This journal is c The Royal Society of Chemistry 2012 Mol. BioSyst., 2012, 8, 879–887 879

Cite this: Mol. BioSyst., 2012, 8, 879–887

A fluorescent amino acid probe to monitor efficiency of peptide

conjugation to glass surfaces for high density microarrays

Yongfeng Zhao,aMichael C. Pirrung

bcand Jiayu Liao*

ac

Received 15th November 2011, Accepted 9th December 2011

DOI: 10.1039/c2mb05471j

Using a fluorescent NBD amino acid, new protease substrates were developed that are attractive

because of the excellent chemical stability and long wavelength of excitation (480 nm) of the

NBD fluorophore. The fluorescent peptides are synthesized by Fmoc solid-phase peptide

synthesis. An example peptide was efficiently immobilized onto a microarray surface using click

chemistry, and its proteolysis was monitored by fluorescence imaging. Excellent site specificity was

achieved for the protease. Fluorescent peptides are also used to monitor the conjugation efficiency

onto a surface using a standard microarray scanner.

Introduction

Proteases are pervasive and essential for cellular function,

via the hydrolysis of their substrates, peptides and proteins.

Numerous physiological processes, such as cell growth and

differentiation, cell–cell communication, and cell death, are

dependent on the actions of proteases. Furthermore, proteases

are involved in diverse diseases, such as cardiovascular disease,

cancer, AIDS, and neurodegenerative diseases. Thus, they are

important targets for diagnosis and drug discovery,1 and

considerable effort has been invested to develop the tools

necessary to study them.

Peptide microarrays are an excellent example of a tool used

to study proteolytic processing. They have been intensively

investigated as reliable and efficient methods for the rapid

analysis of peptide-based biomolecular interactions.2,3 Peptides

conjugated to microarrays allow literally thousands of peptides

to be evaluated in a single assay with minimal quantities of

samples and reagents. The highly scalable nature of these

assays is very valuable in biological studies and especially in

pharmaceutical screening.

In fabricating biosensor microarrays, the immobilization of

biomolecules onto solid surfaces (conjugation) is a key step.

High immobilization efficiency is crucial to the use of surface

biosensors as quantitative tools for bioassays, as is knowledge

of the surface density. Many methods have been developed to

conjugate molecules to surfaces for a variety of biological

assays, diagnostic tests, and high-throughput screening. The

most common methods use reductive amination,4 amide bond

formation,5 Diels–Alder reaction,6Michael addition,7 Staudinger

ligation,8,9 and 1,3-azide–alkyne cycloaddition (click chemistry).10,11

In addition to highly efficient conjugation chemistry, a robust

method for quantitative determination of the ligands immobilized

on a surface is needed. Several strategies have been used to

determine the density of immobilized ligands. The first uses

surface plasmon resonance (SPR) spectroscopy.7,12,13 In this

approach, the surface must be gold or gold-coated to obtain

an SPR signal. A second approach is to conjugate fluorescent

organic molecules, such as Texas Red, to ligand molecules so

that their immobilization can be assessed via fluorescence

imaging. A third strategy uses fluorescent-labeled antibodies

against the ligand to determine the density of conjugation.8,14,15

Recently, the fluorophore 7-amino-4-carbamoylmethyl

coumarin (ACC) was incorporated into protease substrates on

a microarray surface.16 These peptidyl coumarins enable the use

of solid-phase synthesis to prepare fluorogenic substrates for

protease specificity profiling.17 This technology has even been

extended to the assay of other enzyme classes.18 However,

there are several disadvantages limiting applications of these

approaches. First, ACC is incorporated into peptides with an

anilide rather than a peptide bond, which has a different leaving

group potential than a native peptide, and therefore may not be

truly representative of the substrate activity with a particular

protease. Second, because peptidyl coumarins generate fluorescence

upon cleavage at the C-terminus, this method can only be used

to determine P-side (N-terminal) substrate specificity.19 In

addition, ACC is excited at a short wavelength (350 nm).

For fluorescent peptide substrates for microarray applications,

long excitation/emission wavelengths are desired because they

show less interference with the auto-fluorescence of drug

candidates, biological samples, or some array substrates,20,21

aDepartment of Bioengineering and Center for BioengineeringResearch, Bourns College of Engineering, University of California atRiverside, 900 University Avenue, Riverside, CA 92521, USA.E-mail: jiayu.liao@ucr.edu; Fax: +1 951-827-6416;Tel: +1 951-827-6240

bDepartment of Chemistry and UCR Stem Cell Center,University of California at Riverside, 900 University Avenue,Riverside, CA 92521, USA

c Institute for Integrative Genome Biology, University of California atRiverside, 900 University Avenue, Riverside, CA 92521, USA

MolecularBioSystems

Dynamic Article Links

www.rsc.org/molecularbiosystems PAPER

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880 Mol. BioSyst., 2012, 8, 879–887 This journal is c The Royal Society of Chemistry 2012

and they better suit the available fluorescence imaging

equipment.

