the support and the resin was transferred to a syringe
with a frit and rinsed with H2O (20 ml), 0.05 M
ethylenediamine tetraacetate (EDTA) (aq, 20 ml), acetoni-
trile (10 ml) and DCM (3 ml). The support was transferred
to an Eppendorf tube with a screw cap and 0.5 ml of 28%
NH3 (aq) was added. The tube was then heated to and
kept at 55ꢂC over night. Purification was done by
RP-HPLC using a linear gradient of buffer B in A from
0% to 64% for 20 min, detection at 260 nm and oven tem-
perature set at 50ꢂC.
involves bond formation between the copper acetylide and
coordinated azide (31), this could also be expected. We
found that activation of propynoic acid gave side reac-
tions and colored byproducts. To increase the reaction rate
of the click reaction but at the same time have a cleaner
reaction when attaching it to amino groups, we developed
a linker with an activated triple bond donor, ꢀ-(N-
propynoylamino)-p-toluic acid (11, PATA, Scheme 1D).
The electron withdrawing carbonyl of the amide group
vicinal to the triple bond makes it considerably more
reactive in the Cu(I) catalyzed cycloaddition. At the
same time, this reagent has a handle to readily attach it
to oligonucleotides carrying aminolinkers and is UV
visible, which is convenient upon preparation and purifi-
cation. This PATA-linker is straightforward to make from
(Scheme 1D) propynoic acid (9) and aminomethylbenzoic
acid (10). Some side products are formed but the yield
after purification is quite acceptable and since the
starting materials are inexpensive, the cost for the
reagent is quite low. The PATA linker can be made in a
relatively large scale (grams, RT and column chromatog-
raphy) and is stable upon storage.
For the first studies on synthesis of POCs, we used
DNA and LNA oligonucleotides, purchased still attached
to CPG or polystyrene support (PS) and carrying either
2-(aminoethoxy)ethyl phosphate (aminomodifier Am5) or
aminohexyl phosphate (aminomodifier C6) as amino-
linkers (initially MMT protected). The PATA reagent
was then coupled to the solid supported aminolinker
oligonucleotide, and after washing and subsequent Cu(I)
catalyzed cycloaddition under mild conditions with a
peptide-azide the corresponding oligonucleotide peptide
conjugate is formed (Scheme 1E).
Conjugation reactions were initially performed with an
azido derivative of the tripeptide MIF1 (AcMIF1-
L1-azide, 4, Scheme 1B). Attachment of this peptide to
solid-supported oligonucleotide LNA-Am5-PATA in
0.1 mmol scale (of oligonucleotide on support), using 2
equivalents of peptide at different dilutions (down to
2 mM of peptide which would correspond to 1 mM of
oligonucleotide if this was in solution, Supplementary
Data, Figure 1) in the presence of CuSO4/ascorbate gave
conversion to the corresponding conjugate (Figure 1A).
After conjugation of the PLG-tripeptide (Ac-MIF1) to
CPG supported LNA, a series of additional POCs were
produced (Table 1, entries 2–4, 6–9) using the same
approach and conditions. With the MIF peptide, similar
results were obtained upon conjugation to polystyrene-
supported DNA (entry 6, Table 1). This indicates that
although Cu(II)/ascorbate has been reported to cause oxi-
dative damage and cleavage to DNA (32,33), this is not
significant enough to pose any major problem in the syn-
thesis, which could perhaps be expected since the active
alkyne linker allows for relatively mild conditions at RT.
Reaction of the same supported oligonucleotide sequences
with an N-terminal linked peptide-azide from C-myc
(C-myc-L2-azide, 7) was then investigated. With the
LNACPG-Am5, conversion was good using the unprotect-
ed C-myc-L2-azide (entry 2, Table 1), but conjugation to a
DNA fragment on PS gave, although still respectable,
somewhat lower conversion (entry 7, Table 1). The free
The 18-merRNAOMe-AcMIF1 (entry 10, Table 1):
tR = 15.1 min, ES–MS, calcd (M) 6570, found 6570;
18-merRNAOMe-C-myc
(entry
11,
Table
tR = 14.0 min, ES–MS, calcd (M) 7306, found 7304.
