Synthesis of a photo-caged aminooxy alkane thiol

Article abstract of DOI:10.1039/b904195h

A photo-caged aminooxy alkane thiol synthesized in 7 steps and 15% overall yield was used to form a self-assembled monolayer (SAM). Photo-deprotection on the surface was confirmed by FT-IR spectroscopy and contact angle goniometry. Conjugation of a small molecule ketone, ethyl levulinate, further confirmed the presence of aminooxy groups on the surface. The Royal Society of Chemistry 2009.

Full text of DOI:10.1039/b904195h

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis of a photo-caged aminooxy alkane thiol†
Rock J. Mancini, Ronald C. Li, Zachary P. Tolstyka and Heather D. Maynard*
Received 2nd March 2009, Accepted 8th September 2009
First published as an Advance Article on the web 6th October 2009
DOI: 10.1039/b904195h
A photo-caged aminooxy alkane thiol synthesized in 7 steps and 15% overall yield was used to form a
self-assembled monolayer (SAM). Photo-deprotection on the surface was confirmed by FT-IR
spectroscopy and contact angle goniometry. Conjugation of a small molecule ketone, ethyl levulinate,
further confirmed the presence of aminooxy groups on the surface.
Introduction
Improved efficiency of biomarker discovery has resulted in a
demand for high-throughputprotein screeningmethods toidentify
disease at early onset, as well as to monitor the progression
of treatments through biosignature detection.1–4 Self-assembled
monolayers (SAMs) are a viable option for the fabrication of
bioactive arrays required for high-throughput screening. This
is because SAMs present well defined surfaces for protein
immobilization and subsequent investigation of biomolecules
at biologically relevant concentrations.5 The ability to pattern
bioactive arrays of proteins at the micron or nanoscale has been
comprehensively examined.6,7 Surface immobilization at this scale
allows for detection of multiple biomarkers on the same surface,
resulting in a more resolved biosignature and a more robust protein
array.7,8 As such, we sought to provide a method for fabrication of a
photo-activated surface for site-specific conjugation of molecules
of biological interest via oxime bonds. In particular we report
the synthesis of a 2-(2-nitrophenyl)propyloxycarbonyl (NPPOC)
protected aminooxy alkane thiol with subsequent conjugation of
ethyl levulinate to photo-deprotected surfaces (Scheme 1).
Several methods of immobilization have been utilized to at-
tach biomolecules to surfaces. One method involves nonspecific
adsorption onto a surface; however, this often results in major
conformational changes.9–11 Alternatively, biomolecules may be
covalently linked to the surface. Early routes included carbodi-
imide coupling of the free amine groups of the protein to a
surface bearing terminal carboxylic acid moieties or reversible
coupling with surface carbonyl groups via imine chemistry.12,13
These methods however, are not site-selective and often result in
reduction of protein bioactivity.13
To achieve site-specific coupling, free cysteines have been
targeted. However, while effective, free cysteines are rare in natural
proteins and additional steps of adding a surface reactive amino
acid through techniques such as mutagenesis may be required.14,15
Alternatively, Huisgen cycloaddition or “click” chemistry has
been used by modifying the biomolecule to contain an azide or
alkyne moiety with an alkane thiol of complementary reactivity
patterned on the surface.16–18 Phosphonate protein interactions as
Scheme 1 Deprotection and conjugation of a NPPOC aminooxy SAM.
