Biotin-functional oligo(p-phenylene vinylene)s synthesized using click chemistry

Article abstract of DOI:10.1016/j.tetlet.2008.08.080

Using Cu(1)-catalyzed [3+2] Huisgen 'click' cycloaddition, a rigid rod - like oligo(p-phenylene vinylene) (OPV) was functionalized at both ends with biotin ligands, combining the valuable electro-optical properties of conjugated organic molecules with the biological recognition capability of biotin. Biotin can be placed at variable distances from the oligomer via appropriate length of a hydrophilic spacer, which also serves to regulate the binding capabilities of the two terminal biotin units. To demonstrate this binding potential, networks were formed with streptavidin-coated quantum dots. The synthetic conditions are presented, together with representative optimizations of the key reactions. The organic compounds were analyzed by means of ATR/FTIR, ¹H NMR (200 or 600 MHz), ¹³C NMR, 2D NMR (HMBC, HMQC experiments), MS (ESI or MALDI-TOF), and optical spectroscopy. Networks were imaged with TEM.

Full text of DOI:10.1016/j.tetlet.2008.08.080

Tetrahedron Letters 49 (2008) 6386–6389
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Biotin-functional oligo(p-phenylene vinylene)s
synthesized using click chemistry
Neeraja Vundyala a, Chivin Sun a, Francoise Sidime b, Wei Shi b,
William L’Amoreaux b, Krishnaswami Raja a,b, Ralf M. Peetz a,b,
*
a CUNY/College of Staten Island, Center for Engineered Polymeric Materials, 2800 Victory Boulevard, Staten Island, NY 10314, USA
b CUNY/College of Staten Island, Institute of Macromolecular Assemblies, 2800 Victory Boulevard, Staten Island, NY 10314, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Using Cu(1)-catalyzed [3+2] Huisgen ‘click’ cycloaddition, a rigid rod – like oligo(p-phenylene vinylene)
(OPV) was functionalized at both ends with biotin ligands, combining the valuable electro-optical pro-
perties of conjugated organic molecules with the biological recognition capability of biotin. Biotin can
be placed at variable distances from the oligomer via appropriate length of a hydrophilic spacer, which
also serves to regulate the binding capabilities of the two terminal biotin units. To demonstrate this bind-
ing potential, networks were formed with streptavidin-coated quantum dots. The synthetic conditions
are presented, together with representative optimizations of the key reactions. The organic compounds
were analyzed by means of ATR/FTIR, 1H NMR (200 or 600 MHz), 13C NMR, 2D NMR (HMBC, HMQC exper-
iments), MS (ESI or MALDI-TOF), and optical spectroscopy. Networks were imaged with TEM.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 16 July 2008
Revised 31 July 2008
Accepted 4 August 2008
Available online 28 August 2008
Oligo(p-phenylene vinylene)s (OPVs) and their substituted
derivatives belong to the family of conjugated materials with valu-
able opto-electronic properties such as electroluminescence,
photoconductivity, photo-luminescence, nonlinear optical re-
sponse, and electrical conductivity after doping.1–4 OPVs are well
defined compared to higher molecular weight poly(phenylene
vinylene)s, and have the advantage of being soluble and inherently
easier to process, making them attractive electronic building
blocks for organic functionalization. Biomolecule–polymer conju-
gates are widely used in medicine and biotechnology,5–7 the bio-
molecule being either a macromolecule, for example, proteins,
lipids, or ligands, such as sugar or biotin. Biotin binds the protein
avidin with Ka = 1015. As a consequence, the strong biotin–avidin
interaction has been extensively utilized in the life sciences.8–10
Biotinylated polymers and copolymers of poly(ethylene oxide)
(PEO), poly(2-(diethylamino)ethyl methacrylate) (PDEAEMA),
poly(ethylene glycol) (PEG), and poly(lactic acid) (PLA) have been
studied for various biotechnological applications. PEG has been
used as a variable spacer for biotin conjugates with avidin binding
capability.11–13
cells,22 synthesize dendrimers,23 conducting polymers,24–30 poly-
mer modified viruses,31 and gold nanoparticle–enzyme conju-
gates.32 Recently, azide-functionalized biotinylated haptens and
mannose-derivatized azide have been synthesized using
a
Kenner-type linker for the preparation of small-molecule-arrays.33
Herein, we report on using the copper catalyzed azide-alkyne
‘click’ cycloaddition reaction for the efficient functionalization of
alkyne modified OPV oligomers with biotin–PEG–azides. The syn-
theses of the respective dialkyne functional OPVs and azide-termi-
nated biotin–PEG derivatives are also presented. The luminescence
properties of the resulting biotin–‘clicked’–OPV conjugates were
probed, and networks with streptavidin coated quantum dots
(Qdots) were produced, and characterized using transmission
electron microscopy (TEM).
