Automated maskless photolithography system for peptide microarray synthesis on a chip

Article abstract of DOI:10.1021/cc100009g

Maskless photolithographic peptide synthesis was performed on a glass chip using an automated peptide array synthesizer system. The peptide array synthesizer was built in a closed box, which contained optical and fluidic systems. The conditions for peptide synthesis were fully controlled by a computer program. For the peptide synthesis on a glass chip, 20 NVOC-protected amino acids were synthesized. The coupling efficiencies of two model peptide sequences were examined on ACA/APTS and PEG/CHI/GPTS chips. PEG/CHI/GPTS chip gave higher average stepwise yields of GIYWHHY (94%) and YIYGSFK (98%) than those of ACA/APTS chip. To quantify peptide-protein binding affinity, HPQ- or HPM-containing pentapeptides were synthesized on a PEG/CHI/GPTS chip and the binding event of Cy3 labeled-streptavidin was quantified. The peptide sequence of IQHPQ showed highest binding affinity with Cy3 labeled-streptavidin. The results demonstrated that the photolithographic peptide array synthesis method efficiently quantified the binding activities of protein-peptide interactions and it can be used for additional biological assay applications.

Full text of DOI:10.1021/cc100009g

J. Comb. Chem. 2010, 12, 463–471
463
Automated Maskless Photolithography System for Peptide Microarray
Synthesis on a Chip
Dong-Sik Shin,† Kook-Nyung Lee,‡ Byung-Wook Yoo,‡ Jaehi Kim,† Mira Kim,†
Yong-Kweon Kim,‡ and Yoon-Sik Lee*,†
School of Chemical and Biological Engineering and School of Electrical Engineering and Computer
Science, Seoul National UniVersity, Kwanak-Gu, Seoul 151-744, Korea
ReceiVed January 19, 2010
Maskless photolithographic peptide synthesis was performed on a glass chip using an automated peptide
array synthesizer system. The peptide array synthesizer was built in a closed box, which contained optical
and fluidic systems. The conditions for peptide synthesis were fully controlled by a computer program. For
the peptide synthesis on a glass chip, 20 NVOC-protected amino acids were synthesized. The coupling
efficiencies of two model peptide sequences were examined on ACA/APTS and PEG/CHI/GPTS chips.
PEG/CHI/GPTS chip gave higher average stepwise yields of GIYWHHY (94%) and YIYGSFK (98%) than
those of ACA/APTS chip. To quantify peptide-protein binding affinity, HPQ- or HPM-containing
pentapeptides were synthesized on a PEG/CHI/GPTS chip and the binding event of Cy3 labeled-streptavidin
was quantified. The peptide sequence of IQHPQ showed highest binding affinity with Cy3 labeled-streptavidin.
The results demonstrated that the photolithographic peptide array synthesis method efficiently quantified
the binding activities of protein-peptide interactions and it can be used for additional biological assay
applications.
Introduction
contact the chip surface homogeneously. The background
signal from the in situ synthesis method is relatively lower
than that produced by spotting pre-synthesized peptides
because the background surface is selectively inert. However,
the quality of peptide from the in situ synthesis method is
lower than that of the spotting method because the peptide
synthesized on a chip cannot be purified. Other than the
SPOT synthesis method,5c light-directed, spatially address-
able peptide array synthesis is a representative in situ
synthesis method and was introduced by combining solid
phase peptide synthesis with a semiconductor fabrication
system.5g A photolabile protecting group on a chip is
deprotected by selective UV irradiation with a photomask.
