Bioconjugate Chem. 2001, 12, 139−142
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A Chemoselective Method for Site-Specific Immobilization of Peptides via Aminooxy Group Maciej Adamczyk,* John C. Gebler, Rajarathnam E. Reddy, and Zhiguang Yu Department of Chemistry, 9NM, Building AP20, Diagnostics Division, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, Illinois 60064-6016. Received October 16, 2000
Site-specific modification of peptides and proteins is an important area of basic research for preparation of well-defined biosensors and probes. The unique properties of aminooxy group present an opportunity for chemoselective site-specific immobilization of peptides to prepare well-defined biosensors. We have prepared FLAG peptide derivatives containing L--aminooxylysine (L--AOLys, 1a) and L-lysine units in their sequence at the C- and N-terminals via solid-phase synthesis. Site-specific modification of peptides through aminooxy group was demonstrated in the preparation of biosensors and selective conjugation in the preparation of biotinylated probes. Effect of the incorporation of L--AOLys (1a) into the peptide sequence and its subsequent labeling on the FLAG epitopic character was measured using a surface plasmon resonance detector. It was found that incorporation of L--AOLys (1a) into the FLAG peptide and site-specific immobilization through aminooxy group preserved the integrity of FLAG epitope.
INTRODUCTION
Recent advances in solid-phase chemistry and molecular biology have spurred the construction of larger peptides and small proteins through stepwise synthesis using automated instruments. A variety of biophysical probes have been produced from peptides and proteins with carefully engineered properties for applications in medicinal and biotechnology fields (1-8). In contrast to the synthesis of low molecular weight natural products, which is aided by plethora of specific reactions and protective groups, site-specific modification of unprotected peptides and proteins to prepare well-defined bioconjugates is challenging. The complexity becomes even more apparent in the preparation of solid supported probes and biosensors, for example, in applications using surface plasmon resonance technology (6, 9). The observed binding properties of a peptide or protein in solution could be compromised upon immobilization depending on its site of attachment to the solid support in a biosensor. A key requirement for site-specific conjugation is the chemoselectivity. It is desired that only the intended site in a peptide or protein be modified in the presence of other functional groups and thus leading to the formation of a well-defined bioconjugate. It is critical to use such conjugates for implementation of quality control (10, 11) and reliable interpretation of biological results. We and others have been exploring the utility of aminooxy group for a variety of applications such as detection of abasic sites in DNA (12, 13), development of reagents for immunoassays (14, 15), preparation of artificial proteins (16-20) and peptide dendrimers (21), and selective fluorescent labeling of N-methylaminooxy peptides (22). The unique properties of aminooxy group suggest that peptides incorporated with an unnatural amino acid bearing this functionality * To whom correspondence should be addressed. Phone: (847) 937-0225. Fax: (847) 938-8927. E-mail: maciej.adamczyk@ abbott.com.
could be chemoselectively conjugated to a solid support of interest (Figure 1) to provide well-defined biophysical probes. In this paper, we describe an efficient method for chemoselective site-specific immobilization of peptides 12 and 14 via primary aminooxy group (-ONH2) incorporated as an L--aminooxylysine residue (L--AOLys, 1a) (23) to a carboxymethyl dextran chip to form a biosensors. Binding properties of these peptides 12 and 14 and a biotinylated conjugate 16 to anti-FLAG MAb using surface plasmon resonance (BIAcore) are presented. RESULTS AND DISCUSSION
Before attempting the site-specific immobilization of peptides through the aminooxy group, competitive reactivity of aminooxy and amino groups in model amino acid derivatives 6 and 7 at different pH values was studied. Thus, a mixture of 6 (1.0 M equiv) and 7 (1.0 M equiv) (Scheme 1) was treated with biotin succinimidyl ester 8 (1.0 M equiv) in DMSO and sodium phosphate buffer at pH 2.0, 3.0, 3.8, 5.0, 6.0, 7.0, 8.2, and 10.3. Any difference in the reactivity of 6 and 7 in the biotinylation to form 9 or 10 under identical conditions must arise from aminooxy or amine groups. As expected (Figure 2), 7 was found to be unreactive at a pH below 6.0, however, 6 was reactive in acidic buffers down to pH 3.0. When the pH of medium was decreased to 2.0, the rate of conjugation of 6 dropped dramatically, presumably due to the protonation of the aminooxy group or hydrolysis of 8. Under more basic conditions (pH >6.0), compound 7 competed with 6 for the active ester 8. When the pH of the medium increased to 10.3, the amine 7 reacted completely and the aminooxy compound 6 remain unmodified. The competitive reactivity of aminooxy and amine groups was then studied by immobilization of compounds 6 and 7 on a carboxymethyl dextran chip and quantitated by streptavidin binding using surface plasmon resonance (SPR) (6, 9, 24). Thus, 6 and 7 were immobilized (2.0 mM, 150 µL, at pH 3.8) onto separate channels of the preactivated chip surface (35 µL of 0.25 M NHS/EDAC in water over 7 min
10.1021/bc0001239 CCC: $20.00 © 2001 American Chemical Society Published on Web 02/28/2001
140 Bioconjugate Chem., Vol. 12, No. 2, 2001
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Figure 2. Competitive reaction of compounds 6 (dark circle) and 7 (normal circle) with succinimidyl ester 8 in DMSO, sodium phosphate buffer (4:96 ratio), 23 °C, 16 h at different pHs (2.0, 3.0, 3.8, 5.0, 6.0, 7.0, 8.2, and 10.3) as shown in Scheme 1. Scheme 2: Solid-Phase Synthesis of Peptides with Incorporation of L-E-AOLys (12, 14) and L-Lys (13, 15) in Their Sequence, and the Site-Specific Conjugation of Peptide 14 and 15 with Active Ester 8a
Figure 1. General strategy for construction of peptides containing L--AOlys (1a) residue in their sequence via phase synthesis and preparation of biophysical probes by site-specific immobilization through aminooxy (-NH2) group. Scheme 1. Biotinylation of 6 and 7 at Different pH Valuesa
a Reagents and conditions: (a) biotinamidocaproate-N-hydroxysuccinimide ester (8, 1.0 M equiv), DMSO, sodium phosphate buffer (4:96 ratio, v/v), 23 °C, 16 h at 2.0, 3.0, 3.8, 5.0, 6.0, 7.0, 8.2, 10.3 pH.
at 5.0 µL/min). Efficiency of the immobilization process was determined by passing a solution of streptavidin (10 µg/mL in HBS buffer) over both the channels. The signal from the channel immobilized with 6 was 2500 RU, while the signal for channel with 7 was 20 RU (