The general synthetic strategy involves preparing two interme-
diates first, dialkyne functional oligomer 2 and azide functional
biotin–PEG units 7 or 8 (Scheme 1), which then are subsequently
combined by means of ‘click’-chemistry. The approach incorpo-
rates flexibility: the conjugated oligomer can be chosen from a
large library of oligomers or other molecules, depending on the
desired electrooptical characteristics, or the targeted aggregation
behavior. Compounds 7 and 8 represent a second platform for a
library of potentially valuable reactions, involving a straight-
forward functionalization with biotin or other ligand segments of
varying hydrophilicity via ‘click’-chemistry; biotin may be placed
at a defined distance from the conjugated oligomer by simply
choosing a PEG segment of the desired chain length, depending
on the specific requirements of the potential application. The
chain length of the PEG segment also controls the biotin binding
The copper catalyzed azide-alkyne 1,2,3–triazole forming ‘click’
reaction14–18 is a very popular coupling reaction that demonstrates
the concept of click chemistry; the reaction has been used in a vari-
ety of fields, for example, drug discovery,19 materials science,20 and
biology.21 The chemoselective ‘click’ reaction is tolerant to the pres-
ence of a wide range of functional groups; it has been used to label
* Corresponding author. Tel.: +1 718 982 3928; fax: +1 718 982 3910.
E-mail address: peetz@mail.csi.cuny.edu (R. M. Peetz).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.08.080

N. Vundyala et al. / Tetrahedron Letters 49 (2008) 6386–6389
6387
3
HO
N3
OR
OR
OR
OR
OR
OR
O
OR
OR
N
N
OR
OR
2
N
O
O
OR
N
H2N
O
OH
N
OR
OR
OR
OR
OR
OR
N
N
N
HO
O
rt, THF
O
OR
O
N
N
O
4
1
2
NH
H
HN
OR
OR
"Click"
H
OR
N
S
R = -C7H15
Spacer
OR
OR
N
Spacer
OR
N
S
3
O
H
H
O
HO
N3
O
NH
HN
NH
O
HN
S
2
O
N3
N
N
H
H
2
O
EDC.HCl, 60oC
DMF/CH2Cl2
H
HN
H
NH
O
O
N
S
S
O
OH
7
2
Biotin-Ester
9
O
O
H
NH
H
HN
O
S
6
O
Spacer
N3
H2N
O
5
O
NH
HN
O
O
6
N
N
N
N
H
N3
H
N
H
H
CDI, rt, DMF
6
H
H
S
NH
HN
8
Biotin-Amide
10 N
O
6
O
Scheme 1. ‘Click’ functionalization of oligo(p-phenylene vinylene).
capability as well as the overall hydrophobic (conjugated oligo-
mer)/hydrophilic (biotin–PEG spacer) balance of the building
blocks.
Specifically, the conjugated oligomer OPV, trimer dialdehyde 1,
was synthesized and characterized in analogy to literature,34,35 and
subsequently reacted with propargylamine under Schiff base for-
mation conditions to produce dialkyne functionalized trimer OPV
2 in quantitative yields (Scheme 1).
facile purification of the target ester. The reaction conditions were
further optimized: increased yields could be obtained at higher
reaction temperatures and longer reaction times, and/or with the
solvent mixture DMF/CH2Cl2 (Table 1, entries 2, 3, and 7).