well as Diels–Alder reactions have also been exploited.19,20 Another
approach involved reaction of an aminooxy moiety with a protein
containing a ketone or aldehyde to form oxime bonds.21–23
Surface immobilization of proteins via oxime chemistry was
first demonstrated by Boncheva in 1999,24 and we have used light
and a photo-acid generator to liberate surface aminooxy groups
on polymer films.25 We have also directly generated nanofeatures
of aminooxy groups with electron beam lithography.26 Chan
and coworkers were able to immobilize ketone-decorated gold
colloids onto aminooxy SAMs on a gold surface.27 Yousaf and
coworkers have produced a hydroquinone SAM that once oxidized
to the corresponding quinone reacts to immobilize aminooxy
RGD peptides for cell patterning.28 The same group has also
produced a photo-caged aminooxy SAM that has been shown
to immobilize ligands for cell adhesion after removal of the
nitroveratryloxycarbonyl (NVOC) group by ultra-violet (UV)
exposure; in this case a semicarbazide solution was required to
remove the aldehyde generated upon deprotection to prevent
oxime bond formation with the photobyproduct.29 We present
an alternate strategy involving the NPPOC moiety which does
not require a scavenger because an aldehyde photobyproduct is
not produced upon photo-deprotection.30,31 SAMs of a NPPOC
Department of Chemistry and Biochemistry, University of California, Los
Angeles, 607 Charles E. Young Drive South, Los Angeles, CA 90095-1596,
U. S. A. E-mail: maynard@chem.ucla.edu; Fax: +1-310-206-3403; Tel: +1-
310- 267-5162
† Electronic supplementary information (ESI) available: 1H NMR spectra
of compounds 2–5, 7–9. See DOI: 10.1039/b904195h
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Scheme 2 Synthesis of photo-caged aminooxy alkane thiol 7
protected aminooxy alkane thiol were fabricated. Subsequent
deprotection and conjugation to a ketone-containing biologically
relevant small molecule were demonstrated.
Results and discussion
Scheme 3 Synthesis of activated carbonate 9.
Synthesis of photo-caged aminooxy alkane thiol
in 69% yield, to avoid use of this toxic compound. The carbonate
was then added to 4 to give 5 in 97% yield.
The synthetic strategy employed for the formation of the
photolabile alkanethiol is outlined in Scheme 2. The triethy-
lene glycol alkene was synthesized from triethylene glycol and
11-bromo-1-undecene following a literature procedure.32 After
purification, product 2 was isolated in 67% yield. The yield was
lowered by formation of dialkylated triethylene glycol byprod-
uct. Compound 2 was subjected to Mitsunobu conditions with
N-hydroxyphthalimide.33 Using triphenylphosphine and diiso-
propyl azodicarboxylate (DIAD), the terminal alcohol of 2
was substituted. The triphenylphosphine oxide byproduct was
precipitated from the reaction mixture with hexanes. This method
removed the side product, enabling efficient chromatography and
isolation of the desired product 3 in quantitative yield.
Compound 5 was then subjected to thioacetic acid and AIBN
using thermal activation. Chromatography was employed to
remove starting material and byproducts. Again, separation of the
alkene starting material 5 and the resulting thioacetate 6 proved
difficult. Therefore 6 was not isolated prior to removal of the
acetate group.
The final step required deprotection of the thioacteate to reveal
the thiol. Hydrochloric acid in ethanol was used, and the product
was purified. The yield was 28% for the two step conversion of 5 to
7. Overall, the desired NPPOC-protected aminooxy product was
synthesized in seven convergent steps, with a yield of 15%.
The phthalimide protecting group of 3 was removed with
hydrazine.33 The reaction was monitored by TLC to ensure com-
plete consumption of the starting material. Additional hydrazine
was added over the course of the reaction to complete the
deprotection. Purification of 4 by flash column chromatography
resulted in a yield of 83%.
To install the thiol end group, we first attempted to modify
4 with thioacetic acid using 2,2¢-azobisisobutyronitrile (AIBN) as
the radical initiator and light. While this thioene reaction typically
proceeds with good yields,34 the isolation of the thioacteate alka-
nethiol bearing the aminooxy end group proved to be challenging.
To circumvent this problem, we opted to install the NPPOC group
first followed by the formation of thioacetate.
SAM formation, deprotection, and conjugation
Upon obtaining 7, SAM formation, deprotection, and subsequent
surface conjugation were attempted. A piranha cleaned gold wafer
was incubated in a 5 mM ethanolic solution of 7 for 24 h.
The resulting SAM 7 was evaluated by contact angle (76 2◦)
and IR (Fig.1a). In particular, the peak at 1130 cm-1 confirmed
the presence of PEG (C–O stretching) while the signals at 1260
(N–CO–O), 1530 (NO2) and 1720 cm-1 (C O) showed that the
=
NPPOC protecting group was present on the surface. Ellipsometry
also indicated successful formation of the SAM, giving a surface
thickness of 2.4 0.3 nm.
Following SAM formation, photo-deprotection of SAM 7 was
investigated by flood exposure to 365 nm UV light. Deprotection
was monitored by IR via the disappearance of the signal at
1260 cm-1 corresponding to the aminooxy carbamate moiety, with
approximately 50% deprotection occurring after 10 to 15 minutes.