1):
RESULTS AND DISCUSSION
Clickable peptides
The first phase of working out the method was develop-
ment of an efficient way, involving minimum manipula-
tion, for conversion of peptides into clickable derivatives.
Non-supported fully deprotected peptides can be
purchased and easily converted into azido derivatives by
intermediate TFA protection and subsequent attachment
of an azide containing linker, as exemplified for the highly
basic SV-40-derived NLS peptide (24) (Scheme 1A).
Amino groups of peptide 1 were converted into trifluoroa-
cetamides (2) and subsequent coupling to 2-azidoethyla-
mine (linker L1-azide) was performed to obtain the
desired peptide 3 (TFA-SV40-L1-azide), from which a
deprotected derivative could also readily be made (25).
In addition, a short non-charged tripeptide, Ac-MIF1
[N-acetylated PLG which is a blood–brain barrier pene-
trating peptide (26)], was similarly converted to the azide
derivative 4 (Ac-MIF-L1-azide, Scheme 1B). An N-terminal
azido derivatized [C-myc-L2-azide (7, Scheme 1C) less basic
peptide (from the C-myc cell penetrating peptide (27)]
could be readily made from a solid support bound
peptide (5) by condensation with 2-(2-azidoethyl)-
ethoxyacetic acid (linker L2-azide) to give (6) followed
by cleavage from support. Cold ether washing of the pep-
tide removes most impurities and it can then be reacted
with ethyl trifluoroacetate to form TFA-C-myc-L2-azide
(8, Scheme 1C). If a more hydrophilic derivative is desired,
the crude unprotected azidopeptide 7 can be directly
HPLC purified and used for cycloaddition.
POCs from commercial aminolinker oligonucleotides
Thus having ready access to peptide-azides, we attempted
to synthesize POCs by Cu(I) catalyzed 1,3-dipolar cyclo-
addition of an oligonucleotide 4-pentynoic amide deriva-
tive, but this failed totally as no reaction was observed
(Supplementary Data, Scheme 1). Non-catalyzed Huisgen
1,3-dipolar cycloaddition with an azide is accelerated by
electron-withdrawing substituents on the dipolarophile
(28,29) and it seems to be the case also for the Cu (I)
catalyzed case (16,17). In view of the most recent mech-
anistic suggestions (30,31) and in particular the experi-
mentally supported claim that the rate-limiting step

Nucleic Acids Research, 2011, Vol. 39, No. 20 9053
Scheme 1. (A–C) Synthesis of azido-functionalized peptides (D) Synthesis of active alkyne linker, PATA. (E) Scheme for POC synthesis on solid
support using commercial oligonucleotides with aminolinkers. Reagents and conditions: (i) ethyl trifluoroacetate, methanol, 3 days, RT;
(ii) preactivation with HBTU and NMM in DMF 0.5 h, RT; (iii) azidoethylamine, 2 h, RT; (iv) 2-(2-azidoethoxy)ethoxyacetic acid (preactivated
with HBTU and NMM in DMF, 0.5 h, RT), DMF, 2 h, RT; (v) 90% TFA, 4% TIS, 4% H2O, 2% 3,5-dioxo-1,8-octanedithiol, 4 h, RT;
(vi) preactivation of 9 with DCC, in acetonitrile, 0ꢂC, then addition of 10; (vii) preactivation of 11 with HBTU, and NMM, DMF, 0.5 h, RT,
then addition to solid supported oligonucleotide 2 h; (viii) 1.2 eq. CuSO4, 3 eq. sodium ascorbate, 2 eq. of peptide-azide; overnight; (ix) NH3, (aq) sat.
55ꢂC overnight (HBTU, O-benzotriazole-N,N,N0,N0-tetramethyl-uronium-hexafluoro-phosphate; azide, N3; Ac, acetyl; TIS, triisopropyl silane; TFA,
trifluoroacetic acid).