The amidation proved easier than the esterification and pro-
ceeded smoothly at ambient temperatures to give 8 with signifi-
cantly higher yields than ester 7 under analogous conditions
(Table 1, entries 4 and 6), using a commercial NH2-functional
PEG azide, and making further optimizations unnecessary. It
should be noted that the commercial NH2-functional PEG azide
(ꢀ90% purity) was used without any further purification.
In a next step, we synthesized azide functional biotin–PEG con-
jugates 7 and 8 (biotin–PEG–N3) with different PEG spacers using
either 1,3-dicyclohexylcarbodiimide (DCC), carbonyldiimidaz-
ole (CDI), or 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide
hydrochloride (EDCÁHCl) mediated coupling. Two coupling reac-
tions, that is, esterification and amidation, were employed. In order
to vary the distance of biotin from the conjugated oligomer and
overall hydrophobic/ hydrophilic balance (see above), we used
PEG segments of different lengths for the esterification and amida-
tion, the latter using four additional repeat units in the PEG seg-
ment. Representative optimization reactions are summarized in
Table 1 and show satisfactory yields.
Using DCC in DMF or DMSO/CH2Cl2 resulted in no reaction or
trace yields of the biotin–ester 7 after about 20 h at room temper-
ature, as did attempts using EDC.HCl in DMF or CDI in DMSO under
similar conditions (not shown in Table 1). The reaction worked
when CDI or EDC.HCl together with 4-dimethylaminopyridine
(DMAP) in DMF was employed; modest yields of 7 could be
achieved after 18 h at ambient conditions (Table 1, entries 5 and
1, respectively). The coupling reagent EDCÁHCl was selected
because its urea byproduct is water-soluble, hence enabling more
Finally, in the key step, intermediate azide functional PEG–bio-
tins 7 and 8 were ‘clicked’ onto the dialkyne functional OPV 2,
yielding the target conjugates 9 and 10 (Scheme 1). Representative
synthesis results are summarized (Table 2).
Initial ‘clicking’ attempts using CuSO4Á5H2O/sodium ascorbate
mixtures were unsuccessful in various solvent mixtures (not
shown in Table 2), resulting in the degradation of the two imine
functions in 2 and the re-formation of the corresponding aldehyde
functional 1. Model reactions of alkyne 2 and PEG–azide 3 via [3+2]
Huisgen cycloaddition in the presence of commercially available
Cu(CH3CN)4PF6 resulted in very good yields of the corresponding
PEG-functional OPV 4 (Scheme 1) (Table 2, entries 1–3). Inciden-
tally, 4 represents a potentially valuable material in its own rights.
We then proceeded to ‘click’ the dialkyne-functional OPV 2 and
the azide-terminated biotin conjugates 7 and 8 under similar mild
reaction conditions. The reactions were performed in CH2Cl2 using
0.2–0.5 M equiv of Cu(I) catalyst to give the 1,4-triazoles in good to
very good yields (Table 2, entries 4–7). The main challenge
consisted in finding conditions, under which the formation of the
byproduct OPV (di)aldehyde (see above) was minimized, as it
Table 1
Representative synthesis results of biotin–PEG–N3’s 7 and 8b
Table 2
Entry
Coupling reagent/catalyst
Solvent
T (°C)/t (h)
Yielda (%)
Representative click reactions of OPV dialkyne 2a
1
2
3
4
EDCÁHClc/DMAP
DMF
DMF/CH2Cl2
rt/18
60/24
rt/48
20
65
60
80b
Entry
Azide
Product
Cu(CH3CN)4PF6 (mol equiv)
T (h)
Yieldb (%)
1
2
3
4
5
6
7
3
3
3
7
7
8
8
4
4
4
9
9
10
10
1
8
10
18
18
18
18
18
80
90
95
70
85
85
95
0.2
0.2
0.5
0.2
0.5
0.2
5
6
7
CDIc
DMF
rt/18
30
65b
40
80/24
a
Isolated yield.
