Synthesis of biotin linkers with the activated triple bond donor [p-(N-propynoylamino)toluic Acid] (PATA) for efficient biotinylation of peptides and oligonucleotides

Article abstract of DOI:10.3390/molecules171214174

Biotin is an important molecule for modern biological studies including, e.g., cellular transport. Its exclusive affinity to fluorescent streptavidin/avidin proteins allows ready and specific detection. As a consequence methods for the attachment of biotin to various biological targets are of high importance, especially when they are very selective and can also proceed in water. One useful method is Hueisgen dipolar [3+2]-cycloaddition, commonly referred to as "click chemistry". As we reported recently, the activated triple bond donor p-(N-propynoylamino)toluic acid (PATA) gives excellent results when used for conjugations at submicromolar concentrations. Thus, we have designed and synthesized two biotin linkers, with different lengths equipped with this activated triple bond donor and we proceeded with biotinylation of oligonucleotides and C-myc peptide both in solution and on solid support with excellent yields of conversion.

Full text of DOI:10.3390/molecules171214174

Molecules 2012, 17, 14174-14185; doi:10.3390/molecules171214174
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of Biotin Linkers with the Activated Triple Bond
Donor [p-(N-propynoylamino)toluic Acid] (PATA) for Efficient
Biotinylation of Peptides and Oligonucleotides
Martina Jezowska 1, Joanna Romanowska 1,2, Burcu Bestas 3, Ulf Tedebark 4 and
Malgorzata Honcharenko 1,*
1
Department of Biosciences and Nutrition, Karolinska Institutet, Novum, SE-14183 Huddinge,
Sweden; E-Mail: Martina.Jezowska@ki.se (M.J.)
2
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14,
61-704 Poznañ, Poland; E-Mail: joarom@ibch.poznan.pl (J.R.)
3
Clinical Research Center, Karolinska Institutet, Karolinska University Hospital Huddinge, Novum,
SE-14186 Huddinge, Sweden; E-Mail: burcu.bestas@ki.se
4
GE Healthcare Bio-Sciences AB, Björkgatan 30, SE-75184 Uppsala, Sweden;
E-Mail: Ulf.Tedebark@ge.com
* Author to whom correspondence should be addressed; E-Mail: Malgorzata.Honcharenko@ki.se;
Tel.: +46-8-524-81019; Fax: +46-8-524-81034.
Received: 1 November 2012; in revised form: 19 November 2012 / Accepted: 23 November 2012 /
Published: 30 November 2012
Abstract: Biotin is an important molecule for modern biological studies including, e.g.,
cellular transport. Its exclusive affinity to fluorescent streptavidin/avidin proteins allows
ready and specific detection. As a consequence methods for the attachment of biotin to
various biological targets are of high importance, especially when they are very selective
and can also proceed in water. One useful method is Hüisgen dipolar [3+2]-cycloaddition,
commonly referred to as “click chemistry”. As we reported recently, the activated triple
bond donor p-(N-propynoylamino)toluic acid (PATA) gives excellent results when used for
conjugations at submicromolar concentrations. Thus, we have designed and synthesized
two biotin linkers, with different lengths equipped with this activated triple bond donor and
we proceeded with biotinylation of oligonucleotides and C-myc peptide both in solution
and on solid support with excellent yields of conversion.
Keywords: biotin; streptavidin; click chemistry; biotin linker; oligonucleotide conjugates