Table 1. Oligonucleotides, peptide derivatives and linkers used in synthesis of peptide-oligonucleotide conjugates (POCs)
Entry
Oligonucleotide sequence 30 ! 50
Peptide derivative
Peptide
terminus
Linker reagent
(MMT protected)
1
2
3
GLGLALMeCLTL TLTL-Am5 (LNACPG-Am5)
GLGLALMeCLTL TLTL-Am5 (LNACPG-Am5)
GLGLALMeCLTL TLTL-Am5 (LNACPG-Am5)
GLGLALMeCLTL TLTL-Am5 (LNACPG-Am5)
GLGLALMeCLTL TLTL-Am5- L2-azide, (LNACPG-Am5)
dAGAGCCATGAG-Am5 (DNAPS-Am5)
dAGAGCCATGAG-Am5 (DNAPS-Am5)
dAGAGCCATGAG-Am5 (DNAPS-Am5)
dAGAGCCATGAG-C6 (DNACPG-C6)
AcMIF1-L1-azide (4)
C-myc-L2-azide (7)
C
N
N
C
C
C
N
N
N
C
N
Am5-amidite
Am5-amidite
Am5-amidite
Am5-amidite
Am5-amidite
Am5-amidite
Am5-amidite
Am5-amidite
C6-amidite
TFA-C-myc-L2-azide (8)
TFA-SV40-L1-azide (3)
TFA-SV40-PATA (18)a
AcMIF1-L1-azide (4)
C-myc-L2-azide (7)
TFA-C-myc-L2-azide (8)
TFA-C-myc-L2-azide (8)
AcMIF1-L1-azide (4)
TFA-C-myc-L2-azide (8)
4
5a
6
7
8
9
10
11
(20OMe)CCUCUUACCUCAGUUACA-Am5 (RNACPG
(20OMe)CCUCUUACCUCAGUUACA-Am5 (RNACPG
)
)
Am5-H-phosphonate
Am5-H-phosphonate
aSynthesis in solution (see Supplementary Data for details).
XL, locked nucleic acid monomer; dX, deoxynucleotides; (OMe)X, 20-metoxy oligonucleotide. Commercial names of aminolinkers: Am5, amino
modifier 5; C6, aminolinker C6; Ac, acetyl; MeC, 5-methyl cytosine; L1/L2, azide linkers (Scheme 1).

9054 Nucleic Acids Research, 2011, Vol. 39, No. 20
Figure 1. HPLC chromatograms of crude POCs formed after addition of PATA and CuSO4/ascorbate catalyzed peptide attachment. (A)
LNA-Am5-MIF1-Ac conjugate (entry 1, Table 1); (B) DNA(PS)-Am5-C-myc conjugate [entry 7, Table 1, DNA(PS) indicates that this POC was
synthesized on PS]; (C) LNA-Am5-C-myc conjugate (entry 3, Table 1); (D) DNA(CPG)-C6-C-myc conjugate [entry 9, Table 1, DNA(CPG) indicates that
this POC was synthesized on CPG support]. Reaction conditions: support with 0.1 mmol oligonucleotide, 4 eq. peptide sodium ascorbate solution 6 eq.,
2.4 eq. CuSO4 in 50 ml tBuOH–water (45:55), overnight at RT {RP-HPLC: 0–50% linear gradient of B [0.05 M triethylammonium acetate (aqueous,
pH = 6.5) in 50% acetonitrile (aq)] in A [0.05 M triethylammonium acetate (aqueous, pH = 6.5)] over 20 min, detection at 260 nm, at 50ꢂC}.
unprotected side chains of lysines could potentially inter-
fere to some degree, by Michael conjugate addition to the
active linker and perhaps the lower lipophilicity of the
peptide (compared with AcMIF1-azide) could give less
than ideal permeation of the solid support. Conjugation
of the peptide with trifluoroacetyl-protected lysines (TFA-
C-myc-L2-azide, 8) to the polystyrene-bound DNA also
gave better conversion (Figure 1B, entry 8, Table 1). With
this protected peptide good conversions to LNA and
DNA conjugates on CPG was also obtained (Figure 1C
and D, entries 3 and 9, Table 1). In the above conju-
gations, CuSO4 and sodium ascorbate were used. It
has been reported that use of a Cu–TBTA complex
(Cu-([tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine])
as catalyst can improve click reactions (34). However,
little improvement was seen when the Cu(I)-stabilizing
ligand TBTA was used in the reaction with DNA on poly-
styrene (Supplementary Data, Figure 2). It thus seems
like there is not much benefit to use TBTA here, but
it is not excluded that it could be beneficial for some
conjugations not tested here. After deprotection and sim-
ultaneous cleavage from support [with conc. NH3 (aq) at
55ꢂC overnight], the POCs were readily purified by
RP-HPLC.