Peptide microarray can be synthesized by repeated cycles
of photodeprotection and coupling steps. Recently, the
photomasks have been replaced with a digital micromirror
array (MMA) for patterning of biomolecules.5h,7 A method
of using photogenerated acid (PGA) in the maskless pho-
tolithography system was reported by the Gao group to
synthesize oligo-nucleotide and peptide microarrays.5d-f
Instead of using a photolabile protecting group, they used
commercially available, 4,4′-dimethoxytrityl (DMT) pro-
tected nucleotides and tert-butyloxycarbonyl (Boc) protected
amino acids. However, for this method, a physical or
chemical barrier should be constructed on a chip to prevent
the diffusion of PGA to other regions.8
Peptide microarrays have emerged for high-throughput
screening of various biosystems including binding affinity
and enzyme substrate screening.1 Peptide microarrays can
be used as the part of proteins for the elucidation of protein-
biomolecule affinity.2 Moreover, various enzyme substrates,
such as protein kinase, phosphatase, and protease substrates
have been screened using peptide microarrays.3
In general, there are two methods for the preparation of
peptide microarrays: immobilization of pre-synthesized pep-
tide derivatives and in situ synthesis of peptides on a chip.4
An overview of peptide microarray preparation method is
summarized in Table 1.5 The method of spotting pre-
synthesized peptide ensures a high quality chip because
spotting materials can be obtained at high purity. However,
chip design is highly restricted depending on the number of
pre-synthesized peptides. Moreover, spots form with greater
peptide density at the edges than in the middle because
solution flows to the spot edge during solvent evaporation.
The background is prone to contamination during washing
step because of the activated background. The peptide should
be modified to aminooxy acetyl group, cysteine residue, or
cyclopentadiene for the proper chemoselective reaction.6 In
contrast, the in situ synthesis method is flexible for chip
design so that the peptide microarray can be easily prepared
with a well-established synthesis system. The spot shape of
the in situ synthesis method is regular because solutions
The present study focuses on peptide microarray synthesis
on a glass chip using an automated maskless photolithog-
raphy system. Twenty NVOC-amino acid monomers were
synthesized, and peptide synthesis was optimized on a chip.
To evaluate the photolithographic synthesis method, stepwise
* To whom correspondence should be addressed. E-mail: yslee@snu.ac.kr.
Phone: +82-2-880-7073. Fax: +82-2-888-1604.
† School of Chemical and Biological Engineering.
‡ School of Electrical Engineering and Computer Science.
10.1021/cc100009g  2010 American Chemical Society
Published on Web 05/18/2010

464 Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 4
Table 1. Overview of Peptide Microarray Preparation Methods
Shin et al.
in situ synthesis
photolithographic synthesis
photolabile protecting
spotting pre-synthesized peptide
SPOT synthesis
PGA mediated method
group mediated method
chemistry
chemoselective ligation
high
not flexible
Fmoc chemistry
data not available
flexible
Boc chemistry
high
flexible
>40 µm
regular
physical or chemical barrier
NVOC chemistry
moderate
flexible
synthesis efficiency
chip design
spot size
spot feature
background
∼100 µm
∼100 µm
∼25 µm, ∼200 µm (variable)
coffee-ring shape
activated surface
regular
regular
physical barrier
photolabile protecting group
Table 2. List of Chemical Abbreviations Used in the Article
abbreviation
chemical
abbreviation
chemical
ε-ACA
APTS
Boc
ε-aminocaproic acid
3-aminopropyltriethoxysilane
tert-butyloxycarbonyl
benzotriazol-1-yloxy-tris
(dimethylamino)phosphonium hexafluorophosphate
chitosan
2-chlorotrityl chloride resin
dichloromethane
HOBt
HPM
HPQ
1-hydroxybenzotriazole
His-Pro-Met
His-Pro-Gln
methanol
BOP
MeOH
CHI
2-CTC resin
DCM
NMP
NVOC
PEG-SA
N-methylpyrrolidone
6-nitroveryloxycarbonyl
O,O′-bis-(2-aminopropyl)
polypropylene glycol-block-
polyethylene glycol 500-succinic acid
triethylamine
trifluoroacetic acid
tetrahydrofuran
trityl
DIPEA
EtOAc
RBITC
GPTS
diisopropylethylamine
ethyl acetate
rhodamine B isothiocyanate
3-glycidoxypropyltrimethoxysilane
O-(7-azabenzo-triazol-1-yl)-N,N,N′,N′-tetramethyluronium
hexa-fluorophosphate
TEA
TFA
THF
Trt
HATU
peptide coupling yields were checked on various surface-
modified glass chips, and the protein binding assay was
performed on each of the peptide microarray.