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1. Introduction
The streptavidin-biotin complex provides the basis for many important biotechnological
applications and is an interesting model system for studying high-affinity protein-ligand interactions.
Biotin (Btn) is a water soluble vitamin which forms extraordinary stable complexes with avidin
(AVN, Mr = 67,000), neutravidin (NEVN, Mr = 60,000), a deglycosylated form of avidin, and
streptavidin (STV, Mr = 66,000–75,000, isolated from the bacterium Streptomyces avidinii).
Streptavidin is a homotetrameric 159 residue protein where each monomer of the protein binds one
molecule of the vitamin biotin non-covalently with an exceptionally high affinity (Ka ~ 1013 M−1) [1,2].
This fact has been exploited to devise powerful and widely used tools for, e.g., visualization of protein
transport (since fluorophore labeled STV is commercially available), a cell microarray [3], platform for
nuclear import visualization [4] and in many other approaches [5–7].
Thus, quite a few methodologies were reported in which biotin was connected to an attachable
linker. These methods rely on the synthesis of active esters [8], as well as propargyl and azide
derivatives which allows use of “click chemistry” to perform conjugation [9]. The latter method
(Hüisgen 1,3-dipolar cycloaddition) seems to be most attractive since: (1) it can be performed in water;
(2) neither azide nor triple bond groups interfere with protecting-deprotecting strategy during synthesis
of reactive parts; and (3) offers total selectivity. The strategy requires modification of the reactants into
“clickable” ones by incorporating azido and triple bond donors in the respective parts. We have
recently reported [10,11] a convenient method of derivatization of commercially available peptides
into “clickable”-conjugates extending from either the N- or C-terminus. It is also possible to purchase
azido- modified amino acids and nucleotide building blocks that allow the incorporation of this
reactive group at any place in a synthetic peptide or oligonucleotide. Reported “click chemistry”
procedures, that do not use an activated triple bond donor, give good yields provided that at least one
reactant is used in high excess and/or in high overall concentration. From previous experiments in our
own group and as well as by others, it is clear that conjugation by Cu catalyzed Hüisgen dipolar
[3+2]-cycloaddition at a low concentration and using relatively large biomolecules for derivatization,
like oligonucleotides or peptides, is very difficult and often virtually impossible. This is provided that
the triple bond donor is not in activated form as shown in a recent reports, where comparison
of pentynoic acid as triple bond donor versus activated PATA bond donor is discussed [10,11].
In the mentioned study the triple bond donor PATA is an activated linker which can be attached to
various biomolecules like peptides and oligonucleotides and allows conjugation using 1 eq. Cu(I)
(as CuSO4/ascorbate catalyzed or CuI catalyzed cycloaddition) at a low concentration and with
excellent yields. For various studies specially for labeling of proteins it seems valuable to have access
to biotin linkers which are ready for cycloadditions at a low concentration, i.e., equipped with the
activated triple bond donor PATA [12–14].
2. Results and Discussion
We decided to synthesize two linkers, equipped with the PATA moiety, and with different lengths
(compounds 3 and 7, Schemes 1 and 2). For labeling of some larger biopolymers like oligonucleotides
a part of the molecule itself can serve as spacer. The streptavidin recognition pocket is relatively deep

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and biomolecules equipped with biotin typically require a spacer. A longer linker than 1 may be
needed for certain purposes, which is why the longer PATA functionalized biotinylated linker 7 was
also synthesized.
Scheme 1. Synthesis of biotinylated linker 3 carrying the activated triple bond donor PATA.
Scheme 2. Synthesis of the longer biotinylated linker 7 carrying the activated triple bond donor PATA.
2.1. Synthesis of Biotin Linkers Containing (p-(N-propynoylamino)toluic Acid) PATA
Synthesis of the activated triple bond donor PATA (2), necessary for synthesis 3 and 7, was
performed as described earlier [10]. Synthesis of the shorter biotin linker 3 (Figure 1) was initiated by
conjugation of activated (by HBTU/NMM see Supporting Information for details) commercial biotin
with 4,7,3-trioxa-1,13-tridecanediamine. After completion of the reaction the product 1 was purified
by standard column chromatography on silica (due to a lack UV absorption, MS analysis of eluted
fractions was carried out). Synthesis of compound 3 was commenced by activation of the triple bond
donor PATA (2) with HBTU/NMM followed by conjugation with compound 1 which served as an
amine in this reaction. Due to the low UV absorption of the product (3) together with a need for high
purity of the final linker we decided to use RP-HPLC (C-18 column and triethylammonium acetate
buffers, pH 6.5, see Supporting Information for details) for the purification.
It is worth to notice that using a buffer containing triethyl amine requires low temperature
treatment. Heating, even slight, of activated triple bond donor PATA in the presence of triethylamine
(for example for concentration purposes) may result in the formation of a side product which most
likely is a triethylamine adduct (M+ + Et3NH = 732.98 for compound 3 and M+ + Et3NH = 1,082.34 for
compound 7, 10–15%) formed through Michael addition. Although not using heating during HPLC
purification helps to decrease the amount of adduct formed, to avoid it completely we used “buffers”
containing just acetonitrile and water (see experimental section for details).