To address these needs, we sought to utilize the unique

physical and chemical properties of NBD, 7-nitrobenz-2-oxa-1,3-

diazol-4-yl, as a fluorescent marker for immobilized peptides.

Because of the small size of NBD, this fluorophore can be

included in an amino acid that is incorporated into any

position of a peptide via a conventional automated solid-phase

peptide synthesis.22,23 The excitation wavelength of NBD also

fits very well with the commonly used blue laser source. We

used this fluorophore to monitor the efficiency of conjugation

of a peptide onto a glass surface via click chemistry. The ability

to precisely control the surface ligand density is particularly

important for bioassays. Therefore, determining the density of

the immobilized ligand on the surface via the NBD fluorophore

is a key capability to develop valid analytical surfaces. We also

applied a surface-bound peptide incorporating NBD for the

detection of protease activity.

Results

Synthesis of the NBD fluorescent amino acid and the

NBD-containing peptide of a trypsin substrate

Because the fluorescent properties of different NBD amino acids

had not been fully characterized, we examined free and protected

NBD amino acids.22,23 Using basic conditions with commercially

available diaminopropionic acid (Dap) starting materials and

NBD chloride, we synthesized two protected NBD amino acids,

including the Fmoc and Boc versions, and the free amino acid

Dap(NBD) (Fig. 1a). We also prepared an alkyne form of NBD.

The unprotected NBD amino acid was prepared after the Boc

group was removed from Boc–Dap(NBD) (4) with TFA. Yields

of all products were good.

We used the fluorescent amino acid Dap(NBD) in trypsin

substrate peptides that were used in conjugations to glass

surfaces. The protected Dap(NBD) (2) was incorporated into

these peptides via standard Fmoc chemistry. This is a high-

efficiency approach for the preparation of fluorescent peptides

compared to the most common method, where the native

peptide is completely synthesized and then modified with a

fluorescent moiety at a reactive site (e.g. the free amine at the

N-terminus, a side chain amino group or a thiol group).16,24

The problem with the latter method is that several reactive

sites may be simultaneously available on the peptide. Our

approach (Fig. 1b) begins with Fmoc-Gly beads, to which

Fmoc-protected Dap(NBD) is coupled in an automated

peptide synthesizer (CS Bio, Menlo Park, CA). In this method,

the NBD unit can easily be placed in any desired position.

Excitation and emission spectra of the NBD-Cl, NBD

amino acids and NBD-containing peptide

NBD-chloride is non-fluorescent until it is coupled to an

amine group, and then it is strongly fluorescent. The amine-

substituted Dap(NBD) was reported to have two excitation

wavelengths, 340 and 480 nm.22,23 The longer excitation

wavelength would be useful because it avoids much of the

interference from other materials or samples that are often

excited by shorter wavelength light, close to the UV region.

This is particularly important for biochips: the glass slides to

which most biomolecules are conjugated often display auto-

fluorescence when excited by short-wavelength light.

We examined the excitation wavelengths of several different

forms of NBD, including a Dap(NBD)-containing trypsin

substrate peptide, Fmoc-protected Dap(NBD), free Dap(NBD),

a NBD-alkyne, and NBD-chloride (Fig. 2a). In agreement

with the previous report,23 the absorption spectrum of the

H2N–Dap(NBD) shows two excitation wavelength maxima at

340 and 480 nm (Fig. 2b). When excited at 480 nm, the

maximal emission is at 540 nm. Except for NBD-Cl, which

is not fluorescent, all NBD derivatives had very similar

spectra, and in particular the spectrum did not change when

NBD was incorporated into a peptide (Fig. 2c).

Monitoring bioconjugation efficiencies and bioactivities with the

NBD-containing peptide

Our overall strategy for monitoring bioconjugation efficiency

and bioactivity is shown in Fig. 3. We synthesized a bifunctional

peptide with NBD at the C-terminus and an alkyne group at the

N-terminus. The alkyne group can be conjugated onto a glass

surface bearing an azide group using the click reaction;25

surface-bound peptide can be detected by a microarray scanner.