Isolated yield of 8.
b
c
a
EDCÁHCl = 1-(3-(dimethyl amino) propyl)-3-ethylcarbodiimide hydrochloride,
Conditions: [2] = 5.7 mM; [3], [7], and [8] = 13.8 mM; ambient temperature;
CDI = carbonyl diimidazole; conditions: [Biotin] = 80 mM; [PEG–N3] = 100 mM;
solvent CH2Cl2.
b
[EDCÁHCl] = 100 mM; [CDI] = 234 mM; rt = ambient temperature.
Yields based on 1H NMR.

6388
N. Vundyala et al. / Tetrahedron Letters 49 (2008) 6386–6389
1.0
0.8
0.6
0.4
0.2
0.0
1.0
PL
UV
8.8x10-7
4.4x10-7
8.8x10-8
4.4x10-8
8.8x10-9
M
M
M
M
M
(*10-8 Molar)
(*10-5 Molar)
0.8
2 (9.1)
9 (9.4)
10 (8.8)
2 (3.0)
9 (2.2)
10 (2.3)
0.6
0.4
0.2
0.0
250 300 350 400 450 500 550 600 650 700
wavelength/nm
450
500
550
600
650
700
wavelength/nm
Figure 1. UV–vis absorption and emission (normalized) of OPVs 2, 9, and 10 (left), concentration-dependent emission of 10; solutions in DMF, excitation at 438 nm.
Figure 2. TEM images of (a) streptavidin Qdot control, (b) network of streptavidin Qdots and 18
OPV 10.
lM biotin–OPV 10, and (c) network of streptavidin Qdots and 1 lM biotin–
could not be avoided entirely (ꢀ5% by 1H NMR). When larger
amount of Cu catalyst was employed more aldehyde byproduct
was produced (Table 2, entries 1, 4, and 6 vs the respective other
entries). Also, the ester 7 produced more aldehyde than the amide
8, resulting in overall lower yields in 9 vs 10. After workup, the
compound purity was 95%; this level of purity is quite reasonable
if one takes into account the fact that these molecules are oligo-
mers with molecular weights over 1500 D. Attempts to purify the
samples further via column chromatography lead to no additional
improvement. The best and most consistent results were
obtained after 18 h using 0.2 equiv of catalyst (Table 2, entries 2,
5, and 7).
The compounds synthesized were analyzed by means of ATR/
FTIR, 1H NMR (200 or 600 MHz), 13C NMR, and MS (ESI or MAL-
DI-TOF). The Cu-catalyzed cycloaddition typically leads to exclu-
sively 1,4 product, heteronuclear NMR-coupling experiments
confirmed this in the cases of 4, 9, and 10 (see Supplementary
data). The electro-optical properties of the OPV are retained after
the attachment of the biotin–spacer elements, as can be observed
from the very similar absorption and photoluminescence charac-
teristics of 2, 9, and 10 in DMF solution (Fig. 1, left). Due to the high
fluorescence quantum efficiency of the trimeric OPV moiety,36 the
biotin OPV conjugates show fluorescence even at very low concen-
trations. Together with a high molar extinction coefficient,36 the
optical properties make these materials attractive candidates for
imaging/biosensing applications. A broadening of the fluorescence
in the longer wavelengths in the cases of 9 and 10 is most probably
a consequence of enhanced aggregation of the fluorophore moie-
ties in the polar solvent DMF, and supported by concentration-
dependent emission spectra (Fig. 1, right).
on commercially available streptavidin Qdots 655 nm by incubat-
ing the (biotin–OPV–biotin)s 9 and 10 with the Qdots. The Qdots
polyvalently display biotin binding sites; we anticipated the addi-
tion of bivalent biotin–OPV would result in a network of Qdots
connected by OPV linkers. The network formation was observed
using transmission electron microscopy (TEM) (Fig. 2). The Qdot
control sample (Fig. 2a) shows no aggregation. Samples with com-
bined streptavidin-Qdots and biotin–OPV–biotin 10 clearly show
network formation (Fig. 2b and c). In contrast, multiple attempts
with biotin–OPV–biotin 9 (not shown) yielded inconclusive results.