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Figure 1. Schematic representation of biotinylation procedure for oligonucleotides on solid support.
Steps: A: Deprotection of 5'-O-(4-monomethoxytrityl) protecting group followed by reaction with
2-(N-(4-monomethoxytrityl)aminoethoxy)ethyl H-phosphonate, Py, PvCl, 20 min, r.t.; I2, water,
Py; 2% DCA, DCM, 2 min; B: pre-activation of azido acid, HBTU, NMM, DMF, 0.5 h, r.t., and
addition of activated azido acid to solid supported ON, 2 h; C: CuI, DIPEA, overnight, water/DMSO
9/1 (dipolar cycloaddition [3+2] of ON-N3 with PATA-BIOTIN); (Abbreviations: PvCl = pivaloyl
chloride, Py = Pyridine, DCA = dichloroacetic acid, DIPEA = diisopropylethylamine, DMSO =
dimethylsulfoxide, DCM = dichloromethane, HBTU = N,N,N',N'-tetramethyl-O-(1H-benzotriazol-
1-yl)uronium hexafluorophosphate, NMM = N-methylmorpholine, DMF = dimethylformamide).
Successful completion of the synthesis of the “short” biotinylated triple bond donor (3) encouraged
us to synthesize a “long” biotinylated PATA linker (7) in a similar manner. Lack of the possibilities to
detect reaction products using UV absorption can lead to some problems even though staining with
for example ninhydrin or iodine can be used. To simplify purification procedures we introduced an
internal linker having a UV active chromophore (terephthalate) which allowed for more ready
detection of the conjugation products during TLC analysis.
The sequence of reactions which allowed us to synthesize 7 via compound 4 started from activation
of monomethyl terephthalate in the same manner as for biotin (HBTU/NMM see Supporting
Information for details) followed by conjugation with commercial 4,7,3-trioxa-1,13-tridecanediamine.
After completion of the reaction the product 4 was purified using silica gel column chromatography. In
the subsequent step activation of biotin (as described for synthesis of compound 3) followed by
conjugation with compound 4 resulted in the desired product 5. Since the product of that reaction is a
methyl ester, aminolysis was the done with 4,7,3-trioxa-1,13-tridecanediamine (by simply dissolving
compound 5 in excess of amine and leaving the reaction at 44 °C, overnight). This led to the expected
non-symmetrical product 6 with one free amine. To obtain the final product the triple bond donor
PATA (2) was activated by with HBTU and reacted with compound 6. Reaction was completed in 3 h
and the product was pre-purified by silica gel column chromatography. To obtain a high purity of the
final product we used RP-HPLC in a similar manner as for the short linker, avoiding heating, to obtain
the purified “long” biotinylated linker 7.
2.2. Biotinylation of C-myc Peptide and Oligonucleotides Using Biotin Linkers Containing
(p-(N-propynoylamino)toluic Acid) PATA as Activated Triple Bond Donor
Successful synthesis of biotinylated linkers 3 and 7 was followed by their conjugation to
oligonucleotides and C-myc peptide. As oligonucleotides we used commercial 18 and 14-mers (fully
2'-O-methylated in a case of 18-mer and 2'-O-methylated plus two LNA A building blocks used for

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improved hybridization properties of the 14-mer (Table 1)). Additionally cell penetrating peptide
azide: azido- (C-myc) was used to achieve peptide-biotin conjugate synthesis.
Table 1. Substrates and conditions of the biotinylation reaction for linkers 3 and 7.
Substrate
Conditions
Biotin Linker
Product MS calculated/found
18merRNA(2'-OMe) oligonucleotide
5'-CCUCUUACCUCAGUUACA-3'
14merRNA(2'-OMe/LNA) oligonucleotide
5'-AAAUGUAACUGAGG-3'
Azido- (C-myc)
Solid phase
3
6796/6798
Solid phase
7
7
6031/6032
2111/2112
Solution phase
N3-L-GAAKRVKLD a
L = linker see Scheme 2; for the structure and synthesis of peptide-azide see Supplementary Information;
a LNA-A building block.
Labeling with biotin is an effective, step-wise procedure, where both oligonucleotides were
commercially synthesized (thus no need for an synthesizer) and easily converted into their
azido-counterparts. Oligonucleotides and C-myc peptide were all purchased still attached to the solid
support and subjected to reactions which transformed these molecules into “clickable ones” by
addition of an azide linker (Figures 1 and 2). In a case of oligonucleotides biotinylation was performed
on oligonucleotide still attached to the solid support (with the conversion 55–75%, see Table 1)
whereas biotinylation of C-myc peptide was done in solution.
Figure 2. Crude chromatograms depicting: (A) Crude 18mer (2'-OMe) oligonucleotide and
(B) Crude reaction mixture of the synthesis of 18mer (2'-OMe)-BIOTIN conjugate; both
reaction after deprotection cleavage from the support.
Biotinylation of oligonucleotides relies on stepwise derivatization of commercial oligonucleotides
and proceed as follows: (1) amino-linker is attached to the 5'-hydroxyl position of oligonucleotide; (2)
after removal of MMTr group, terminal amino group is conjugated with 2-(2-azidoethoxy)ethoxyacetic
acid; (3) CuI catalyzed Hüisgen dipolar [3+2]-cycloaddition with biotin linker containing activated
triple bond donor PATA followed by ammonia treatment leads with high conversion to the desired
conjugate as it is seen on the crude chromatogram depicted in Figure 2. Reaction on solid support
allows for multistep reactions where excess of reagents can be removed after each step and activated