Furthermore, a fluorescent peptide substrate for trypsin,

which can be cleaved at the carboxyl side of the amino acids

Fig. 1 Synthetic routes to NBD amino acids and peptides. (a) The

synthesis of Fmoc-protected Dap(NBD), free Dap(NBD), and NBD

alkyne. (b) Incorporation of a fluorescent Dap(NBD) residue into

protease substrate peptides by solid-phase peptide synthesis (SPPS).

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lysine and arginine, was conjugated to the surface by this

procedure. Accessibility of the peptide on the surface to

enzymatic reactions was demonstrated by its ready cleavage

by trypsin. No cleavage was detected when BSA was used as a

control. Enzyme activity was easily observed by a decrease of

fluorescence on the surface image. Importantly, no blocking

step was needed after peptide conjugation or before protease

digestion. Most likely, the azide group on the glass surface was

inert under most of the reaction conditions.

Optimizing the reaction conditions for bioconjugation

An azide-derivatized surface was prepared by silanization with

a new synthetic reagent we developed that includes a poly-

ethylene glycol (PEG) linker and a terminal azide group.25 We

next sought to optimize the reaction conditions for peptide

conjugation via a click reaction. To determine the optimal

immobilization time, bifunctional peptides with an alkyne group

and a Dap(NBD) at opposite ends were printed (Fig. 3). The

Huisgen click cycloaddition reaction was performed under

humid conditions for 0.5–6.0 h (Fig. 4). The conjugation

solution was phosphate-buffered saline (PBS, pH 8.0) including

1.0 mM peptide, CuSO4 (0.10 mM), sodium ascorbate (2.0 mM),

and glycerol (10% v/v, to impede evaporation). Slides incubated

for 30 min show high-intensity fluorescence (Fig. 4a and b).

Some studies have suggested that conjugation efficiency can be

improved by prolonging the incubation,10 but we found 30 min

to be optimal for peptide conjugation. In fact, the amount of

peptide conjugated decreases slightly when longer incubations

are used. The copper and sodium ascorbate might adversely

affect the NBD peptides.

We also examined the effects of pH and the concentration of

copper and sodium ascorbate on the immobilization. We

varied the pH from 6.0 to 9.0, keeping the incubation time

fixed at 30 min. We found pHs of 7 and 8 ideal for efficient

immobilization (Fig. 4b and c). This pH range is also optimal

for preserving the biological activities of most peptides. We

examined the effect of copper concentrations of 0.02–0.5 mMwith

20 times the concentration of sodium ascorbate. The fluorescent

intensity representing the immobilized NBD-containing peptide

increases as the copper concentration increases. The highest

fluorescence intensity of conjugated peptides is observed at

0.5 mM copper (Fig. 4c and d).

Determination of protease substrate specificity

Once we had optimized the conjugation protocol, we needed

to ensure that the biological activities of the conjugated

molecules are maintained for bioassays. To determine the

assay sensitivity of the NBD-containing fluorescent peptide

in the microarray format, we used it to determine the

specificity of protease substrates. Three peptides were synthesized

(Fig. 5a), A and C being trypsin substrates, and B containing

the SUMO-specific protease 1 substrate.26,27 Their sequences

are:

peptide A: G7-Gly-Pro-Ala-Arg-Leu-Ile-Gly-Dap(NBD)-Gly

peptide B: G7-Ala-Gln-Thr-Gly-Gly-Ala-Dap(NBD)-Gly

peptide C: G7-Gly-Pro-Ala-Lys-Leu-Ala-Ile-Gly-Dap(NBD)-Gly

where G7 represents a propargylamine succinamide linker.

Fig. 2 Fluorescence excitation and emission spectra of NBD derivatives. (a) NBD derivatives. a, peptide (trypsin substrate); b, Dap(NBD);

c, Fmoc-protected Dap(NBD); d, NBD alkyne; e, NBD-Cl. (b) Excitation spectra of NBD derivatives. (c) Emission spectra of NBD derivatives.

Fig. 3 Strategy to monitor conjugation efficiency and activity of

Dap(NBD)-containing peptides on a glass surface.

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These three peptides were then conjugated onto a glass

surface by the procedure described above. After being subjected

to trypsin digestion in situ, peptides A and C show significant

decreases in fluorescent intensity, but peptide B shows no

obvious decrease in fluorescent intensity (Fig. 5b). Differences

in the decrease of fluorescent intensity were observed between

the Arg- and Lys-containing trypsin substrates (Fig. 5c). This

result is consistent with previous studies showing that arginine

peptides are cleaved more efficiently by trypsin than lysine

peptides.28 The results suggest that NBD-containing peptides

can be used for protease substrate specificity testing and also for

quantitative digestion determination on glass surfaces.

