Use of γ-aminopropyl-coated glass surface for the patterning of oligonucleotides through oxime bond formation

Article abstract of DOI:10.1016/j.bmcl.2008.03.054

The present work reports on the preparation of glass surfaces coated with NPPOC-protected aminooxy groups and their use for the patterning of oligonucleotides on glass slides and in capillary tubes. The method involves the use of surfaces coated with amino groups using (γ-aminopropyl)triethoxy silane and subsequent grafting of the aminooxy groups by using the activated ester 1. The NPPOC-protected aminooxy groups on the surfaces can be cleaved upon irradiation. The free aminooxy groups so obtained are subsequently reacted with aldehyde-containing oligonucleotides to achieve efficient surface patterning.

Full text of DOI:10.1016/j.bmcl.2008.03.054

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2540–2543
Use of c-aminopropyl-coated glass surface for the patterning
of oligonucleotides through oxime bond formation
Nabil Dendane,a,b Antoine Hoang,b Eric Defrancq,a,* Franc¸oise Vinetb and Pascal Dumya
aDCM/I2BM, UMR CNRS 5250, Universite´ Joseph Fourier, BP 53, 38041 Grenoble Cedex 9, France
bLETI-CEA-Grenoble 17, rue des Martyrs, 38054 Grenoble Cedex 9, France
Received 28 January 2008; revised 17 March 2008; accepted 18 March 2008
Available online 22 March 2008
Abstract—The present work reports on the preparation of glass surfaces coated with NPPOC-protected aminooxy groups and their
use for the patterning of oligonucleotides on glass slides and in capillary tubes. The method involves the use of surfaces coated with
amino groups using (c-aminopropyl)triethoxy silane and subsequent grafting of the aminooxy groups by using the activated ester 1.
The NPPOC-protected aminooxy groups on the surfaces can be cleaved upon irradiation. The free aminooxy groups so obtained are
subsequently reacted with aldehyde-containing oligonucleotides to achieve efficient surface patterning.
Ó 2008 Elsevier Ltd. All rights reserved.
The design of micro-arrays of DNA, carbohydrates and
peptides is the subject of intensive research as they rep-
resent a powerful tool for high-throughput screening of
biomolecules with applications in genetic analysis,
molecular diagnostics, and drug discovery. Similarly,
micro-fluidic systems in which the synthesis, purifica-
tion, and analysis are accomplished on chip are of signif-
icant interest. The ‘lab on a chip’ devices along with the
micro-array technologies are expected to make feasible
more rapid, multi-parametric and economical analysis
with minimal sample volumes.
techniques’ and hence benefits from the use of auto-
mated robots. The surface immobilization of biomole-
cules by later approach can be achieved by chemical
reaction between surface-bound reactive groups and
functionalized biomolecules.3 Several strategies includ-
ing maleimide/thiol,4 ‘click chemistry’ involving al-
kyne/azide,5 hydrazide/aldehyde6 have been reported
for covalent attachment of biomolecules on open sur-
faces. However, the patterning inside the micro-channels
for ‘lab on a chip’ preparation is more tedious and there-
fore only few methods have been reported to date. A
photo-generated-surface-bound aldehyde group has
been described earlier for surface immobilization of pro-
teins through the formation of Schiff base.7 Another
method is based on the photo-crosslinking of surface-
bound benzophenone derivatives with biomolecules
through C–H insertion.8 Recently, an elegant method
based on the use of ultraviolet-light emitting diodes
(UV-LED’s) has been proposed for in-situ synthesis of
DNA in capillaries.9
The control of surface chemistry is crucial to achieve
efficient immobilization of biomolecules for preparation
of such micro-devices. One of the most challenging tasks
is to carry out the immobilization of biomolecules with-
out perturbing their intrinsic properties. Two major ap-
proaches are available for surface immobilization and
patterning of biomolecules on surfaces. The first ap-
proach involves the direct on-surface synthesis (in-situ),1
whereas the second approach involves the immobiliza-
tion of prefabricated oligonucleotides on the support.2
The latter approach is more widely used to prepare
low to medium density micro-arrays on account of its
flexibility. Moreover, this approach utilizes ‘spotting
In this context, we have investigated the use of oxime
bond formation for the covalent attachment of the oli-
gonucleotides on glass surface. In our earlier works,
we have demonstrated the efficiency of oxime linkage
for functionalization of planar glass surfaces and inner
wall of glass capillary tubes.10 Others have also reported
the surface immobilization of oligonucleotides on gold
surfaces or polymeric particles using oxime bonds.11
Recently, we reported the efficient surface patterning
Keywords: DNA; Lab on a chip; Micro-array; Oxime; Photo-depro-
tection; Surface patterning.
*
Corresponding author. Tel.: +33 (0)476514433; fax: +33
(0)476514946; e-mail: Eric.Defrancq@ujf-grenoble.fr
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.03.054