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triple bond donor permit for effective conversion in small scale and with minimal excess of biotin
linker (usually 0.1 μmol solid supported oligonucleotide and 4 eq. of linker were used).
Solution phase biotinylation of peptide was also accomplished using linker 7 in a model reaction
leading to required peptide conjugate with high yields and low concentration (below 1 mM) (Figure 3
and Supplementary Materials). Peptides with different cell penetrating properties or nuclear
localization sequences can be easily purchased and converted to “clickable” ones on solid support
using our recently reported method [10,11]. In short, commercial solid supported peptide is N-terminal
deprotected (piperidine) and subjected to conjugation to 2-(2-azidoethoxy)ethoxyacetic acid. The
reaction is followed by standard deprotection condition using TFA and HPLC purification (see
Supplementary Materials for details). Such purified peptide is easily converted to biotin conjugate
using “click” reaction condition depicted in Figure 3 for PATA containing biotin linker 7.
Figure 3. Schematic representation of biotinylation procedure for peptides.
Steps: A: Deprotection of N-terminal Fmoc protecting group (20% piperidine in DMF); B: reaction
with 2-(2-azidoethoxy)ethoxyacetic acid (pre-activated with HBTU and NMM in DMF, 0.5 h, r.t.),
DMF, 2 h, r.t.; C: 90% TFA, 4% TIS, 4% H2O, 2% 3,5-dioxo-1,8-octanedithiol, 4 h, r.t.; D: CuI,
DIPEA, water/DMSO 9/1, overnight (dipolar cycloaddition [3+2] of PEPTIDE-N3 with
PATA-BIOTIN); (Abbreviations: DIPEA = diisopropylethylamine, DMSO = dimethylsulfoxide,
HBTU = N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate, NMM =
N-methylmorpholine, DMF = dimethylformamide).
3. Experimental
3.1. General Materials and Methods
Acetonitrile (HPLC grade, Merck), methanol (MeOH) and dichloromethane (DCM, Fisher
Scientific, Analytical Grade) were of commercial grade and used as received. Dimethylformamide
(DMF) and pyridine (both from Merck and analytical grade) were additionally dried over 4A
molecular sieves. Silica gel column chromatography were performed on Merck G60, TLC-analysis
was carried out on precoated 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). The oligonucleotide, 5'-ODMT-2'-OMe-CCUCUUACCUCAGUUACA
[18merRNA(2'-OMe)], was purchased from Rasayan Inc. (Encinitas, CA, USA), and 5' ODMT-
AAAUGUAACUGAGG-3'[14merRNA(2'-OMe/LNA)] (Ribotask, Odense, Denmark) still attached on solid
support. Solid supported peptide GAAKRVKLD (C-myc) was obtained from Eurogentech (Seraing,

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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-butyloxycarbonyl (Boc) for lysine,
t-butyl ester (O-tBu) for aspartic acid and 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl (Pbf)
for arginine. Nucleobase protecting groups were benzoyl for cytosine and adenine and isobutyryl for
guanine. Reversed phase HPLC was carried out on a Jasco HPLC system using the following columns:
Hypersil 5 μm (250 × 4.6 mm) with 1 mL/min flow rate, Kromasil 100-5-C18, 5 μm (250 × 4.6 mm)
with 1 mL/min flow rate and Kromasil 100-5-C18, 5 μm (250 × 10 mm) at 4 mL/min flow rate.
Buffers for reversed phase chromatography were as follows: A: H2O, B: H2O:CH3CN (1:1), C: 50 mM
TEAA pH 6.5; D: 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) instrument. Molecular weights of the
conjugates were reconstructed 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.
N-methyl morpholine (NMM), biotin, 4,7,10-trioxa-1,13-tridecanediamine, were purchased from
Aldrich and used without further purification.
Biotin-(3-(2-[2-(3-aminopropoxy)ethoxy]ethoxy)propyl)amide (1). The reaction flask was charged with
biotin (1 eq., 1 mmol, 240 mg) and HBTU (1.05 eq., 1.05 mmol, 398 mg), evacuated on a pump and
then flushed with N2. The solid substrates were dissolved in DMF (13 mL) and NMM (10 eq.,
10 mmol, 1.1 mL) was added with a syringe. The reaction mixture was stirred for 30 min. Next
4,7,10-trioxa-1,13-tridecanediamine (2 eq., 2 mmol, 438 µL) was added and the solution was stirred
for an additional 3h. After this time the reaction mixture was evaporated to dryness and the crude
product was purified by column chromatography (gradient: 0–23% MeOH in DCM; due to lack of UV
visibility reaction was monitored by mass spectrometry). Collected fractions containing pure product
1
were combined and evaporated and the product was obtained as yellow oil (150 mg, 34%). H-NMR
(CD3OD): δ 4.40–4.38 (1H, m), 4.22–4.19 (1H, m), 3.55–3.38 (12H, m), 3.21–3.09 (3H, m), 2.83 (1H,
½ ABq, H: J = 4.9 Hz, J = 14.5 Hz), 2.60 (1H, ½ ABq, H: J = 12.7 Hz, J = 0 Hz), −2.59 (2H, m),
13
2.70 (2H, t, J = 6.8 Hz), 2.10 (2H, t, J = 7.3 Hz), 1.68–1.41 (8H, m), 1.38–1.32 (2H, m). C-NMR
(CDCl3): δ 27.3, 29.9, 30.2, 30.8, 33.0, 37.3, 38.2, 40.5, 41.5, 57.4, 62.1, 63.8, 70.3, 70.9, 71.60, 71.65,
71.91, 71.94. ES-MS, calc m/z (M-H)+1 447.3, found 447.5.
N-Biotin-(3-(2-[2-(3-aminopropoxy)ethoxy]ethoxy)propyl)amide-N’-4-(propynoylamino-methyl)-benzamide
(3). The reaction flask was charged with the PATA linker (2) (1.1 eq., 0.22 mmol, 45 mg) and HBTU
(1.05 eq., 0.21 mmol, 80 mg), evacuated on a pump and then flushed with N2. The solid substrates
were dissolved in DMF (3.5 mL) and NMM (10 eq., 2 mmol, 220 µL) was added with a syringe. The
reaction mixture was stirred for 30 min. Compound 1 (1 eq., 0.2 mmol, 90 mg) was then added and the
solution was stirred for an additional 2 h. After this time mass spectrometry showed completion of
reaction and the mixture was evaporated to dryness. The crude product was dissolved in water and
purified by RP-HPLC using a linear gradient of buffer B in A from 15%–65% in 25 min, detector at
1
220nm, tR = 22.2 min. Concentration of pure fractions gave 64 mg (52%) of 3. H-NMR (CDCl3): δ
7.78 (2H, d, ArH, J = 8.0 Hz), 7.40 (2H, d, J = 8.0 Hz), 4.50–4.42 (3H, m), 4.30–4.18 (2H, m), 3.67–3.43
(14H, m), 3.27–3.05 (3H, m), 2.86 (1H, m), 2.72 (1H, m), 2.19–2.15 (2H, m), 1.91–1.88 (2H, m),
13
1.79–1.52 (6H, m), 1.46–1.36 (2H, m). C-NMR (CDCl3): δ 26.0, 28.2, 28.4, 28.8, 29.2, 35.8, 37.1,