High-density peptide arrays conjugated by click chemistry

Traditional biochemical assays have provided invaluable

knowledge about activities of enzymes, such as proteases, in

diverse organisms. However, those assays require relatively

large amounts of material for each analysis and can generally

only be performed one-at-a-time. Microarray platforms

function in a highly parallel format and require only small

amounts of material. The development of peptide substrate

microarrays described here could provide novel solutions

amenable to systems biology and systematic disease diagnosis.

Fabricating high density protein or peptide arrays is

challenging.8,17,29,30 Using our NBD-containing fluorescent

peptide, we printed about 4000 individual spots on a glass slide

with a standard microarray spotter (Fig. 6). With a delivered

solution volume of 1 nL, the spot size was ca. 200 mm in

diameter, and the distance between spots was 500 mm. The

fluorescent peptide array is ready for evaluation immediately

following fabrication. This provides an important improvement

over other approaches because the conjugation efficiency for

each spot can be monitored by its fluorescence density, and

therefore, amount of conjugated peptides/proteins may be

easily determined before or after biological reactions. Our

results show the feasibility of this system for high-throughput

screening of the sequence selectivity of proteases, limited only

by the numbers of synthetic peptides available.

Fig. 4 Optimization of click chemistry-based conjugation conditions using the Dap(NBD)-containing peptide. (a) Time course of the conjugation

reaction. (b) Quantitation of conjugated peptide in (a). (c) Conjugation efficiencies at different pH. (d) Quantitation of conjugated peptide in (a).

(e) Conjugation efficiencies at different Cu2+ concentrations. (f) Quantitation of conjugated peptide in (e). The standard deviation is based on

twelve replicate data measurements.

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Dap(NBD) can be used as an imaging tool to compare

conjugation methods

The efficiency of conjugation is a critical factor in making

microarrays. Various chemistries have been developed to bind

biomolecules to solid surfaces. We compared the efficiencies of

conjugations with click chemistry and two traditional methods,

amide bond conjugation and hydrazide conjugation.31 Each

peptide was prepared with a functional group appropriate

for the conjugation chemistry at its N-terminus. Their

sequences are:

peptide A: G7-Gly-Pro-Ala-Arg-Leu-Ile-Gly-Dap(NBD)-Gly

peptide D: Leu-Gly-Pro-Ala-Arg-Leu-Ile-Gly-Dap(NBD)-Gly

peptide E: keto-Gly-Pro-Ala-Arg-Leu-Ile-Gly-Dap(NBD)-Gly

Three different surfaces and conjugation procedures were

compared (see Materials and Methods). After washing, the

slides were scanned, and the fluorescent intensities of the

conjugated peptides were evaluated. They are shown with

different threshold settings of the fluorescent scanner

(Fig. 7). At the filter threshold setting of zero, fluorescent

signals were detected in all three conjugation methods. The

fluorescent image of peptides conjugated by click chemistry

showed a solid spot, while those prepared by the other

methods showed circular shapes. When the filter threshold

setting was increased to 280, the fluorescent image of amine-

conjugated peptides lost most of its signal, and the keto-

conjugated peptides lost signal completely. However, the click

chemistry-conjugated peptide still had a strong signal. When

the filter threshold setting was increased to 520, only the click

chemistry-conjugated peptide still gave strong fluorescent

signals. Clearly, the conjugation by click chemistry had the

highest fluorescent signals.

Discussion

Here we report a powerful strategy for monitoring the efficiency

of immobilizing a fluorescent peptide onto glass surfaces and for

conducting protease assays with high-throughput microarray

technology. The key element of this technology is the NBD

fluorescent amino acid. With its small size, it can be incorporated

into a peptide at any position by automated solid-phase peptide

synthesis, and it serves as a marker for monitoring conjugation

densities and for bioassays on solid surfaces. Both non-specific

and site-specific immobilizations of biomolecules have been

performed on solid surfaces.32

Several chemistries have been used previously for assays on

microarrays. Determination of the density of conjugatedmolecules

has depended mainly on SPR technology or fluorescent-labeled

large biomolecules.7,8,12–15 However, to obtain SPR signals, the

surface must be gold or coated with gold, an expensive and time-

consuming process. For fluorescence analysis of density, the signal

comes from a fluorescent-labeled antibody (e.g. Cy5)8,14,15 against

the immobilized biomolecules or bound molecules, or fluorescent

proteins (e.g. GFP),33 or fluorescent dye-labeled conjugates

(e.g. Texas Red),34 most of which are attached to assay probes

after synthesis. In another method, the fluorogenic molecule

7-amino-4-carbamoylmethyl coumarin was incorporated into

protease substrates at their C-terminus.17 However, specialized

solid-phase synthesis beads are needed for the peptide synthesis.