N. Dendane et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2540–2543
2541
of oligonucleotides in capillary tubes as well as on glass
surfaces through oxime bond formation by using sur-
faces grafted with reactive aminooxy groups masked
with photo-cleavable protected group, 2-(2-nitrophenyl)
propyloxycarbonyl group (NPPOC).12 NPPOC groups
were removed by brief irradiation to unmask the
reactive aminooxy group on surfaces and subsequently
reacted with the aldehyde-containing oligonucleotides.
The masked-aminooxy moieties were grafted on the
glass surfaces by using a triethoxysilane derivative bear-
ing the NPPOC-protected aminooxy group. The trieth-
oxysilane derivative was prepared in four steps from
11-bromo-1-undecene. In the present work, we report
an alternative method for grafting NPPOC-protected
aminooxy moieties on the glass surfaces. The current
strategy involves coupling reaction of activated ester 1,
bearing the NPPOC-protected aminooxy group, with
surface-bound amino groups. The preparation of sur-
faces coated with amino groups using (c-aminopro-
pyl)triethoxy silane (APS) is well documented in
literature.13,14 We report that current approach allows
also an efficient surface immobilization and patterning
of oligonucleotides on glass slides and in capillary tubes.
aqueous solution for different time durations (10, 15,
and 20 s). It should be noted that the presence of base
is required for the photolysis to proceed. Oligonucleo-
tide immobilization was accomplished by dipping the
glass slides in a solution of 0.4 M ammonium acetate
buffer containing oligonucleotide aldehyde (20 lM) for
2 min. The oligonucleotides containing aldehyde func-
tionality at 50-terminus were prepared as reported
earlier.18
The efficiency of this method was further evaluated by
investigating the ability of surface-bound oligonucleo-
tides to hybridize with the complementary strand
labeled with Cy3 fluorescent probes. After hybridiza-
tion and subsequent washings, the glass slides were
scanned under a fluorescent scanner. Herein, the sig-
nal intensity directly reflects the efficiency of surface
immobilization by current approach. The representa-
tive scanned images from the hybridization experi-
ments are shown in Figure 1.
A
pattern of
fluorescent and non-fluorescent areas with the shape
of the mask was visualized. The ratio signal/noise S/
N was evaluated for the different time durations (10,
15, and 20 s) to 2, 3, and 4, respectively. An irradia-
tion time of 15 s was thus found enough to obtain a
good signal to noise ratio.
The activated ester 1 was prepared in two-steps from
carboxymethoxyamine hydrochloride (Scheme 1). The
aminooxy moiety was protected with photolabile
NPPOC chloride derivative and N-hydroxysuccinimide
was introduced next using the standard procedure.15
Compounds 1 and 2 were characterized by NMR and
mass analysis.16