A tentative explanation could be that the longer PEG spacer in the
case of 10 is necessary for the biotin moieties to access the strep-
tavidin binding sites.
The electro-optical properties of 9, 10, as well as the networks
with two interacting electronic systems are currently under further
investigation and will be presented elsewhere. Also, the fabrication
of biotin–OPV monolayers on silica chips is underway for arraying
Qdots in two dimensions for bio-sensing applications.
In conclusion, we have developed a highly efficient, novel
approach to combine the electro-optical properties of conjugated
organic molecules, such as OPVs, with the biotechnological func-
tionalities of biotin, using the Cu(I)-catalyzed [3+2] Huisgen cyclo-
addition. This click chemistry route for synthesizing conducting/
conjugated oligomer-based biomolecules described here will be
useful for the design of a variety of biologically active molecules,
biosensors, and biomedical device coatings.
Acknowledgments
This work was supported by a PSC-CUNY Grant, a CUNY
collaborative grant and postdoctoral fellowship. F.S. acknowledges
a stipend from the LSAMP (Louis Stokes Alliance for Minority
Participation). The authors would like to thank Dr. Hsin Wang for
help with the NMR experiments.
One objective for synthesizing soluble biotin–OPV was to see if
they could be used as ‘adaptors’ to connect streptavidin coated
quantum dots (Qdots). Preliminary studies were performed to
evaluate the abilities of 9 and 10 to bind the biotin binding sites

N. Vundyala et al. / Tetrahedron Letters 49 (2008) 6386–6389
6389
16. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int. Ed.
2002, 41, 2596–2599.
Supplementary data
17. Bock, V. D.; Hiemstra, H.; van Maarseveen, J. H. Eur. J. Org. Chem. 2006, 51–68.
18. Gupta, S. S.; Kuzelka, J.; Singh, P.; Lewis, W. G.; Manchester, M.; Finn, M. G.
Bioconjugate Chem. 2005, 16, 1572–1579.
19. Kolb, H. C.; Sharpless, K. B. Drug Discovery Today 2003, 8, 1128–1137.
20. Hawker, C. J.; Wooley, K. L. Science 2005, 309, 1200–1205.
21. Agard, N. J.; Baskin, J. M.; Prescher, J. A.; Lo, A.; Bertozzi, C. R. ACS Chem. Biol.
2006, 1, 644–648.
22. Beatty, K. E.; Xie, F.; Wang, Q.; Tirrell, D. A. J. Am. Chem. Soc. 2005, 127, 14150–
14151.
23. Wu, P.; Feldman, A. K.; Nugent, A. K.; Hawker, C. J.; Scheel, A.; Voit, B.; Pyun, J.;
Frechet, M. J.; Sharpless, K. B.; Fokin, V. V. Angew. Chem., Int. Ed. 2004, 43, 3928–
3932.
24. Such, G. K.; Quinn, J. F.; Quinn, A.; Tjipto, E.; Caruso, F. J. Am. Chem. Soc. 2006,
128, 9318–9319.
25. Nandivada, H.; Chen, H.; Bondarenko, L.; Lahann, J. Angew. Chem., Int. Ed. 2006,
45, 3360–3363.
26. Gao, H.; Matyjaszewski, K. Macromolecules 2006, 39, 4960–4965.
27. Riva, R.; Schmeits, S.; Stoffelbach, F.; Jerome, C.; Jerome, R.; Lecomte, P. Chem.
Commun. 2005, 42, 5334–5336.
28. van Steenis, D. J. V. C.; David, O. R. P.; van Strijdonck, G. P. F.; van Maarseveen, J.
H.; Reek, J. N. H. Chem. Commun. 2005, 34, 4333–4335.