Furthermore, in this approach, the fluorogenic 7-amino-

4-carbamoylmethyl coumarin must be at the C-terminus

Fig. 5 Determination of protease substrate specificity using NBD-

containing fluorescent peptides. (a) Trypsin substrate and control peptides.

A: trypsin substrate peptide 1; B: SENPprotease substrate peptide; C: trypsin

substrate peptide 2. (b) Cleavage of surface-conjugated peptide substrates by

trypsin. (c) Quantitation of peptide fluorescence after trypsin cleavage. The

standard deviation is based on six replicate data measurements.

Fig. 6 High-density peptide array conjugated using click chemistry.

A total of 3960 spots of NBD-containing peptide was generated on a

glass slide. The spot pitch is 500 mm.

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of substrate. The fluorescent peptide array can only be used

for substrate profiling at the P positions (N-terminus), but not

the P0 positions (C-terminus). All of these previous methods

require extra steps to label conjugated molecules or detecting

molecules, and they are often hindered by inefficient labeling

and the multiple steps involved. Therefore, these methods are

difficult to develop into reliable tools.

In contrast, our fluorescent amino acid-containing peptide

allows the direct monitoring of the process of immobilization

by 1,3-dipolar cycloaddition of azides with an alkyne (click

chemistry). Conventional solid-phase synthesis provides 100%

incorporation of the fluorescent amino acid label into the

target peptide, which is readily purified following normal

procedures. Using trypsin substrate peptides flanked by an

alkyne and Dap(NBD), optimal conditions for click chemistry

conjugation of the peptide onto the azide-derivatized glass

surface were elucidated (Fig. 4). With this method, we achieved

a very high density of peptides, 1.3 � 1014 peptides cm�2 on

the glass surface.25 The peptide density is comparable to

those of DNA chips and is one of the highest reported

densities for peptide or protein chips.35 Accessibility and

bioactivity of the peptides immobilized on the glass surface

were demonstrated by their selective cleavage with trypsin

(Fig. 5 and 7).

Click chemistry has been applied for conjugations of

various biomolecules onto surfaces, such as proteins10,36 and

carbohydrates.37 However, the conjugation efficiencies have

not been carefully studied. In addition, peptide ligands

containing fluorescent amino acids, which are promising tools

for bioassays and diagnostics, have not been conjugated onto

glass surfaces at high efficiency. We have accomplished both of

these tasks in this work.

In addition, we demonstrated the advantages of site-specific

immobilization of peptides using an alkyne group, which can

be easily incorporated into any peptide using solid-phase

synthesis. When the peptide was conjugated to the surface in

this manner, the active site of peptide was preserved; thus, its

bioactivity is maximal, which cannot be assured with random

amide bond formation. Our site-specific conjugation of

fluorogenic substrates to glass surfaces should permit continuous

kinetic analysis of protease activity and should be useful for

screening potential protease inhibitors.24,38 These substrates

can be used to determine substrate specificity, to provide

valuable information about biological function, and also to

help in the design of potent and selective substrates and

inhibitors.39

Long excitation and emission wavelengths were a high

priority for this microarray application since long wavelengths

are less compromised by the auto-fluorescence of drug candidates,

biological samples and some array substrates.20 Among

numerous available fluorophores, NBD chloride reacts with

amines to form highly stable fluorescent derivatives.22

Furthermore, the NBD group has a compact structure and

shows a long excitation and emission wavelength.23 The

excitation wavelength fits well with commonly used sources.

Our strategy is applicable to other methods that involve

peptide ligands immobilized on a solid surface. Given the

straightforward method of this site-specific modification

approach which relies on the robust click chemistry and

fluorescent amino acid, our procedure should be well-suited to

prepare peptide and protein microarrays using well-developed

DNA array facilities. This approach should make it possible to

fabricate peptide microarrays for quantitative research and

biomedical applications.

Fig. 7 Comparison of the efficiency of different surface conjugation methods. (a) Peptide substrates used for different conjugation chemistries. All

peptides have the same trypsin substrate sequence and differ only in the functional group at the N-terminus. Click chemistry: G7-Gly-Pro-Ala-Arg-

Leu-Ile-Gly-NBD-Gly. G7 contains an alkyne. NHS slide: Leu-Gly-Pro-Ala-Arg-Leu-Ile-Gly-NBD-Gly. Leu contains an amine group. Hydrazide

slide: Keto-Gly-Pro-Ala-Arg-Leu-Ile-Gly-NBD-Gly. Keto is keto amino acid containing a keto group. (b) Fluorescence images of surface-conjugated

peptides at different threshold settings of the scanner.