The strategy was used next for surface immobilization of
oligonucleotides into the inner wall of capillary tubes.
Capillary tubes are extensively used in many biological
applications, they are inexpensive and provide higher
surface-to-volume ratio. The glass capillary tubes were
silanized with (c-aminopropyl)triethoxy silane and the
coupling with activated ester 1 was performed in trichlo-
roethylene instead of DMF. DMF was found to cause
the degradation of the fused capillary and hence not
used. The photo-deprotection of the aminooxy moieties
was carried out by irradiation at 365 nm for varying
time periods (from 5 to 30 s) through a slit aperture ad-
justed at 150 lm. This allowed the unmasking of amino-
oxy moieties only at preselected portions of the tube.
The surface immobilization was achieved by filling the
capillary tubes with aqueous solution of aldehyde-con-
taining oligonucleotides. The hybridization experiments
with complementary sequence labeled with Cy3 were
performed at 100 nM concentrations. SSC buffer was
used to remove unhybridized complementary oligonu-
cleotides and the capillary tube was scanned under a
fluorescent scanner (Fig. 2). Fluorescence spots were
found localized at the irradiated portion of the tube.
The photo-deprotection was rapid and an irradiation
time of 5 s was sufficient to obtain a measurable
response.
The oligonucleotide immobilization was accomplished
first on glass slides and then in capillary tubes.17 The
glass slides were activated by treatment with sodium
hydroxide solution. The slides were then dipped at room
temperature in a solution of (c-aminopropyl)triethoxy
silane in toluene overnight to achieve chemisorption of
amino groups on surfaces. This was followed by curing
at 110 °C for 3 h to stabilize the silane layer. The silan-
ization step was followed by overnight coupling reaction
with activated ester 1 in DMF at room temperature. The
photo-cleavage of the NPPOC-protecting group of the
aminooxy moieties on the glass surface was next carried
out by irradiation at 365 nm through a mask containing
holes (diameter of the holes = 100 lm) in a 20% pyridine
H
N
Me NO
2
a
HO
NH Cl
3
O
HO
O
O
O
O
O
2
b
In conclusion, an alternative protocol for the surface
patterning of oligonucleotides was developed. The effec-
tiveness of the method was verified by accomplishing oli-
gonucleotide immobilization on surfaces of glass slides
and capillary tubes. Although the present method shows
a signal to noise ratio slightly less favorable than our
previously reported strategy, it presents the main advan-
tage that the NPPOC-protected aminooxy surfaces can
be prepared easily from (c-aminopropyl)triethoxy
H
N
Me NO
2
O
O
O
O
N
O
O
O
1
Scheme 1. Reagents and conditions: (a) 2-(2-Nitrophenyl)propyl
chloroformate, Na2CO3 10% H2O, rt, 3 h, 90%; (b) DCC, N-
hydroxysuccinimide, CH2Cl2, rt, 4 h, 67%.