The activated photolabile group was synthesized from 2-
(2-nitrophenyl)propanol (Scheme 3). Typical conditions to acti-
vate this group involve the use of phosgene.31 Instead, disuccin-
imidyl carbonate (DSC) was coupled to the alcohol of 8 to form 9
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Fig. 1 Infrared spectroscopy of (a) SAM 7, (b) SAM 7 after 3 h exposure to a hand-held 365 nm UV light (c) DP-SAM 7 after 3 h exposure to a 3 mM
ethyl levulinate solution at 60 ◦C.
Although recent investigations report more rapid removal of the
NPPOC protecting group with a UV laser at a dose of 1.2 J/cm2,35
our deprotection scheme employs a hand-held UV lamp, as a less
intense source of light, applied at a distance of 1.5 cm from the
surface. While this is a more convenient method for UV exposure, it
accounts for theincreased exposure time required for deprotection.
SAM 7 was exposed to a hand held UV lamp for 3 hours to ensure
maximal surface deprotection. The resulting photo-deprotected
surface, DP-SAM 7 was subsequently examined. The contact
angle (62 7◦) decreased as expected. The peaks at 1260, 1530,
and 1720 cm-1 in the IR spectrum (Fig. 1b) were no longer
visible.
Taken together these data showed that successful deprotection
and conjugation of a ketone to the SAM via oxime bond formation
occurred. This suggests that the aminooxy moiety present in this
photo-caged molecule may be used to immobilize proteins and
other bio-molecules with ketones onto surfaces. This site selec-
tive conjugation, in conjunction with standard photolithography
strategies, may be employed to pattern biomolecules for a range
of applications, including oriented protein arrays.
Conclusions
A photo-caged aminooxy surface reactive moiety was synthesized
in seven steps in 15% yield. This molecule was used to form a SAM
that was subsequently shown to reveal surface aminooxy groups
when exposed to 365 nm light. No scavenging additives were
required. Conjugation of the resulting aminooxy terminated SAM
to the biologically relevant molecule ethyl levulinate via oxime
bond formation was confirmed by IR spectroscopy. Application
of this alkane thiol to immobilize biomolecules on surfaces is
underway.
Reaction with DP-SAM 7 was subsequently examined using
ethyl levulinate, a small molecule with a ketone moiety (Scheme 1).
This molecule was chosen because it is widely used to modify
proteins with ketone groups.36 The photo-deprotected aminooxy
SAM was rinsed with 5 mL of ethanol and incubated with a
3 mM ethanolic solution of ethyl levulinate at 60 ◦C. The contact
angle of the conjugate was measured as 64 4◦. This was only
a slight change compared to the deprotected aminooxy surface.
Covalent conjugation of ethyl levulinate to the aminooxy surface
was confirmed by observation of the oxime bond stretch in the IR
Experimental
spectrum (Fig. 1c) at 1640 cm-1 (C N). The ester carbonyl stretch
=
Materials and methods
at 1715 cm-1 was also observed. Absorption by the oxime bond is
typically weak, yet it was found to be comparable to that of the
carbonyl stretch; this was likely due to surface enhancement.
All chemicals were purchased from Sigma–Aldrich unless other-
wise noted. The products 232 and 831 were synthesized according
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to literature procedures. 1H and 13C NMR spectra were obtained
on either a Bruker ARX 500 MHz or AVANCE 500 MHz spec-
trometer. J values are given in Hz. Mass spectra were obtained on
either a Applied Biosystems Voyager-DE-STR MALDI-TOF or
a high resolution ESI Applied BioSystems Q-Star Elite supported
by Grant Number S10RR024605 from the National Center For
Research Resources. The spectra are solely the responsibility of
the authors and do not necessarily represent the official views
of the National Center For Research Resources or the National
Institutes of Health. Chemical infrared spectra were recorded on
a Perkin-Elmer Spectrum One FT-IR spectrophotometer fitted
with an ATR accessory. Surface infrared spectra were obtained on
a Bio-Rad FTS 175C Dynamic Alignment FT-IR Spectrometer.
Ellipsometry was performed using a Gaertner LSE ellipsometer
equipped with a 633 nm HeNe laser fixed at a 70◦ incidence angle.