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Methods

Materials

Tetrahydrofuran (THF), CH2Cl2 and toluene were dried before

use. All other reagents and solvents were obtained from Aldrich

and used without any further purification unless stated

otherwise. Fmoc-L-4-acetylphenylalanine (keto amino acid) was

purchased from 3B Scientific Corporation (Libertyville, IL).

Reactions were routinely carried out under an atmosphere of

dry argon with magnetic stirring. Flash chromatography was

generally performed on silica gel. The yield was calculated after

isolation. Thin-layer chromatography (TLC) was performed on

EMD silica gel 60 F254 precoated plates of 250 mm thickness.

The spots were visualized by UV light or by staining in a jar of

iodine. 1H NMR spectra were recorded on Varian VRX 300 and

400 spectrometers, and chemical shifts were expressed in parts

per million (d) relative to internal CDCl3. Bovine serum albumin

(BSA) and trypsin were purchased from Sigma. The solutions of

peptides were printed on the surface at a spacing of 500–1000 mmwith an OmniGrid Accent Microarrayer from GeneMachines

(San Carlos, CA). Cover slips were purchased from Thermo

Fisher Scientific (Portsmouth, NH). Fluorescence intensities of

peptides on the microarray were obtained with a ScanArray

Express from PerkinElmer Life Sciences (Boston, MA).

Quantitation of imaging was performed by software included with

the machine. The excitation and emission wavelengths employed

for detecting peptide were lex = 488 nm and lem = 530 nm.

Fmoc–Dap(NBD)–OH (2)

To a stirred solution of Na-Fmoc-L-diaminopropionic acid

(1, 0.500 g, 1.53 mmol) in water/acetonitrile (20 mL of a 1 : 1

mixture) was added 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole

(NBD-Cl, 0.367 g, 1.836 mmol) and sodium bicarbonate

(0.154 g, 1.83 mmol). The reaction mixture was stirred for 6 h

at RT. Then, water (10 mL) was added, the mixture was washed

with ethyl acetate/hexanes (1 : 5) twice. The aqueous phase was

acidified by 1 M HCl until a pH of 5–6 was achieved. The

product was extracted from the aqueous solution with ethyl

acetate. The combined organic phases were dried over

anhydrous sodium sulfate, filtered, and the solvent was removed

by rotary evaporation. Rf = 0.37 (CH2Cl2 : MeOH = 10 : 2).1H NMR (300 MHz, DMSO-d6): d 3.80 (br s, 2H), 4.15

(t, J = 6.8 Hz, 1H), 4.28 (d, J = 6.9 Hz, 2H), 4.37 (m, 1H),

6.48 (d, J=8.7 Hz, 1H), 7.24 (t, J=7.2 Hz, 2H), 7.35 (m, 2H),

7.60 (m, 2H), 7.63 (d, J = 7.8 Hz, 1H), 7.85 (d, J = 7.5 Hz,

2H), 8.48 (d, J=9.0 Hz, 1H). This compound is known and the

NMR data match the published data.23 IR (neat): 3362, 1705,

1622, 1576, 1495, 1447, 1296, 1127, 738 cm�1. HRMS (ESI+):m/z

calcd for C24H19N5O7 [M + H]+ 490.1363, found 490.1345.

Boc–Dap(NBD)–OH (4)

To a stirred solution of Na-Boc-L-diaminopropionic acid

(3, 0.50 g, 2.45 mmol) in water/acetonitrile (14 mL of a 1 : 1

mixture) was added NBD-Cl (0.7 g, 3.5 mmol) and sodium

bicarbonate (0.25 g, 3.0 mmol). The reaction mixture was stirred

for 6 h at RT. Then, water (10 mL) was added, the mixture was

washed with ethyl acetate/hexanes (1 : 5) twice. The aqueous

phase was acidified by 1 M HCl until a pH of 5–6 was achieved.