Slides # 2948) were purchased from Corning. Chemical
abbreviations are summarized in Table 2.
Instruments. A microarray scanner (Axon Instrument,
GenePix 4000B) was used for analyzing fluorescence
intensity. NMR spectra were recorded on a JNM-LA300
spectrometer (Jeol Inc.) in deuterated solvents and were
referenced to TMS (δ scale). For maskless photolithography,
UV light passed through 365 nm band-pass filter from an
illuminator (Hamamatsu Photonics, Mercury-Xenon lamp
# L2570).
Experimental Section
Materials. 3-Aminopropyltriethoxysilane (APTS), 3-gly-
cidoxypropyltrimethoxy-silane (GPTS), chitosan (CHI)
(75-85% deacetylated chitin, MN ) 50,000-190,000), 4,5-
dimethoxy-2-nitrobenzyl alcohol, biotin, 1-hydroxybenzot-
riazole (HOBt), diisopropylethylamine (DIPEA), triethy-
lamine (TEA), trifluoroacetic acid (TFA), benzotriazol-1-
yloxy-tris(dimethylamino)phosphonium hexafluorophosphate
(BOP), O-(7-azabenzo-triazol-1-yl)-N,N,N′,N′-tetramethyl-
uronium hexa-fluorophosphate (HATU), succinic anhydride,
magnesium sulfate (anhydrous), ninhydrin, phenol, pyridine,
chloroform-d, methanol-d,4 dimethylsulfoxide-d,6 amino acid,
and their derivatives were purchased from Aldrich. Strepta-
vidin-Cy3 conjugates, Rhodamine B isothiocyanate (RBITC),
ε-aminocaproic acid (ε-ACA), and polyoxyethylene sorbitan
monolaurate (Tween 20) were purchased from Sigma. O,O′-
Bis-(2-aminopropyl) polypropylene glycol-block-polyethyl-
ene glycol-block-polypropylene glycol 500 (Jeffamine ED-
600) was purchased from Fluka. 2-Chlorotrityl chloride resin
(2-CTC resin), Fmoc-amino acid, and their derivatives were
purchased from BeadTech Inc. Sodium hydroxide, disodium
hydrogen phosphate, and sodium dihydrogen phosphate were
purchased from Oriental Chemical Industry. N-Methylpyr-
rolidone (NMP), chloroform, dichloromethane (DCM), di-
oxane, methanol (MeOH), tetrahydrofuran (THF), ethyl
acetate (EtOAc), and hexane were purchased from Junsei
Chemical Co. All solvents were purified by methods reported
in the literature.9 The glass slides (75 mm ×25 mm, Micro
Peptide Array Synthesizer. The peptide array synthesizer
consists of a UV light source, MMA, projection lenses, a
UV bandpass filter, an optical stop, a chemical reaction
chamber, and control valves for flow system as shown in
Figure 1. MMA was fabricated with single crystalline silicon
by micromachining a spatial UV light modulator.10 The
micromirrors were 210 µm × 210 µm of single crystalline
silicon. The micromirrors were 16 × 16 arrayed and had a
5 µm gap from the bottom electrode (Figure 2). The reagents
for amino acid coupling were injected through the inlet port
by controlling valves and the pressure of nitrogen stream.
The reaction chamber was sealed by a slide glass, on which
surface peptides were synthesized. UV irradiation, solution
delivery, reaction time, and washing condition were con-
trolled by programming language, LABVIEW (National
Instruments Co.) (Figure 3).