UV–vis spectra were recorded on a Thermospectronic Biomate
5 spectrophotometer using MeOH as the solvent. An FTA 135
Version 2.0 was used for contact angle measurements. An Entela
UVGL-25 4 Watt UV lamp was operated at 365 nm for the photo-
deprotection.
1.97 (m, 2 H, CH2CH2O) 1.57–1.46 (m, 2H, CH2CH2O), 1.38–
1.16 (m, 12H, CH2CH2CH2) ppm; dC(125 MHz; CDCl3) 163.6,
139.3, 134.5, 129.1, 123.6, 114.2, 76.9, 71.6, 70.9, 70.7, 70.6, 70.1,
69.4, 33.9, 29.7, 29.7, 29.6, 29.2, 29.0, 28.7, 25.9, 21.5 ppm; IR:
n
max/cm-1 2924, 2854, 1790, 1731, 1639, 1611, 1524, 1467, 1374,
1325, 1292, 1257, 1186, 1109, 1083, 1033, 996, 978, 953, 908,
877, 787, 699; UV–vis: lmax = 310 nm (e = 455 cm-1 M-1);
m/z (Electrospray) Found: MNa+ (sodium adduct), m/z 470.2491;
Calc. for C25H37NO6Na: 470.2519.
Synthesis of 4. Hydrazine hydrate (0.9 mL, 20 mmol) was
added to product 3 (1.64 g, 3.66 mmol) dissolved in 20 mL of
DCM. The solution was stirred vigorously for 8 h. Monitoring the
reaction by TLC indicated that starting material remained and
additional hydrazine (0.9 mL, 20 mmol) was added. The solution
was stirred for an additional 24 h. The reaction mixture was con-
centrated and the product purified by FCC (hexanes:ethyl acetate,
2:1, Rf = 0.2) to give a clear oil (0.96 mg, 3.0 mmol, 83% yield).
NMR dH(500 MHz; CDCl3) 5.85–5.77 (m, 1H, CHCH2), 5.01–
4.91 (m, 2H, CHCH2), 3.85–3.84 (m, 2H, CH2ON), 3.69–3.63 (m,
8H, OCH2CH2O), 3.59–3.57 (m, 2H, OCH2CH2O), 3.44 (t, J =
6.8 Hz, 2H, CH2CH2O), 2.05–2.01 (m, 2H, CH2CH2O), 1.60–1.54
(m, 2H, CH2CH2CH2), 1.39–1.27 (m, 12H, CH2CH2CH2) ppm;
dC(125 MHz; CDCl3) 139.2, 114.1, 74.8, 71.6, 70.6, 70.6, 70.1,
69.6, 33.8, 29.6, 29.5, 29.5, 29.4, 29.1, 28.9, 26.1 ppm; IR: nmax/cm-1
2924, 2854, 1640, 1591, 1458, 1349, 1458, 1349, 1296, 1245, 1200,
1106, 1041, 993, 908, 846, 723; UV–vis: lmax = 252 nm (e =
28 cm-1 M-1); m/z (Electrospray) Found: M + 1, m/z 318.2585;
Calc. for C17H35NO4: 318.2644.
Synthesis
Synthesis of 2. 2 was synthesized according to literature
procedure.32 NaOH (0.49 mL, 50%) was added to triethylene glycol
(8.00 mL, 60.0 mmol) in a 2-necked round bottom flask equipped
with a water-jacketed condenser. The mixture was heated to
100 ◦C for 30 min before adding 11-bromo-1-undecene (2.66 mL,
12.3 mmol) dropwise. The reaction was allowed to proceed for 12 h
before being diluted with 50 mL of water, and the resulting aqueous
layer was washed with hexanes (3 ¥ 50 mL). The organic layers
were combined and dried over MgSO4 and the solvent removed
under reduced pressure. 2 was isolated following flash column
chromatography (FCC) (hexanes: ethyl acetate, Rf = 0.2) as a clear
oil (2.48 g, 8.21 mmol, 67% yield). NMR dH(500 MHz; CDCl3)
5.82–5.72 (m, 1H, CHCH2), 4.99–4.86 (m, 2H, CHCH2), 3.73–
3.52 (m, 12H), 3.41 (t, J = 6.6 Hz, 2H), 2.80 (bs, 1H, OH), 2.02–
1.95 (m, 2H, CH2CH2O), 1.59–1.48 (m, 2H, CH2CH2CH2), 1.38–
1.19 (m, 12H, CH2CH2CH2) ppm; dC(125 MHz; CDCl3) 139.2,
114.2, 72.6, 71.6, 70.7, 70.7, 70.4, 70.1, 33.8, 29.6, 29.6, 29.5, 29.5,
29.2, 29.0, 26.1 ppm; IR: nmax/cm-1 3456, 2923, 2854, 1640, 1458,
1350, 1295, 1248, 1106, 1069, 993, 908, 722, 675; UV–vis: lmax =
265 nm (e = 18 cm-1 M-1); (m/z (Electrospray) Found: MNa+
(sodium adduct), m/z 325.2346; Calc. for C17H34O4Na: 325.2355.