The product was extracted from the aqueous solution with ethyl

acetate. The combined organic phases were dried over anhydrous

sodium sulfate, filtered, and the solvent was removed by rotary

evaporation. The crude residue was purified by flash chromato-

graphy on silica gel (CH2Cl2 : MeOH= 10 : 2) to give a brown

solid (0.11 g, 61%). Rf = 0.24 (CH2Cl2 : MeOH = 10 : 2).1H NMR (300 MHz, DMSO-d6): d 1.39 (s, 9H), 2.70 (m, 1H),

3.00 (dd, J = 4.8, 11.7 Hz, 1H), 3.15 (br s, 2H), 3.59 (m, 1H),

6.15 (m, 1H), 8.20 (br s, 1H). IR (neat): 3316, 1687, 1620, 1578,

1495, 1294, 1243, 1157, 1021, 998 cm�1. [a]25D = +33.3 (c 1.0,

MeOH/CHCl3 = 1 : 10). HRMS (ESI+): m/z calcd for

C14H18N5O7: 390.1026 [M + Na]+, found 390.1046.

H2N–Dap(NBD)–OH (5)

Boc–Dap(NBD)–OH (4, 0.30 g, 0.82 mmol) was dissolved in

dichloromethane (4 mL) and TFA (1.5 mL) was added slowly

drop-wise at 0 1C. The reaction mixture was stirred at RT for

2.5 h. The solvent was removed under vacuum to afford the

title compound, 3-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-

L-alanine, as a brown solid (0.21 g, 0.74 mmol, 90.2%).1H NMR (300 MHz, DMSO-d6): d 4.01 (m, 2H), 4.30

(m, 1H), 6.63 (d, J = 8.7 Hz, 1H), 8.70 (br s, 2H). HRMS

(ESI+): m/z calcd. for C9H10N5O5 [M+H]+ 268.0681, found

268.0676.

7-Nitro-N-(prop-2-ynyl)benzo[c][1,2,5]oxadiazol-4-amine (6)

To a stirred solution of propargylamine hydrochloride

(0.092 g, 1 mmol) in the mixture solvent of THF and ethanol

(2 mL, 1 : 1), sodium bicarbonate (0.336 g, 4 mmol) and

4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (0.2 g, 1 mmol) were

added sequentially. After the mixture was stirred at RT for

12 h, the solvent was removed. The remaining solid was

partitioned between water and dichloromethane. The organic

phase was dried over sodium sulfate, filtered, and the solvent

removed by rotary evaporation. The crude residue was purified

by flash chromatography on silica gel (hexanes : ethyl

acetate = 2 : 1) to give the title compound as a brown solid

(0.12 g, 0.55 mmol, 55%). Rf = 0.4 (hexanes : ethyl acetate =

2 : 1). 1H NMR (400 MHz, CDCl3): d 2.44 (t, J = 2.8 Hz, 1H),

4.31 (q, J=2.4 Hz, 2H), 6.30 (br s, 1H), 6.35 (d, J=6.4 Hz, 1H),

8.54 (d, J = 6.4 Hz, 1H). IR (neat): 3360, 3321, 3238, 2120,

1620, 1574, 1488, 1290, 1276, 1116, 1705, 1622, 1576, 1495,

1447, 1296, 1127, 738, 679 cm�1. Mp: 140.5–142.0 1C. HRMS

(ESI+): m/z calcd. for C9H6N4O3 [M + H]+ 219.0513, found

219.0519.

Peptides were synthesized by CS Bio Co. (Menlo Park, CA).

The peptide sequences in this work are tabulated below.

G7 represents the linker with a terminal alkyne group, and

Dap(NBD) represents diaminopropionic acid with the NBD

(7-nitrobenz-2-oxa-1,3-diazole) fluorophore.

Sequence Peptide

G7-Gly-Pro-Ala-Arg-Leu-Ile-Gly-Dap(NBD)-Gly A

G7-Ala-Gln-Thr-Gly-Gly-Ala-Dap(NBD)-Gly B

G7-Gly-Pro-Ala-Lys-Leu-Ala-Ile-Gly-Dap(NBD)-Gly C

NH2-Gly-Pro-Ala-Lys-Leu-Ala-Ile-Gly-Dap(NBD)-Gly D

NH2-Leu-Gly-Pro-Ala-Arg-Leu-Ile-Gly-Dap(NBD)-Gly E

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886 Mol. BioSyst., 2012, 8, 879–887 This journal is c The Royal Society of Chemistry 2012

Measurement of fluorescent properties of Dap(NBD) and

derivatives

The spectra of Dap(NBD) and its derivatives were measured

on a FlexStation 3 (Molecular Devices, Sunnyvale, CA). All

Dap(NBD) derivatives and peptides were dissolved in aceto-

nitrile at a concentration of 5 mmol. The excitation spectrum

was obtained by measuring the emission at 540 nm. The

emission spectrum was obtained by exciting at 480 nm.