When conjugates between polyanionic oligonucleotides
and polycationic peptides are fully deprotected, problems
with aggregation can occur but a solution has been sug-
gested where a conjugate with a protected peptide is mixed
with a complementary target strand and only then depro-
tected (35). Direct block coupling of TFA-protected
TFA-SV40-L1-azide (3) with the solid-supported oligo-
nucleotide LNA-Am5 (entry 4, Table 1) gave the desired
conjugate (this was clearly the major product as evidenced
by MS of the crude mixture after NH3 deprotection).
Unfortunately, any handling that included evaporation,
redissolving, freeze drying and filtering of the product
resulted in severe aggregation, which made it difficult to
isolate the deprotected conjugate by HPLC. In order to
synthesize a POC with unprotected oligonucleotide but
protected peptide, we developed
a solution phase

Nucleic Acids Research, 2011, Vol. 39, No. 20 9055
Figure 2. HPLC chromatograms of crude peptide oligonucleotide products synthesized according to Scheme 3 (coupling of H-phophonate linker,
addition of PATA and CuI catalyzed peptide conjugation): (A) after performing click reaction of solid-supported oligonucleotide-PATA with
azido-modified peptide AcMIF1-L1-azide in the presence of CuI/ascorbate/DIPEA. (B) After performing click reaction of solid-supported
oligonucleotide-PATA with azido-modified peptide TFA-C-myc-L2-azide in the presence of CuI/ascorbate/DIPEA. Reaction conditions: support
with 0.2 mmol oligonucleotide, 4 eq. peptide sodium ascorbate solution 3 eq., 1 eq. CuI in 100 ml tBuOH–Water–DMSO–DIPEA (43.5:43.5:10:3),
overnight at RT {RP-HPLC: 0–80% linear gradient of B [0.05 M triethylammonium acetate (aqueous, pH = 6.5) in 50% acetonitrile (aq)] in A
[0.05 M triethylammonium acetate (aqueous, pH = 6.5)] over 20 min, detection at 260 nm, at 50ꢂC}.
conjugation method between a protected peptide PATA
derivative and 50-azido-functionalized oligonucleotide
(Supplementary Data). This is more cumbersome and
gives more loss of material than the solid phase procedure,
but it shows that PATA derivatives can be useful in
solution conjugation as well.
manually followed by conjugation to the peptide, thus
still enabling POC synthesis without the need to employ
a DNA/RNA synthesizer.
Aminolinker H-phosphonates
Since we have extensive practical experience in
H-phosphonate handling and preparation, and because
of which the monoesters are stable and can be stored for
months without changes in quality of coupling reactions,
it seemed natural to make linkers that would be attached
with this chemistry (37–41). We synthesized protected
2-(aminoethoxy)ethyl linkers (commonly known as
amino modifier 5, Am5) as H-phosphonate monoesters
(Scheme 2). Two different N-protecting groups were
chosen, the most commonly used monomethoxytrityl
group and the 2-(biphenyl)-2-propyloxycarbonyl (Bpoc)
previously used in online step wise POC synthesis (8).
Both linkers are readily made, especially the MMT pro-
tected H-phosphonate 13, because it can be synthesized in
a one-pot, three-step reaction [monomethoxytritylation in
pyridine to 12 followed by addition of diphenyl-H-
phosphonate and subsequent hydrolysis (42,43)]. The
Bpoc derivative involves protection with 14 to give 15
and then phosphonylation with H-pyrophosphonate
(42,43) to give 16. The two new linkers were used in an
overall approach with subsequent attachment of H-phos-
phonate linker, PATA and peptide-azide (Scheme 3)
whereby POCs were made from an 18-mer 20-O-methyl
RNA [a splice-correcting sequence we used in a luciferase
assay to estimate nuclear delivery (44)].