Syntheses of NVOC-Amino Acids.11 Amino acids (2
mmol) were dissolved in 2 N aq. NaOH solution (1 mL).
6-Nitroveratryloxycarbonyl chloride (NVOC-Cl; 662 mg, 2.4
mmol) in 1,4-dioxane (1 mL) and DCM (4 mL) and 2 N aq.
NaOH solution (1.1 mL) were added to the amino acid in
NaOH solution in turns for 10 times in an ice bath. After

Maskless Photolithographic Peptide Synthesis
Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 4 465
Figure 1. Photograph of peptide array synthesizer system.
Figure 2. (a) Single crystalline silicon MMA (16 × 16 mirrors), (b) enlarged view of MMA (210 µm × 210 µm).
Figure 3. Reaction cycle of peptide synthesis.
stirring for 1 h, the ice bath was removed, and the solution
was vigorously stirred for 4-12 h. The progress of the
reaction was monitored by TLC. The organic layer was
removed, and the aqueous layer was acidified to pH 3-4
using 5 N aq. HCl solution. Side chain-protected amino acids
were acidified by acetic acid. The product was extracted by
EtOAc and dried by MgSO4, and the solvent was evaporated.
When acetic acid was added, the remaining acetic acid
residue in the product was evaporated azeotropically with
hexane three times.
Synthesis of NVOC-PEG-SA. 6-Nitroveratryloxycarbo-
nyl-Jeffamine ED-600-succinic acid (NVOC-PEG-SA) was
synthesized using solid phase chemistry. 2-CTC resin (10
g, 1.0 mmol/g) was swollen in TEA (50 mmol, 7.0 mL)/
DCM (100 mL), and Jeffamine ED-600 (100 mmol, 60 g)
was added. The reaction mixture was stirred at 25 °C for
12 h. The solution, which contained excess reagents, was
filtered out, and the resin was washed with DCM (×3) and
MeOH (×3). The resin was swollen in DIPEA (25 mmol,
4.4 mL) and DCM (100 mL). The solution of DIPEA (25

466 Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 4
Shin et al.
Scheme 1. Synthesis of NVOC-Amino Acida
mmol, 4.4 mL) and NVOC-Cl (20 mmol, 5.5 g) in DCM
was added into the resin mixture and stirred at 25 °C for
12 h. After washing with the above method, 5% (v/v) TFA
in DCM (120 mL) was added and stirred at 25 °C for 30
min. The resin was filtered, and the filtrated solution was
collected. After the solvent was completely evaporated, the
remaining residue was dissolved in DIPEA (15 mmol, 2.6
mL)/THF (25 mL). Succinic anhydride (15 mmol, 1.5 g) in
THF (25 mL) was added to the solution, and the solution
was magnetically stirred. The product was separated by flash
column (chloroform/MeOH ) 5:1).
a Reagents and conditions: (a) NVOC-Cl (1.2 equiv), 2 N aq. NaOH
(2.1 equiv) in (1:4) 1,4-dioxane and DCM, 0 °CfRT, 4-12 h.
Table 3. Yield of NVOC-Amino Acids
NVOC-amino acid
yield (%)
NVOC-amino acid
yield (%)
Surface Modification of Glass Slides.12 The glass slides
were precleaned in a mixture of H2SO4 and H2O2 (4:1) for
10 min and rinsed with H2O, ethanol, and dried in vacuo.