Functionalization and peptide immobilization on glass surfaces

by 1,3-azide–alkyne cycloaddition

Step 1. Preparation of the surface: glass slides were immersed in

piranha solution (5 : 1 H2SO4 : H2O2) overnight and rinsed

with deionized water, absolute ethanol, THF and toluene.

(Caution: piranha solution is a strong oxidizing reagent and

should be handled with extreme care.) The slides were dried

under argon.

Step 2. Silanization: a solution of the azido-PEG–triethoxy-

silane in toluene was filtered with a PTFE filter (Fisherbrand,

0.45 mm). Glass slides were immersed in a 10 mM toluene

solution of this silane overnight at RT. The slides were washed

with toluene, ethanol, THF, deionized water, THF, ethanol,

and toluene. The slides were cured under vacuum at 80 1C

for 3 h.

Step 3. Conjugation of peptide on an azide-derivatized glass

surface: conjugation solutions consisted of phosphate buffered

saline (PBS, pH 8.0) including peptide (1.0 mM), CuSO4

(0.10 mM), and sodium ascorbate (2.0 mM). Glycerol (10%, v/v)

was added to the solutions to minimize evaporation. After

spotting, slides were incubated under humid conditions for

0.5 h. They were washed successively with copious amounts of

deionized water, 0.5% Triton X-100 solution in PBS buffer

(pH 7.5), and water. The slides were finally dried with argon.

Preparation of a NHS-derivatized glass surface and peptide

conjugation

The glass slides were cleaned in piranha solution overnight as

described above and rinsed with deionized water, absolute

ethanol, THF and toluene. (Caution: piranha solution is a

strong oxidizing reagent, and should be handled with extreme

care.) Slides were dried under argon. Slides were immersed

in a solution of 3-aminopropyltriethoxysilane (3 mL) in toluene

(40 mL) at RT for 7 h. Slides were washed with toluene, ethanol,

THF, deionized water, THF, ethanol, and toluene. Slides were

cured under vacuum at 90 1C for 2 h. The amine-coated glass

slides were shaken in a solution of succinic anhydride (3%, w/v)

in DMF for 7 h. Slides were washed with DMF and THF and

were immersed in a solution of diisopropylcarbodiimide (DIC,

3%, v/v) and N-hydroxysuccinimide (NHS, 3%, w/v) in DMF

for 12 h. After being washed with DMF and THF, the slides

were dried under argon.

Peptides were dissolved in phosphate buffered saline

(PBS, pH 7.5) containing glycerol (10%, v/v). The peptides

were spotted on NHS-derivatized slides by an OmniGrid Accent

Microarrayer (San Carlos, CA). Following 3 h incubation under

moisture conditions at RT, slides were washed with copious

amounts of water and immersed in a solution of PBS (pH 8.0)

containing 600 mM ethanolamine. After shaking for 1 h at RT,

the slides were washed with copious amounts of deionized water,

0.5% Triton X-100 solution in PBS buffer (pH 7.5), and water.

The slides were finally dried under argon.

Preparation of a hydrazide derivatized glass surface and peptide

conjugation

NHS activated slides were immersed into a solution of hydrazine

monohydrate (3%) in DMF with gentle shaking for 5 h. Then

the slide was washed with DMF and dried with flux of argon.

Peptides were dissolved in phosphate buffered saline (PBS,

pH 5.0) containing glycerol (10%, v/v). The peptides were

spotted on hydrazide-derivatized slides by an OmniGrid

Accent Microarrayer (San Carlos, CA, USA). Following

18 h incubation with humidity at room temperature, the slides

were washed with copious amounts of water. The slides were

finally dried with argon.

Cleavage of peptides by protease

The peptide array area was covered with a cover slip. Hydrolysis

reactions were initiated by pipetting either trypsin solution or a

control solution between the slide and the cover slip. The trypsin

solution consisted of 0.5 mg mL�1 trypsin in Tris saline (20 mM

Tris-HCl, 50 mM NaCl, pH 7.5). The control solution was Tris

saline or BSA (0.5 mg mL�1) in Tris saline. After allowing

reactions to proceed at RT for 3 h, they were terminated by

washing the slides with deionized water. A fluorescence intensity

ratio (FIR) was obtained from the ratio of fluorescence observed

after trypsin digestion to that observed before digestion.

Competing financial interests

The authors declare no competing financial interests.

Acknowledgements

We would like to thank Glenn Hicks in the Institute of Integrative

Genome Biology at UCR for his valuable assistance with the

fluorescence scanner. This work was partially supported by the

NIH (grant # AI076504) and the Center of Bioengineering

at UCR.

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