Especially, in the solid-supported syntheses conjugates
from the commercial amino-linked oligonucleotides gave
the oligonucleotide with N-acetylated aminolinker as a
side product, in varying amounts depending on the manu-
facturer. This side product has previously been reported,
and avoided, either by removing one capping step and
include morpholine washing when synthesizing POCs
from 50-aminonucleoside-containing oligonucleotides (8)
or use of active esters when conjugating acridine deriva-
tives (36). This is a separate problem, occurring before and
not connected to the conjugation method per se, and the
acetylated fraction will not react further. The presence of
the side product did not affect purification since it did not
overlap with starting material or products in the chro-
matograms. Although the above method is quick, conveni-
ent and suitable for use in many research laboratories, it may
not always be acceptable with this loss of oligonucleotide,
especially if considered for larger scale synthesis. Another
aspect is that, when purchasing amino-linked oligonucleo-
tides, a substantial part of the cost is due to the linker,
while for laboratories that do not produce large volumes,
it is more expensive with in-house synthesis of standard
oligonucleotides than to purchase them from a commer-
cial source. Taking this into account, we opted for an al-
ternative approach for solid-phase conjugation of peptides
to oligonucleotides which would not only lower the cost
but also hoping that this would reduce the amount of
acetylated side product. This is based on purchasing oligo-
nucleotides on support and then attach an aminolinker
POC synthesis from supported oligonucleotides, with ‘in
house’ attachment of aminolinker
The oligonucleotide was purchased with 50-monomethoxy-
trityl group on and attached to the solid support (CPG).

9056 Nucleic Acids Research, 2011, Vol. 39, No. 20
Scheme 2. Synthesis of 2-(2-aminoethoxy)-ethanol based 50-amino modifiers for solid-supported oligonucleotides. (A) Synthesis of H-phosphonate of
monomethoxytrityl protected amino modifier (15): (i) pyridine, monomethoxytrityl chloride, RT, triethylamine, 2 h; (ii) diphenyl-H-phosphonate, 1 h,
RT; (iii) water, RT, overnight; (B) synthesis of H-phosphonate of 2-(p-biphenyl)-2-propyloxycarbonyl (Bpoc) protected amino modifier (18);
(iv) DMF, Bpoc reagent, RT, triethylamine, 3.5 h; (v) 2 M H3PO3 and PvCl, pyridine, overnight, RT.
Scheme 3. Schematic presentation of the synthesis of different POCs by ‘click’ reaction on solid support. (i) (a). N-terminal functionalization of
peptide with linker L2-azide: pre-activation of L2-azide with HBTU, and NMM, DMF, 0.5 h, RT, then addition to peptide 2 h; (b). deprotection
from solid support with trifluoroacetic acid: 90% TFA, 4% TIS, 4% H2O, 2% 3,5-dioxo-1,8-octanedithiol, 4 h, RT; (ii) trifluoroacetyl (TFA)
protection of amino groups: ethyl trifluoroacetate, methanol, 3 days, RT; (iii) functionalization of solid-supported oligonucleotide with Am5: 1,
compound 15, pyridine, PvCl, 20 min, RT, 2, I2/H2O/pyridine; (iv) functionalization of amino-linked oligonucleotide with the active alkyne linker:
preactivation of PATA (11) with HBTU, and NMM, DMF, 0.5 h, RT, then addition to solid-supported oligonucleotide 2 h; (v) CuI [3+2] dipolar
cycloaddition of oligonucleotide PATA with a peptide-azide derivative 8 or 4; (vi) preactivation of peptide with HBTU and NMM in DMF 0.5 h,
RT, and then addition of azidoethylamine, 2 h, RT (HBTU = O-benzotriazole-N,N,N0,N0-tetramethyl-uronium-hexafluoro-phosphate; azide, N3; Ac,
acetyl; TIS, triisopropyl silane; PvCl, pivaloyl chloride; TFA, trifluoroacetic acid).