Molecules 2012, 17
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38.8, 40.7, 43.2, 56.1, 60.0, 62.1, 69.1, 69.9, 70.3, 70.4, 70.5, 70.6, 76.7, 77.8, 126.3, 126.4, 127.4,
127.7, 133.8, 141.5, 162.5, 167.2, 173.6, 177.2. ES-MS, calc m/z (M−H)+1 632.3, found 632.6.
N-(3-(2-[2-(3-Amino-propoxy)ethoxy]ethoxy)propyl)terephthalamic acid methyl ester (4). The reaction
flask was evacuated on a pump and flushed with N2. Monomethyl terephthalate was added
(1 eq., 1 mmol, 180 mg) followed by HBTU (1.05 eq., 1.05 mmol, 398 mg) and DMF (30 mL). NMM
(10 eq., 10 mmol, 1.1 mL) was then added with a syringe and the reaction mixture was stirred for
30 min. After that time 4,7,10-trioxa-1,13-tridecanediamine (2 eq., 2 mmol, 438 µL) was added and
the solution was stirred for an additional 3 h. The reaction mixture was then evaporated to dryness. The
crude product was purified by column chromatography (a linear gradient of MeOH in DCM from 0%
to 20%). Fractions containing product were concentrated giving compound 4 as yellow oil (244 mg, 64%).
1H-NMR (CDCl3): δ 8.08 (2H, d, J = 8.4 Hz), 7.83 (2H, d, J = 8.4 Hz), 7.19 (2H, s), 7.00 (1H, m), 3.94
(3H, s), 3.79 (2H, t, J = 5.2 Hz), 3.68–3.49 (12H, m), 3.35 (2H, q, J = 5.5 Hz), 2.04–1.98 (2H, m),
13
1.89–1.81 (2H, m). C-NMR (CDCl3): δ 27.0, 30.0, 41.0 (2C), 52.8, 69.7, 69.9, 70.02, 70.03, 71.2,
127.7, 130.2. ES-MS, calc m/z (M−H)+1 383.2, found 383.4.
N-(3-(2-[2-(3-Aminopropoxy)ethoxy]ethoxy)propyl)-N'-biotinterephthalamic acid methyl ester (5). To
the reaction flask biotin (1 eq., 0.315 mmol, 77 mg) was added followed by HBTU (1.05 eq.,
0.33 mmol, 125 mg) and the flask was evacuated on a pump and flushed with N2. The solid substrates
were dissolved in DMF (5 mL) and NMM (10 eq., 3.15 mmol, 346 µL) was added with a syringe. The
reaction mixture was stirred for 30 min. 4 (2 eq., 0.63 mmol, 240 mg) dissolved in DMF (10 mL) was
then added and the solution was stirred for an additional 3 h. After completion of the reaction the mixture
was concentrated and the crude product was purified by column chromatography (gradient: 0%–9%
MeOH in DCM). Collected fractions were concentrated and the product was obtained as white solid.
(175 mg, 91%). 1H-NMR (CDCl3): δ 8.08 (2H, d, J = 8.3 Hz), 7.93 (2H, d, J = 8.3 Hz), 7.44–7.43 (1H, m),
4.57–4.55 (1H, m), 4.38–4.37 (1H, m), 3.95 (3H, s), 3.68–3.59 (10H, m), 3.53–3.50 (4H, m), 3.53–3.33
(2H, m), 3.18–3.16 (1H, m), 2.84 (2H, ABq, H1: J = 4.9 Hz, J = 14.8 Hz; H2: J = 13.0 Hz, J = 0 Hz), 2.29
(2H, t, J = 7.3 Hz), 1.95–1.92 (2H, m), 1.78–1.67 (6H, m), 1.47–1.43 (2H, m). 13C-NMR (100 MHz, CDCl3):
δ 26.0, 28.1, 29.2, 36.0, 38.5, 39.0, 42.0, 52.8, 55.7, 61.5, 62.5, 70.3 (2C), 70.5, 70.8, 71.0 (4C), 127.6
(2C), 130.1 (2C), 133.0, 137.0, 163.0, 166.9 (2C), 174.0. ES-MS, calc m/z (M−H)+1 609.3, found 609.1.
N-(3-(2-[2-(3-Amino-propoxy)-ethoxy]-ethoxy)-propyl)-N'-(3-[biotin]-propoxy)-ethoxy)-ethoxy]-propyl)-
terephthalamide (6). 5 (1 eq., 0.24 mmol, 150 mg) was dissolved in 4,7,10-trioxa-1,13-
tridecanediamine (3.8 eq., 0.91 mmol, 200 µL) and the reaction mixture was left to stir overnight. The
next day MS and TLC analysis confirmed completion of reaction and the crude product was purified
by column chromatography (gradient: 0%–23% MeOH in DCM + 0.5% Et3N). Collected fractions
1
were evaporated and the product was obtained as yellow oil (92 mg, 47%) H-NMR (400 MHz,
CDCl3): δ 7.93 (2H, d, J = 8.3 Hz), 7.83 (2H, d, J = 8.3 Hz), 4.41–4.38 (1H, m), 4.24–4.18 (1H, m),
3.60–3.33 (28H, m), 3.20–3.18 (2H, m), 3.04–3.01 (3H, m), 2.74 (2H, ABq, H1: J = 4.0 Hz, J = 12.0 Hz;
H2: J = 13.0 Hz, J = 0 Hz), 2.12–2.08 (2H, m), 1.88–1.81 (6H, m), 1.68–1.41 (8H, m), 1.38–1.31 (4H, m).
13C-NMR (100 MHz, CDCl3): δ 24.6, 25.7, 27.0, 27.7, 27.85, 27.91, 36.2, 36.3, 37.0, 37.6, 38.3, 39.5