2542
N. Dendane et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2540–2543
Figure 1. (Left) Scanned images of the oligonucleotide arrays on the glass slides. Arraying was achieved by irradiation through a mask (hole
diameter: 100 lm) at 365 nm for (a) 10 s, (b) 15 s, and (c) 20 s in a 20% aq pyridine solution, followed by the deposition of a 20 lM solution of
aldehydic oligonucleotide in 0.4 M ammonium acetate buffer for 2 min. Hybridization was carried out at 39 °C for 1 h using a 10 nM solution of
Cy3Ò labeled complementary oligonucleotide. Fluorescence intensities are color-coded, varying from blue (low) to green, yellow, red, and then white
(saturation), (right) profiles intensity for quantification analysis.

N. Dendane et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2540–2543
2543
8. Balakirev, M. Y.; Porte, S.; Vernaz-Gris, M.; Berger, M.;
´
Arie, J.-P.; Fouque, B.; Chatelain, F. Anal. Chem. 2005,
77, 5474.
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9. Blair, S.; Richmond, K.; Rodesch, M.; Bassetti, M.;
Cerrina, F. Nucleic Acids Res. 2006, 34, e110.
10. Defrancq, E.; Hoang, A.; Vinet, F.; Dumy, P. Bioorg.
Med. Chem. Lett. 2003, 13, 2683.
11. Boncheva, M.; Scheibler, L.; Lincoln, P.; Vogel, H.;
Akerman, B. Langmuir 1999, 15, 4317.
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Vinet, F.; Dumy, P. Bioconjugate Chem. 2007, 18, 671.
13. Taylor, S.; Smith, S.; Windle, B.; Guiseppi-Elie, A. Nucleic
Acids Res. 2003, 31, e87.
Figure 2. Scanned images of the oligonucleotides arrayed into the
capillary tube. The arrayed capillary tube was obtained after following
procedure: irradiation at 365 nm in 20% aq pyridine solution for
various time periods; deposition of a 20 lM solution of aldehydic
oligonucleotide in water for 2 min; and hybridization with the
complementary strand labeled with Cy3Ò. Time of irradiation: (a)
5 s, (b) 10 s, (c) 15 s, (d) 20 s, (e) 30 s.
14. Trens, P.;Denoyel, R.;Rouquerol,J. Langmuir1995, 11, 551.
15. Preparation of 1: A solution of 2-(2-nitrophenyl)propyl
chloroformate (2.2 g, 9 mmol) in dioxan (20 mL) was added
drop-wise to a solution of carboxymethoxylamine hydro-
chloride (1 g, 4.6 mmol) in 10% aq Na2CO3 (25 mL) at 0 °C.
The solution was stirred for 3 h in darkness and evaporated
under vacuum. The residue so obtained was re-dissolved in
water and the aqueous layer was washed with Et2O,
acidified with 1 N HCl and extracted from CH2Cl2. The
organic layer was dried over Na2SO4 and solvent removed
under vacuum. The crude product was purified by silica gel
column chromatography using CH2Cl2/methanol (97/3,
v/v) as eluent to obtain compound 2 as white powder (1.2 g,
90%). 2 (1.2 g, 4 mmol) was dissolved in anhydrous CH2Cl2
(15 mL) and dicyclohexylcarbodiimide (0.82 g, 4.4 mmol)
and N-hydroxysuccinimide (0.46 g, 4.4 mmol) were added
to the solution. The reaction mixture was stirred overnight,
filtered and evaporated under vacuum. The crude product 1
was obtained after purification by silica gel column chro-
matography (Eluent: EtOAc) as white powder (1 g, 67%).
16. Characterization data for 2: Mp: 88–90 °C; 1H NMR
(300 MHz, CDCl3): d 1.37 (d, J = 7 Hz, 3H), 3.68 (m, 1H),
4.30 (m, 2H), 4.42 (s, 2H), 7.39 (t, J = 8 Hz, 1H), 7.46 (d,
J = 8 Hz, 1H), 7.56 (t, J = 8 Hz, 1H), 7.75 (t, J = 8 Hz,
1H), 8.25 (br s, 1H), 9.45 (br s, 1H); 13C NMR (75 MHz,
CDCl3): d 17.3 (CH3), 33.1 (CH), 70.4 (CH2), 73.5 (CH2),
124.2 (CH), 127.7 (CH), 128.0 (CH), 132.8 (CH), 136.5
(quat), 150.6 (quat), 158.2 (C@O), 172.0 (C@O). ESIMS
(m/z): 297 (MꢀH)ꢀ. Anal. Calcd for C12H13N2NaO7: C,
45.01; H, 4.09; N, 8.75. Found: C, 45.02; H, 4.29; N, 8.56.
Characterization data for 1: 1H NMR (200 MHz, CDCl3):
d 1.39 (d, J = 6 Hz, 3H), 2.89 (s, 4H), 3.68 (m, 1H), 4.31
(m, 2H), 4.39 (s, 2H), 7.30–7.60 (m, 4H). ESIMS (m/z): 395
(M+H)+.
silane-coated glass. The immobilization protocol
reported herein could be an efficient tool in the design
of ‘labs on a chip’ devices.
Acknowledgments
This work was supported by the ‘Centre National de
la Recherche Scientifique’ (CNRS) and the ‘Commis-
`
sariat a l’Energie Atomique’ (CEA). We are grateful
to NanoBio program for the facilities of the Synthesis
Platform.
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