The MMT was removed just before coupling with
H-phosphonate linkers 13 or 16 in the presence of
pivaloyl chloride whereupon the H-phosphonate was
oxidized with iodine in pyridine water. The PATA linker
was then attached as before whereupon the peptide-azide
was conjugated by Cu (I) catalyzed cycloaddition.

Nucleic Acids Research, 2011, Vol. 39, No. 20 9057
Although results with both linkers are comparable, a
slightly better HPLC profile was obtained with the
MMT protected linker, which then was chosen for synthe-
sis of the POCs. For the solid-supported conjugation
of the 20-OMeRNA to the Ac-MIF-L1-azide peptide
(entry 10, Table 1), we also compared a few conditions
for the ‘click reaction’ using different copper (I) and (II)
salts (CuI, CuI/ascorbate, CuCl and CuSO4/ascorbate
as above). One equivalent of copper catalyst was used
in all cases and the results were nearly comparable
(Supplementary Figure S5) but with somewhat better
results for CuI/ascorbate (Figure 2A). We then also
prepared the C-myc 20-OMeRNA conjugate (Figure 2B,
entry 11, Table 1) using the MMT protected H-phospho-
nate 13 and the TFA-C-myc-L2-azide (7) with CuI/ascor-
bate according to same overall scheme (Scheme 3). When
preparing conjugates with the ‘in house’ attachment of
the different H-phosphonate linkers, we can see no clear
presence of N-acetylated product. We cannot com-
pletely exclude that N-acetylated side product could be
present in minor amounts as well as it is possible that
the presence of this side product when using purchased
aminolinker oligonucleotides is dependent on other factors
than the linker used. Nevertheless, there is clearly not
much N-acylation occurring when following the procedure
in Scheme 3.
The overall procedure, as performed here, involves
transfers of the solid support between different containers.
If this is done with milligram amounts of support, it will
result in some material loss. Then in addition, there is a
typical loss occurring upon HPLC purification (especially
if this is performed by multiple injections). The loss upon
HPLC purification is exemplified by comparing the UV
absorbance of the crude non-conjugated 20-OMeRNA
after removal from support with NH3 with that of the
HPLC-purified isolated non-conjugated 20-OMeRNA.
Doing this (with 5 mg support) gave 53% isolated
non-conjugated oligonucleotide, which is not untypical
after HPLC purification. To investigate what the
isolated yield would be in the worst possible scenario of
material transfer and conversion, synthesis of the C-myc
20-OMeRNA POC was performed in milligram scale (as
described above and depicted in Scheme 3, after which
77% of the mass of support remained). This gave an
overall isolated yield of 27% of HPLC-purified POC
after all reaction steps, transfers and HPLC purification.
Judging from the HPLC profile, we could expect a higher
percentage for the MIF conjugates. This is then to be
compared with the 53% obtained of HPLC-isolated
non-conjugated oligonucleotide.
applications, the POC would be used in considerably
lower concentration and would be non-toxic for human
intake. However, there may be situations where even low
amounts of copper ions could have an influence, e.g. when
conjugates to chelating agents are prepared such as those
used in oligonucleotide-based artificial nucleases (45–47)
and PNAzymes (48,49). An estimation of copper ions in
the crude product (entry 17 in Supplementary Data) gives
that after cleavage from support, the POC has largely
retained the copper [about 85–90% of the concentration
expected if all copper added during reaction remained
with the POC (the theoretical max concentration)].
However, an additional advantage of performing the
‘click’ reaction on solid support is that the support can
be conveniently washed with EDTA solution after conju-
gation but before cleavage of POC from support, which
gave a substantial reduction in copper ion concentration
[to ꢃ20% of the theoretical max concentration and at the
same level as the drinking water (at a POC concentration
of 30 mM) that we have in the Stockholm area]. This level
(ꢃ1 mM Cu if diluting the POC to 4 mM concentaration)
should be sufficiently low, even for most applications
involving oligonucleotide chelates. It is certainly low
enough for human intake and should probably not, in
general, cause acute toxicity for cell experiments where
3 mM hardly affected astrocytes (50) and concentrations
up to 100 mM did not affect the growth of HeLa cells
(51). However, effects on the metabolism of cells can be
found for low micromolar concentrations (50,51) which
suggest that caution should be exerted, especially if a
higher concentration of the POC is to be used. Thus, if
an ultra low concentration is needed for some particular
application, it may be advisable to include more extensive
EDTA washing and perhaps evaluate the use of lower
amounts of Cu (I) than used here.