The amination or epoxidation process was performed on the
glass surface by two different routes. Silanizations with
3-aminopropyltriethoxysilane (APTS) or 3-glycidoxypropy-
ltrimethoxysilane (GPTS) was carried out at 45 °C in 5%
(v/v) chloroform solution for 2 or 12 h, respectively. To
remove the non-covalently adsorbed silane molecules, soni-
cation in chloroform was performed for 10 min. The glass
slides were rinsed with ethanol and blown dry with nitrogen
gas. The attachment of the hydrophilic polymer, chitosan
(CHI), was carried out on a GPTS treated glass slide with
1% (w/v) CHI in 1% AcOH-H2O at 25 °C for 12 h. The
slides were then sonicated in H2O for 10 min, rinsed
extensively with H2O, and blown dry with nitrogen gas. To
generate a photolabile NVOC-protected surface, the aminated
surface or CHI-treated surface was exposed to a 5 mM
solution of NVOC-ε-aminocaproic acid (NVOC-ε-ACA-OH)
or NVOC-PEG-SA, BOP, HOBt, and DIPEA in DMF at 25
°C for 2 h. The samples were then rinsed with DMF and
DCM, and dried by a nitrogen stream.
NVOC-Gly-OH
NVOC-Ala-OH
NVOC-Leu-OH
NVOC-Ile-OH
NVOC-Phe-OH
NVOC-Val-OH
NVOC-Pro-OH
NVOC-Trp-OH
NVOC-Met-OH
NVOC-Arg(Pbf)-OH
NVOC-Tyr(tBu)-OH
76
82
88
78
74
83
90
96
64
45
93
NVOC-Ser(tBu)-OH
NVOC-Thr(tBu)-OH
NVOC-Cys(Trt)-OH
NVOC-His(Trt)-OH
NVOC-Gln(Trt)-OH
NVOC-Asn(Trt)-OH
NVOC-Glu(tBu)-OH
NVOC-Asp(tBu)-OH
94
54 (92a)
21
88 (94a)
52 (94a)
25
23 (94a)
94
NVOC-Lys(Boc)-OH 24
NVOC-ε-ACA-OH 91
a Products were acidified with acetic acid before extracting with
EtOAc.
Preparation of Peptide Microarray. UV light was
irradiated for 20 min on the NVOC-protected surface placed
in the peptide array synthesizer. The exposed surface amino
groups were coupled with 5 mM NVOC-amino acid, HATU,
and DIPEA in DMF at 25 °C for 20 min. The slide was
then rinsed with DMF and DCM, and it was dried by a
nitrogen stream. After repeating UV irradiation, coupling of
monomer, and surface washing, the side chain-protecting
groups of each amino acid were removed by Reagent K (85%
TFA, 5% phenol, 5% thioanisol, 2.5% ethandithiol, 2.5%
H2O) at 25 °C for 30 min, and the slide was rinsed and dried.
Binding of Cy3-Conjugated Streptavidin on Peptide
Microarray. The peptide microarray was exposed to the
phosphate buffer solution of Cy3-conjugated streptavidin (1
µg/mL) at 25 °C for 30 min. The glass slide was rinsed with
0.5% (v/v) Tween 20 dissolved in the phosphate buffer and
dried by a nitrogen stream. The protein-patterned images
were observed using a microarray scanner.
Optimization of Peptide Synthesis on Glass Slides. The
reaction chamber was prefilled with 5 mM sulfuric acid/
dioxane to reduce side reactions during the photocleavage
reaction.13 By using MMA, UV light was irradiated on the
specific region of the NVOC-protected glass surface for 20
min, as previously reported.10 Stepwise coupling efficiencies
of the synthesized peptides on the glass slides were calculated
from the ratios of fluorescence intensities of RBITC using
the following procedures.14 After UV light was irradiated
on the NVOC-protected glass surface using MMA, a 5 mM
solution of NVOC-amino acid, HATU, and DIPEA in NMP
were injected and coupled to the exposed free amino groups
on the surfaces for 20 min. The unreacted amino groups were
capped by 10% acetic anhydride/10% pyridine in NMP for
10 min. As the number of on-state micromirrors was reduced
through proper direction of the UV irradiation, the coupling
and capping steps were repeated. Finally, droplets of 5 mM
DIPEA and RBITC/NMP were added to the free amino
groups to quantify stepwise peptide coupling efficiency by
the equation below:15
Results and Discussion
Synthesis of NVOC-Amino Acid. The 6-Nitroveratryl-
oxycarbonyl (NVOC) group is a representative photolabile
protecting group in solid phase peptide synthesis. Twenty
NVOC-protected amino acids and NVOC-ε-aminocaproic
acid were synthesized from NVOC-Cl and the corresponding
amino acids under the conventional Scho¨tten-Baumann
conditions (Scheme 1). After introducing the NVOC-group,
extraction with EtOAc gave good results of final products,
especially for hydrophobic side chain-containing amino acids.