Molecules 2012, 17
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(2C), 54.6, 58.9, 60.8, 68.59, 68.67, 68.78 (2C), 68.85, 69.0, 69.1 (2C), 69.29 (2C), 69.30 (2C), 126.2
(4C), 126.5 (2C), 136.0 (2C), 165.9, 172.5. ES-MS, calc m/z (M−H)+1 797.5, found 797.0.
N-(3-[2-(2-(Biotin)-pentanoylamino]-propoxy}-ethoxy)-ethoxy]-propyl)-N'-(3-[2-(2-{3-[4-(propynoyl-
aminomethyl)benzoylamino]propoxy)ethoxy)ethoxy]propyl)terephthalamide (7). The reaction flask
was charged with PATA (2) (1 eq., 0.027 mmol, 5 mg) and HBTU (1.05 eq., 0.028 mmol, 11 mg),
evacuated on a pump and then flushed with N2. The solid substrates were dissolved in DMF (1 mL) and
NMM (10 eq., 0.27 mmol, 29 µL) was added with a syringe. The reaction mixture was allowed to stir
for 30 min. 6 (2 eq, 0.053 mmol, 42 mg) was then added and the solution was stirred for an additional
3 h. After this time TLC analysis showed completion of reaction and the mixture was concentrated to
dryness. The crude product was purified first by column chromatography (gradient: 0%–50% MeOH
in DCM +0.5% Et3N) and then dissolved in water and purified by RP-HPLC using a linear gradient of
buffer B in A from 15%–100% in 40 min, detector at 220nm, tR = 25.6. Collected fractions gave 8mg
1
(14%) of 7. H-NMR (CD3OD): δ 7.78 (4H, s), 7.67 (2H, d, J = 8.0 Hz), 7.26 (2H, d, J = 8.0 Hz),
4.71–4.33 (2H, m), 4.38–4.34 (2H, m), 4.20–4.17 (1H, m), 3.54–3.33 (30H, m), 3.12–3.07 (3H, m),
2.71 (2H, ABq, H1: J = 4.2 Hz, J = 12.3 Hz; H2: J = 12.7 Hz, J = 0 Hz), 2.08 (2H, t, J = 7.3 Hz),
13
1.84–1.73 (6H, m), 1.67–1.43 (H, m), 1.37–1.27 (2H, m). C-NMR (CDCl3): δ 27.3, 29.9, 30.2, 30.8
(3C), 37.3, 38.2, 39.2 (3C), 41.5, 44.3, 57.4, 62.0, 63.8, 62.0, 63.8, 70.3 (2C), 70.65 (2C), 70.73 (2C),
71.63 (2C), 71.67 (2C), 71.9 (2C), 128.90 (8C), 128.99, 129.1 (2C), 129.3, 135.3, 138.9, 143.4, 166.5,
169.5, 176.4. ES-MS, calc m/z (M−H)− 980.5, found 980.9.
3.2. General Method for Biotinylation of Oligonucleotides on Solid Support
The reaction with solid supported oligonucleotides was carried out in the Eppendorf tube with
screw cap. Each reagent, unless stated differently, was added to the tube with a syringe, vortexed,
centrifuged and the solution was carefully removed from the solid support using a syringe. The
washing steps following each reaction were carried out in the similar manner.
3.2.1. Synthesis of 18-mer-(2′-OMe)-Biotin Conjugate
The commercially available solid-supported oligonucleotide (2.5 mg, 0.1 µmol CPG supported
18-mer RNA (2'-OMe)), placed in a sealed Eppendorf was treated with 2% dichloroacetic acid (DCA)
solution in DCM (2 × 1 mL for 1 min) and subsequently washed with DCM (4 × 1 mL) and MeCN
(2 × 1 mL). The protected MMT-aminolinker H-phosphonate was dissolved in anhydrous pyridine
(60mM coupling solution, used 16.3 mg, 0.03 mmol in 0.5 mL pyridine) and added to the supported
oligonucleotide followed by pivaloyl chloride (PvCl) in acetonitrile (1.1 eq. towards amino linker, 50 µL
form stock solution: 808 µL PvCl in 10 mL MeCN) and the reaction mixture was shaken on vortex for
20 min. After this time, the coupling solution was removed and the crude product on solid support was
oxidized with I2 (20 mg) in 1 mL pyridine/water (98:2) on vortex at RT over 15 min. The support was
then washed extensively with pyridine/water (9:1) (5 × 1 mL), MeCN (5 × 1 mL), DCM (5 × 1 mL).
The MMT group protecting the H-phosphonate amonolinker was then removed by treatment with
2%DCA in DCM (2 × 1 mL for 2 min) and the support was washed with DCM (5 × 1 mL), MeCN
(2 × 1 mL) and DMF (2 × 1 mL).