CONCLUSIONS
Alternative methods for the synthesis of POCs by use of
‘click chemistry’ enhanced by the PATA moiety have been
developed. LNA conjugates with the N-terminal of a
C-myc-derived peptide as well as with the C-terminal of
the MIF peptide were prepared on solid support (CPG).
DNA–peptide conjugates with the C-myc peptide were
made on both polystyrene and CPG supports. Apart
from the oligoether aminolinker (aminomodifier Am5),
an aminohexylphosphate linker (aminolinker C6) was
also used.
Peptides made by standard Fmoc synthesis can be
purchased on solid support [e.g. the acid-labile Wang
resin which gives release of carboxylic acid at the
C-terminus or Rink amide resin to obtain a C-terminal
amide (for N-terminal conjugation only)]. This makes
N-terminal attachment of an azide-containing linker a
convenient procedure which just involves coupling of the
linker to the support-bound peptide, washing and then
removal from the support. There is also no apparent limi-
tation with respect to amino acid sequence for this
N-terminal attachment approach, and lysines can be sub-
sequently protected for increased lipophilicity. Peptides
Residual amounts of copper ions
The use of an activated alkyne in combination with
solid-phase conjugation enables the use of just one equiva-
lent of copper salt, which makes the possible contamin-
ation of the product with this toxic metal ion less of an
issue. A simple calculation reveals that even if all copper
(equimolar amounts) used in the reaction remains with the
POC, this would be at the maximum limit for drinking
water at ꢃ30 mM concentration of POC. For most

9058 Nucleic Acids Research, 2011, Vol. 39, No. 20
containing hydroxyl functions, e.g. serine, threonine and
tyrosine have not been included in this study, but it is
unlikely that these would pose any major problems.
Hydroxyls would not be expected to be protected using
ethyl trifluoroacetate in methanol and there should also be
no need for protection of these in the click reaction. For
C-terminal attachment, non-supported fully deprotected
peptides can be readily converted into both azide and
activated alkyne derivatives by intermediate TFA protec-
tion (one would expect that hydroxyl containing amino
acids can be incorporated also in this case since the
activated carboxyl should react preferentially with the
aminolinker and if necessary the coupling could be per-
formed in water). The peptide should preferably have a
C-terminal glycine or other non-chiral linker to avoid
racemization upon coupling of the peptide to the linker.
Although the approach with use of a commercial
aminolinker oligonucleotide is a convenient and viable
approach, that in some cases gave acceptably low levels
of concomitant N-acetylation, we would consider the
overall approach in Scheme 3 to be the preferred method.
A variety of oligonucleotides, including modified ones,
can be purchased on solid support (with or without a
number of different aminolinkers) but are in general more
available and at a lower cost without the aminolinker.
Both the H-phosphonate aminolinker and the PATA
reagent are readily prepared, made from inexpensive ma-
terials and stable upon storage. Subsequent coupling of
these two reagents to a solid-supported oligonucleotide
occur in high yields and when used together in the
overall approach, the N-acetylated side product is not
observed. This may be due to these aminolinkers, but we
cannot exclude that the approach of attaching the linker
and PATA consecutively at the same occasion with
minimal time in between is a major reason as well. We
consider the overall conversion from oligonucleotide to
POC to be excellent, especially since there is no need to
isolate and purify any oligonucleotide intermediate
(neither aminolinked nor the alkyne containing one) but
only the final POC.
highly suitable for the parallel synthesis of libraries of
POCs in which either or both components are varied.
Use of the 1,3-dipolar cycloaddition with an activated
alkyne should also make the approach suitable for conju-
gation with other biomolecules or labels, thus enabling the
use of a single type of conjugation method in the synthesis
of different conjugates.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
FUNDING
Swedish Research Council, VINNOVA and European
Council (FP7:ITN-2008-238679). Funding for open
access charge: VINNOVA and Swedish Research Council.
Conflict of interest statement. None declared.
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