Their yields were 64-96% (Table 3). Some amino acids with
acid-sensitive side chain-protecting groups (e.g., trityl or
t-butyl containing amino acids) were hardly extracted by
EtOAc after acidification by aq. HCl solution. They formed
insoluble materials, which were suspended in solution after
treatment with aq. HCl solution. As a result, the normal
extraction procedure produced poor yields. In particular,
Cys(Trt), Asn(Trt), Glu(tBu), and Lys(Boc) gave yields of
%Yield (step n) ) (In/In-1) × 100
where In ) fluorescence intensity for peptide oligomer of
length n and In-1 ) fluorescence intensity for peptide
oligomer of length n - 1.

Maskless Photolithographic Peptide Synthesis
Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 4 467
a
Scheme 2. Synthesis of NVOC-PEG-SA
a Reagents and conditions: (a) Jeffamine ED-600 (10 equiv), TEA (5 equiv) in DCM, 25 °C, 12 h; (b) NVOC-Cl (2 equiv), DIPEA (2.5 equiv) in DCM,
25 °C, 12 h; (c) 5% (v/v) TFA in DCM, 25 °C, 30 min; (d) succinic anhydride (1.5 equiv), DIPEA (1.5 equiv) in THF, 25 °C, 12 h.
Scheme 3. Coupling of NVOC-Spacers to the Aminated Glass Surfacea
a Reagents and conditions: (a)( i) 5% APTS/chloroform, 45 °C, 2 h, (ii) 5% GPTS/chloroform, 45 °C, 12 h and then 1% (w/v) CHI in 1% AcOH-H2O,
25 °C, 12 h; (b) 5 mM NVOC-ε-ACA-OH or NVOC-PEG-SA, BOP, HOBt, DIPEA in DMF, 25 °C, 12 h.
approximately 20%. As side chain-protecting groups were
acid labile, the strong acid probably caused some side
reaction during the workup. However, by acidifying them
with acetic acid, the products were extracted more easily
than with hydrochloric acid. All yields increased to greater
than 90%.
synthesis efficiency to that of the CHI surface. In addition,
the resulting aminated surfaces were introduced by two types
of spacers, NVOC-ε-ACA-OH and NVOC-PEG-SA. NVOC-
ε-ACA-OH was coupled as a hydrophobic spacer and
NVOC-PEG-SA as a hydrophilic spacer (Scheme 3). The
surfaces were named ACA/APTS and PEG/CHI/GPTS,
respectively.
Synthesis of NVOC-PEG-SA. The 6-nitroveratryloxy-
carbonyl-O,O′-bis-(2-aminopropyl)polypropylene glycol-
block-polyethylene glycol 500-succinic acid (NVOC-PEG-
SA) was synthesized via two steps; monoprotection of
diamino PEG (Jeffamine ED-600) and subsequent succiny-
lation of the remaining amino group (Scheme 2). First,
monoprotection of the diamino group was performed on the
2-chlorotritylchloride (2-CTC) resin. It was reported that
solution phase synthesis produced the monoprotected di-
amino group at less than 50% yield because the isolation
and purification steps were troublesome. However, the
efficiency of monoprotection via the solid phase method
turned out to be higher than the solution phase method when
an excess of diamino PEG was used for attachment to the
2-CTC resin. By virtue of the mild acid cleavage conditions
(1 to 5% TFA in DCM) of 2-CTC resin, monoprotection of
the bifunctional group can be achieved even with an acid
labile Boc group. Excess reagent (Jeffamine ED-600) could
be reused after evaporation of the solvent. After succinyla-
tion, the product was separated by flash column chromatog-
raphy, and the overall yield was 91%.