29. Englert, B. C.; Bakbak, S.; Bunz, U. H. F. Macromolecules 2005, 38, 5868–5877.
30. Punna, S.; Diaz, D. D.; Li, C.; Sharpless, K. B.; Fokin, V. V.; Finn, M. G. Polym.
Preprints 2004, 45, 778–779.
31. Gupta, S. S.; Raja, K. S.; Kaltgrad, E.; Strable, E.; Finn, M. G. Chem. Commun.
2005, 34, 4315–4317.
32. Brennan, J. L.; Hatzakis, N. S.; Tshikhudo, T. R.; Dirvianskyte, N.; Razumas, V.;
Patkar, S.; Vind, J.; Svendsen, A.; Nolte, R. J. M.; Rowan, A. E.; Brust, M.
Bioconjugate Chem. 2000, 17, 1373–1375.
33. Kohn, M.; Wacker, R.; Peters, C.; Schroder, H.; Soulere, L.; Breinbauer, R.;
Niemeyer, C. M.; Waldmann, H. Angew. Chem., Int. Ed. 2003, 42, 5830–5834.
34. Wang, B.; Wasielewski, M. R. J. Am. Chem. Soc. 1997, 119, 12–21.
35. Peetz, R. M.; Strachota, A.; Thorn-Csanyi, E. Macromol. Chem. Phys. 2003, 204,
1439–1450.
Supplementary data (experimental and analytical details) asso-
ciated with this article can be found, in the online version, at
doi:10.1016/j.tetlet.2008.08.080.
References and notes
1. Dai, L.; Winkler, B.; Dong, L.; Tong, L.; Mau, A. W. H. Adv. Mater. 2001, 13, 915–
925.
2. Burroughes, J. H.; Bradley, D. D. C.; Brown, A. R.; Marks, R. N.; MacKay, K.;
Friend, R. H.; Burn, P. L.; Holmes, A. B. Nature 1990, 347, 539–541 and
references cited therein.
3. Bernius, M. T.; Inbasekaran, M.; O’Brien, J.; Wu, W. Adv. Mater. 2000, 12, 1737–
1750.
4. Fumitomo, H.; Maria, A. D-G.; Schwartz, B. J.; Heeger, A. J. Acc. Chem. Res. 1997,
30, 430–436.
5. Hoffman, A. S. Clin. Chem. 2000, 46, 1478–1486.
6. Duncan, R. Nat. Rev. Drug Discovery 2003, 2, 347–360.
7. Delair, T.; Charles, M.-H.; Cros, P.; Laayoun, A.; Mandrand, B.; Pichot, C. Polym.
Adv. Technol. 1998, 9, 349–361.
8. Lue, R. Y.; Chen, G. Y.; Hu, Y.; Zhu, Q.; Yao, S. Q. J. Am. Chem. Soc. 2004, 126,
1055–1062.
9. Ashraf, S. S.; Benson, R. E.; Payne, E. S.; Halbleib, C. M.; Gron, H. Protein Exp.
Purif. 2004, 33, 238–245.
10. Chen, I.; Howarth, M.; Lin, W.; Ting, A. Nat. Methods 2005, 2, 99–104.
11. Tan, J. F.; Ravi, P.; Too, H. P.; Hatton, T. A.; Tam, K. C. Biomacromolecules 2005, 6,
498–506.
12. Salem, A. K.; Cannizzaro, S. M.; Davies, M. C.; Tendler, S. J. B.; Roberts, C. J.;
Williams, P. M.; Shakessheff, K. M. Biomacromolecules 2001, 2, 575–580.
13. Marek, M.; Kaiser, K.; Gruber, H. J. Bioconjugate Chem. 1997, 8, 560–566.
14. Huisgen, R. In 1,3-Dipolar Cycloaddition Chemistry; Padwa, A., Ed.; Wiley: New
York, 1984; pp 1–176.
15. Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004–
2021.
36. Narwark, O. Dissertation, University of Hamburg, 2002.