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In a separate Eppendorf tube, 2-(2-azidoethoxy)ethoxyacetic acid (4.7 mg, 245 eq.) was dissolved
in anhydrous DMF (280 µL) and mixed with HBTU (9 mg, 240 eq.). NMM (55 µL, 5,000 µmol) was
added and the mixture was shaken on a vortex for 30 min at RT. This preactivation solution was then
transferred to the Eppendorf containing oligonucleotide on support and left to react at RT on vortex for 2 h.
After this time, the resulting solid supported azide-modified oligonucleotide was washed with DMF
(3 × 1 mL) and tBuOH/water (1:1) (2 × 1 mL). In new Eppendorf tube 4 eq. of 3 (0.25 mg) were
dissolved in 70 µL of tBuOH/water (1:1) and added into the reaction vial followed by 1 eq. of
diisopropyl ethyl amine (DIPEA) in 15 µL of water (from a stock solution of 2.3 µL/mL; 1 eq.,
0.034 µL) and 1 eq. of CuI in 15 µL of DMSO (from stock solution of 10.1 mg CuI in 1 mL DMSO).
The Eppendorf was then sealed, placed of on vortex and left for reacting at RT overnight. Next day
after removing the solution from the support, the resin was washed with tBuOH/water (1:1) (3 × 1 mL),
0.05 M ethylenediaminetetraacetic acid (EDTA) disodium salt, dihydrate (2 × 1 mL) and DCM
(2 × 1 mL). Next the 0.5 mL of MeOH/NH3 was added and the reaction mixture was stirred on a
vortex at RT overnight. The reaction vial was centrifuged and the solution containing crude product
was taken up carefully from the solid support and concentrated. The purification was done by
RP-HPLC using a linear gradient of buffer D in C from 0% to 80% for 40 min, detection at 254 nm
and oven temperature set at 50 °C. tR = 22.9, ES-MS, calcd (M) 6796, found 6798.
3.2.2. Synthesis of 14-mer-(2'-OMe)-Biotin Conjugate
The commercially available solid-supported oligonucleotide (2.5 mg, 0.1 µmol CPG supported),
placed in sealed Eppendorf was treated with 2% dichloroacetic acid (DCA) solution in DCM (2 × 1 mL for
1 min) and subsequently washed with DCM (4 × 1 mL) and MeCN (2 × 1 mL). The protected
MMT-aminolinker H-phosphonate was dissolved in anhydrous pyridine (60mM coupling solution, used
16.3 mg, 0.03 mmol in 0.5 mL pyridine) and added to the supported oligonucleotide followed by
pivaloyl chloride (PvCl) in acetonitrile (1.1 eq. towards amino linker, 50 µL form stock solution: 808 µL
PvCl in 10 mL MeCN) and the reaction mixture was shaken on vortex for 20 min. After this time, the
coupling solution was removed and the crude product on solid support was oxidized with I2 (20 mg) in
1 mL pyridine/water (98:2) on vortex at RT over 15 min. The support was then washed extensively
with pyridine/water (9:1) (5 × 1 mL), MeCN (5 × 1 mL), DCM (5 × 1 mL). The MMT group protecting
the H-phosphonate aminolinker was then removed by treatment with 2%DCA in DCM (2 × 1 mL for
2 min) and the support was washed with DCM (5 × 1 mL), MeCN (2 × 1 mL) and DMF (2 × 1 mL).
In a separate Eppendorf tube, 2-(2-azidoethoxy)ethoxyacetic acid (4.7 mg, 245 eq.) was dissolved
in anhydrous DMF (280 µL) and mixed with HBTU (9 mg, 240 eq.). NMM (55 µL, 5,000 µmol) was
added and the mixture was shaken on a vortex for 30 min at RT. This preactivation solution was then
transferred to the Eppendorf containing oligonucleotide on support and left to react at RT on vortex for 2 h.
After this time, the resulting solid supported azide-modified oligonucleotide was washed with DMF
(3 × 1 mL) and tBuOH/water (1:1) (2 × 1 mL). In new Eppendorf tube 4 eq. of 7 (0.39 mg) were
dissolved in 70 µL of tBuOH/water (1:1) and added into the reaction vial followed by 1 eq. of
diisopropyl ethyl amine (DIPEA) in 15 µL of water (from a stock solution of 1.2 µL/mL; 1 eq., 0.017 µL)
and 1 eq. of CuI in 15 µL of DMSO (from stock solution of 5 mg CuI in 1 mL DMSO). The Eppendorf
was then sealed, placed of on vortex and left for reacting at RT overnight. Next day after removing