Two peptide sequences were synthesized to assess the
stepwise coupling efficiency during peptide synthesis on a
glass chip. Two model peptides, GIYWHHY and YIYGSFK,
which are active substrates for tyrosine kinase p60c-src, were
synthesized on the ACA/APTS and PEG/CHI/GPTS glass
surfaces. Figure 4 shows the procedure of coupling efficiency
measurement. UV light was irradiated on a specific surface
region from MMA. The NVOC-amino acid was coupled,
and the remaining free amino groups were acetylated. The
irradiation, coupling, and capping steps were repeated as the
number of on-state micromirrors was reduced through the
proper direction during the UV irradiation step. Finally, after
all the protecting groups were removed, RBITC was coupled
to the remaining amino groups. The resulting surfaces
provided information for the stepwise coupling efficiency,
and the ratios of fluorescence intensities were calculated. In
GIYWHHY synthesis, the coupling efficiency was promi-
nently lowered, particularly in the step where the surface
amino groups were coupled with NVOC-His(Trt) which
possessed a bulky trityl group. On the ACA/APTS glass
surface, the overall coupling yield was 25%, and the average
stepwise coupling yield was 82% (Figure 5 (a)). The PEG/
CHI/GPTS surface gave a higher yield than the ACA/APTS
glass surface (overall coupling yield: 62%, average coupling
yield: 94%) (Figure 5 (b)). Since the amino groups of the
ACA/APTS glass surface were distributed more densely than
Surface Modification and Peptide Coupling Efficiency.
We previously reported that a hydrophilic polymer (e.g.,
chitosan (CHI)) grafted surface reduced nonspecific bind-
ing.12 In this study, CHI was grafted on an epoxy group
modified surface, which was produced by GPTS. The APTS
modified surface was also prepared to compare peptide

468 Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 4
Shin et al.
Figure 4. Method of coupling efficiency measurement.
Figure 5. RBITC-labeled surface for checking coupling efficiency of GIYWHHY: (a) fluorescence image and graph on the ACA/APTS
glass surface, (b) fluorescence image and graph on the PEG/CHI/GPTS glass surface.

Maskless Photolithographic Peptide Synthesis
Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 4 469
Figure 6. RBITC-labeled surface for checking coupling efficiency of YIYGSFK: (a) fluorescence image and graph on the ACA/APTS
glass surface, (b) fluorescence image and graph on the PEG/CHI/GPTS glass surface.
those of PEG/CHI/GPTS, there would have been more steric
hindrance when synthesizing the peptide on the ACA/APTS
glass surface. In YIYGSFK synthesis, the coupling efficiency
was relatively higher than that of GIYWHHY. On the ACA/
APTS glass surface, the overall coupling yield was 59%,
and the average stepwise coupling yield was 93% (Figure 6
(a)). The PEG/CHI/GPTS glass surface also gave a higher
yield than the ACA/APTS glass surface (overall coupling
yield: 84%, average coupling yield: 98%) because of the
same reason as above (Figure 6 (b)). It can be concluded
that peptide synthesis on a glass chip is more favorable on
the PEG/CHI/GPTS glass surface. However, the efficiency
of peptide coupling on a chip was still lower than that of
conventional solid phase peptide synthesis on polymer beads.