Synthesis of 8. 8 was synthesized according to a literature
procedure.31 Triton B (3.3 mL, 8 mmol) was added to nitroethyl
benzene (1.08 mL, 8 mmol). Paraformaldehyde (245 mg, 8.1 mmol)
was added, and the reaction was heated to 60 ◦C for 6 h. The
reaction was concentrated in vacuo and neutralized with 5%
aqueous HCl followed by extraction with ethyl acetate (3 ¥ 10 mL).
The material was dried over MgSO4 and the solvent was removed
under reduced pressure. 8 was isolated following FCC (CH2Cl2,
Rf = 0.2). NMR dH(500 MHz, CDCl3) 7.75 (dd, J = 8.3, 1.2 Hz,
1H, Ar-H), 7.60–7.55 (m, 1H, Ar-H), 7.50 (dd, J = 8.1, 1.5 Hz,
1H, Ar-H), 7.365 (m, 1H, Ar-H), 3.84–3.76 (m, 2H, CH2), 3.56–
3.48 (m, 1H, CH), 1.70 (br s, 1H, OH), 1.33 (d, J = 6.8 Hz, 3H,
CH3) ppm; dC(125 MHz; CDCl3) 138.2, 132.8, 128.5, 128.3, 127.3,
124.2, 67.9, 36.5, 17.7 ppm; IR: nmax/cm-1 3375, 3073, 2973, 2877,
1719, 1607, 1577, 1519, 1480, 1465, 1454, 1351, 1299, 1244, 1194,
1164, 1085, 1055, 1034, 1011, 976, 954, 875, 851, 782, 746, 709,
680, 662; UV–vis: lmax = 250 nm (e = 4126 cm-1 M-1), 390 nm
(e = 471 cm-1 M-1); m/z (MALDI-TOF) Found: MNa+ (sodium
adduct), m/z 204.47; Calc. for C27H44N2O8Na: 204.06.
Synthesis of 3. Alkane–PEG 2 (2.7 g, 8.9 mmol) was dissolved
in 90 mL of dry dichloromethane (DCM). To that solution, N-
hydroxyphthalimide (1.74 g, 10.6 mmol) was added, followed by
triphenylphosphine (2.79 g, 10.6 mmol). After ensuring complete
dissolution of the reagents, DIAD (1.9 mL, 9.8 mmol) was added
dropwise over 5 min, and the reaction was allowed to stir over 18 h.
The solvent was removed in vacuo, and the residue resuspended
in hexanes. The triphenylphosphine oxide byproduct crystallized
and was isolated by filtration. The solvent was evaporated in vacuo.
A white residue (4.0 g, 8.9 mmol, >99% yield) was obtained
after purification by FCC (hexanes:ethyl acetate, 3:1, Rf = 0.7).