Molecules 2012, 17
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the solution from the support, the resin was washed with tBuOH/water (1:1) (3 × 1 mL), 0.05 M
ethylenediaminetetraacetic acid (EDTA) disodium salt, dihydrate (2 × 1 mL) and DCM (2 × 1 mL).
Next the 0.5 mL of ammonia was added and the reaction mixture was placed in the oven at 55 °C for
overnight. Next day the solution containing crude product concentrated and purified by RP-HPLC
using a linear gradient of buffer D in C from 0% to 80% for 40 min, detection at 254 nm and oven
temperature set at 50 °C. tR = 25.4, ES-MS, calcd (M) 6031, found 6032.
3.2.3. Synthesis of Peptide (C-myc)-Biotin Conjugate
The peptide azide was prepared according to our previous report [10] see the supporting information
for the procedure. The biotinylation reaction was accomplished using following method: the peptide
azide derivative (0.975 µmol, 1.1 mg) was dissolved in water (20 µL). The biotin linker 7 (2 eq., 1.95 µL)
was dissolved in 70 µL tBuOH/water (1:1) and added to Eppendorf tube containing the peptide,
followed by 1 eq. of diisopropyl ethyl amine (DIPEA) in 5 µL of water (from a stock solution of
33.6 µL/mL; 1 eq., 0.168 µL) and 2 eq. of CuI in 10 µL of DMSO (from stock solution of 45.6 mg/mL
DMSO). The reaction was placed on vortex and stirred for 3 h at RT. The conjugate was purified by
RP-HPLC using a linear gradient of buffer D in C from 0% to 80% for 40 min, detection at 220 nm
and oven temperature set at 50 °C. tR = 28.5, ES-MS, calcd (M) 2111, found 2112.
4. Conclusions
Use of the extraordinarily stable biotin/streptavidin complex pair in research has advanced steadily
over the past decades. Development of new tools that take advantage of emerging technologies is
imperative to accelerate progress in the field. Given the rapid influx of techniques in biochemistry and
chemical biology that use the powerful [3+2] cycloaddition between azides and alkynes (“click
chemistry”) for chemoselective conjugation, arming biotin with a new activated click tag provides a
platform for developing novel more versatile technologies. This in turn can facilitate new approaches
for exploring biological properties and important processes as well as therapeutic applications. We
have shown that presented linkers are effective tool in achieving biotinylated both peptides and
oligonucleotides and this reaction is effective on solid support as well as in solution. We are now using
synthesized linkers and biotin-oligonucleotide conjugates for labeling of bioactive macromolecules in
duplexes and the study of their action, in vitro, is under investigation.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/17/12/14174/s1.
Contains: Mass spectra of biotin linkers 3, and 7, mass spectra of oligonucleotide-biotin conjugates
and peptide-biotin conjugate, description of synthesis of azido peptide (N3-C-myc); crude chromatograms
of Biotin-oligonucleotide conjugates and crude chromatogram of peptide (C-myc)-biotin conjugate.
Acknowledgments
This work was supported by grants from Signhild Engkvist foundation, VINNOVA and EU
(EuroNanoMed).

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Sample Availability: Samples of the compounds 3 and 7 as well as purified conjuagets are available
from the authors.
© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).