The solution of NVOC-amino acid activated by HATU was
unstable for long storage time in the automated synthesis
system. The problem of low coupling efficiency could be
overcome by using more stable NVOC-amino acid pen-
tafluorophenyl esters, which will be reported in the near
future.11
Synthesis of Protein Binding Peptide Microarray. A
peptide microarray was prepared on the glass chip using
NVOC-amino acids. To measure binding affinity between
peptide and protein, streptavidin was chosen as a model
protein to study the binding assay, since HPQ- or HPM-
containing pentapeptides are known to bind streptavidin.16
Thirteen NVOC-amino acids (Ala, Gly, Ile, Met, Gln, Pro,
His, Glu, Val, Asn, Trp, Phe, Arg) were used for synthesizing
peptides on the PEG/CHI/GPTS glass surface. UV light was
irradiated on the NVOC-protected surface with a proper
pattern generated from MMA. Then, NVOC-amino acid was
coupled to the irradiated site by using a coupling agent. UV
irradiation and NVOC-amino acid coupling steps were
repeated until the predesigned peptide sequences were built

470 Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 4
Shin et al.
Figure 7. Synthesis of streptavidin-binding peptide sequence: (a) mask design and NVOC-amino acid coupling, (b) resulting peptide array
and Cy3-streptavidin binding.
Figure 8. Fluorescence analysis of surface-bound Cy3-streptavidin on the peptide microarray; (a) fluorescence image, (b) analysis of
fluorescence intensity.
up (Figure 7 (a)). Eight sequences of HPQ- or HPM-con-
taining pentapeptides were synthesized on the glass chip,
and biotin was coupled as a control. Thereafter, the glass
surface was treated with Reagent K to remove the side chain-
protecting groups from the peptides. After treating the
prepared peptide microarray with Cy3-streptavidin, fluores-
cence intensity was measured in the region of each peptide
(Figure 7 (b)).
> HPQIG.17 The assay result from the peptide chip was the
same as the one from resin beads. As these results show,
the peptide microarray synthesis method in this research is
quite efficient for quantifying the binding activities of various
peptides to a protein, and it is easy to apply other bioassay
systems.
Conclusion
The fluorescence image of streptavidin binding is depicted
in Figure 8. The binding affinity with Cy3-streptavidin was
in the following order: Biotin > IQHPQ > IGHPQ > IHPQG
> > REHPQ > FHPQG . VHPMA ≈ HPQIG ≈ WNHPM.
In our previous study, the binding affinity of streptavidin-
alkaline phosphatase conjugate was spectrophotometrically
quantified on TentaGel resin with four kinds of ligands, and
the order of binding affinity was Biotin > IGHPQ > IHPQG
The maskless photolithographic synthesis method using
micromirror array (MMA) was used on a glass chip. For
the preparation of peptide microarray, an automated peptide
array synthesizer was built in a closed box. A computer
program controlled photolithography, monomer injection, and
washing conditions. For the peptide synthesis on a glass chip,
the ACA/APTS glass surface and the CHI/GPTS glass
surface were prepared, and NVOC-protected 20 amino acids

Maskless Photolithographic Peptide Synthesis
Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 4 471
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and their analogues were synthesized as building blocks using
the photolithographic synthesis method. The coupling ef-
ficiencies of GIYWHHY and YIYGSFK were verified to
determine efficient peptide synthesis conditions on a glass
chip. Peptide synthesis on a glass chip was more favorable
on the PEG/CHI/GPTS surface than on the ACA/APTS
surface. To examine peptide-protein binding profile, HPQ-
or HPM-containing pentapeptides were synthesized on a glass
chip and a Cy3-streptavidin binding assay was performed.
The peptide sequence of IQHPQ showed the highest binding
affinity with Cy3 labeled-streptavidin. From these results,
we have proven that photolithographic peptide synthesis is
an efficient method to quantify the binding activities of
various peptide ligands.
Acknowledgment. This work was supported by the Nano-
Systems Institute-National Core Research Center (NSI-
NCRC) program and Korea Biotech R&D Group of Next-
generation growth engine project of the Ministry of Education,
Science and Technology (F104AB01005-07A0201-00510).
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Supporting Information Available. Screen capture of the
LABVIEW program and characterization of NVOC-amino
acids by 1H NMR. This material is available free of charge
via the Internet at http://pubs.acs.org.
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