NMR dH(500 MHz; CDCl3) 7.85–7.79 (m, 2H, Ar-H), 7.76–
7.71 (m, 2H, Ar-H), 5.85–5.77 (m, 1H CHCH2), 4.97–4.87 (m,
2 H, CH2CH), 3.86–3.80 (m, 2H), 3.66–3.59 (m, 2H), 3.57–3.44
(m, 6H), 3.41 (t, J = 7.0 Hz, 2H), 2.80 (bs, 1H, OH) 2.02–
Synthesis of 9. Disuccinimidyl carbonate (390 mg, 1.5 mmol)
was dissolved in 5 mL of dry DMF. 8 (180 mg, 0.99 mmol)
was added followed by triethylamine (0.77 mL, 5.5 mmol). The
reaction was stirred for 18 h and concentrated in vacuo. A dark
red oil 9 (220 mg, 0.68 mmol, 69% yield) was isolated following
FCC (hexanes:ethyl acetate, 1:1, Rf = 0.4). NMR dH(500 MHz;
CDCl3) 7.82 (dd, J = 8.0, 1.3 Hz, 1H, Ar), 7.63–7.60 (m, 1H,
Ar), 7.50 (dd, J = 7.9, 1.2 Hz, 1H, Ar), 7.43–7.40 (m, 1H, Ar),
4.56–4.48 (m, 2H, CH2), 3.82–3.78 (m, 1H, CH), 2.85 (s, 4H, Su),
1.43 (d, J = 7.0 Hz, 3H, CH3) ppm; dC(125 MHz; CDCl3) 168.7,
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151.5, 150.0, 135.9, 133.1, 128.6, 128.4, 128.0, 124.6, 74.5, 33.4,
25.5, 17.5 ppm; IR: nmax/cm-1 2936, 1812, 1788, 1736, 1668, 1609,
1577, 1523, 1458, 1430, 1386, 1355, 1257, 1199, 1088, 1048, 1022,
991, 942, 853, 813, 786, 750, 712, 658; UV–vis: lmax = 251 nm
(e = 3855 cm-1 M-1), 372 nm (e = 458 cm-1 M-1); m/z (MALDI-
TOF) Found: MK+ (potassium adduct), m/z 361.12; Calc. for
C27H44N2O8K: 361.04.
Self assembled monolayer (SAM) formation and reactivity
SAM 7. A gold wafer was immersed in hot piranha solution
(1:3v/v H2O2:H2SO4) for 5 min CAUTION: PIRANHA SOLUTION
REACTS VIOLENTLY WITH ORGANIC MATERIALS, rinsed with Milli-
Q water and ethanol, and dried under a stream of argon. The
wafer was then exposed to a 5 mM ethanolic solution of 7 at
23 ◦C for 36 h. The surface was then rinsed with 5 mL of ethanol
and dried under argon before analysis via contact angle (76 2◦)
and IR spectroscopy to confirm SAM 7. IR: nmax/cm-1 2915 and
Synthesis of 5. The hydroxylamine 4 (230 mg, 0.73 mmol)
was dissolved in 10 mL of DCM. The activated ester 9 (250 mg,
0.77 mmol) was added followed by triethylamine (0.41 mL,
2.9 mmol). The reaction was allowed to stir over 18 h in the dark.
The reaction was concentrated and purified by FCC (hexanes:ethyl
acetate, 2:1, Rf = 0.4) with a gradient solvent system from 2:1 to
1:1 hexanes:ethyl acetate to afford a pale yellow oil 5 (370 mg,
71 mmol, 97% yield). NMR dH(500 MHz; CDCl3) 7.96 (bs, 1H,
NH), 7.74 (dd, J = 8.1, 1.0 Hz, 1H, Ar), 7.58–7.55 (m, 1H,
Ar), 7.48–7.46 (m, 1H, Ar), 7.38–7.35 (m, 1H, Ar), 5.81–5.80
(m, 1H, CHCH2), 5.00–4.96 (m, 1H, CHCH2), 4.93–4.91 (m,
1H, CHCH2), 4.33–4.26 (m, 2H, CH2CHCH3), 3.95–3.94 (m,
2H, CH2ON), 3.71–3.61 (m, 9H, OCH2CH2O, CHCH3), 3.56–
3.54 (m, 2H, OCH2CH2O), 3.42 (t, J = 6.8 Hz, 2H, CH2CH2O),
2.05–2.01 (m, 2H, CH2CH2O), 1.57–1.54 (m, 2H, CH2CH2CH2),
1.36–1.27 (m, 15H, CH2CH2CH2) ppm; dC(125 MHz; CDCl3)
156.9, 150.5, 139.2, 137.0, 132.6, 128.1, 127.4, 124.1, 114.1, 75.4,
71.5, 70.6, 70.5, 70.4, 70.0, 69.2, 69.1, 33.8, 33.3, 29.5, 29.5,
29.4, 29.4, 29.1, 28.9, 26.0, 17.6 ppm; IR: nmax/cm-1 3252, 2925,
2855, 1740, 1639, 1609, 1578, 1524, 1460, 1352, 1298, 1242,
1103, 1033, 995, 909, 852, 784, 749, 710, 662; UV–vis: lmax =
252 nm (e = 3338 cm-1 M-1), 330 nm (e = 503 cm-1 M-1); m/z
(Electrospray) Found: MNa+ (sodium adduct), m/z 547.2846;
Calc. for C27H44N2O8 Na: 547.2995.
=
2845 (CH), 1720 (C O), 1530 and 1350 (NO2), 1490 (NH), 1260
(N–CO–O), 1130 (C–O). Ellipsometry measurements gave a
surface thickness of 2.4 0.3 nm.
Photo-deprotection of SAM 7. SAM 7 was exposed to a 365 nm
hand-held UV lamp for 3 h at a distance of 1.5 cm under ambient
conditions before washing with 5 mL of ethanol and drying under
a stream of argon. Deprotection of SAM 7 was confirmed by
contact angle (62 7◦) and IR: nmax/cm-1 2920 and 2850 (CH),
1490 (NH2), 1130 (C–O).
Ethyl levulinate conjugation to DP-SAM 7. DP-SAM 7 was
exposed to a 3 mM ethanolic solution of ethyl levulinate at 60 ◦C
for 3 h. The resulting surface was washed with 5 mL of ethanol
and dried under a stream of argon. Conjugation to the surface was
confirmed by contact angle (64 4◦) and IR: nmax/cm-1 2920 and
=
=
2850 (CH), 1715 (C O), 1640 (C N), 1130 (C–O).
Acknowledgements
This research was funded by the National Science Foundation
Career Award CHE-0645793. RJM acknowledges a fellowship
from the NSF IGERT: Materials Creation Training Program
(MCTP)-DGE-0654431 and the California Nanosystems Insti-
tute. ZPT thanks the NIH sponsored Biotechnology Training in
Biomedical Sciences and Engineering Program and the Jonsson
Comprehensive Cancer Center (JCCC) for fellowships. HDM
thanks the Alfred P. Sloan Foundation for additional funding.
The authors would like to thank Steven Jonas for his aid with the
ellipsometry measurements.
Synthesis of 6. Thioacetic acid (0.26 mL, 4 mmol) was added
to a solution of 5 (370 mg, 0.71 mmol) in 2.6 mL of methanol.
AIBN (5 mg, 0.03 mmol) was added, and the reaction mixture
◦
was heated to 70 C for 9 h. The solution was concentrated and
purified by FCC (hexanes:ethyl acetate, 2:1, Rf = 0.2) to give
impure product 6 as a pale yellow oil. This was used directly in the
next step.
Synthesis of 7. A solution of 6 (320 mg, 0.53 mmol) in 20 mL
of 0.1 M HCl in EtOH was prepared and refluxed for 3 h. The
solution was cooled and stirred for an additional 3 h. The reaction
mixture was concentrated and purified by FCC (hexanes:ethyl
acetate, 2:1, Rf = 0.4) to give 7 (110 mg, 0.20 mmol, 28% yield over
two steps). NMR dH(500 MHz; CDCl3) 7.95 (bs, 1H, NH), 7.74
(dd, J = 8.1, 1.2 Hz, 1H, Ar), 7.58–7.55 (m, 1H, Ar), 7.48–7.46 (m,
1H, Ar), 7.38–7.35 (m, 1H, Ar), 4.31–4.20 (m, 2H, CH2CHCH3),
3.95–3.86 (m, 2H, CH2ON), 3.78–3.50 (m, 11H, OCH2CH2O,
CH2CHCH3), 3.42 (t, J = 6.8 Hz, 2H, CH2CH2O), 2.52–2.50 (m,
2H, CH2SH), 1.67–1.51 (m, 4H, CH2CH2O, CH2CH2SH), 1.42–
1.15 (m, 17H, CH2CH2CH2) ppm; dC(125 MHz; CDCl3) 156.9,
150.5, 137.0, 132.6, 128.1, 127.4, 124.1, 84.7, 76.1, 75.4, 71.5, 70.5,
70.5, 70.4, 70.0, 69.2, 69.1, 34.0, 33.8, 33.5, 33.3, 29.5, 29.5, 29.5,
29.4, 29.0, 28.3, 28.2, 26.0, 24.6, 24.5, 17.6 ppm; IR: nmax/cm-1
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