Synthesis of Alkyne-Terminated PCDA Linker for Applying Click Chemistry on PDA Layers

Abstract

An alkyne-terminated PCDA (10,12-pentacosadiynoic acid) linker, which can undergo a simple click reaction with azide compounds, was synthesized for the purpose of immobilizing various kinds of biomolecular surface ligands on PDA (polydiacetylene) layers. In order to confirm the ability of the linker compound to anchor azide-modified surface ligands, we carried out a model ­reaction with benzyl azide under typical click-reaction conditions. We were able to confirm that the model reaction proceeded smoothly by observing the color change of PDA, including its thermochromic reversibility.

alkynes - azides - solid-phase synthesis - conjugation - self-assembly

References and Notes

<A NAME="RU08509ST-1">1</A> Cheng Q. Stevens RC. Adv. Mater.  1997,  9:  481 

<A NAME="RU08509ST-2">2</A> Cheng Q. Stevens RC. Langmuir  1998,  14:  1974 

<A NAME="RU08509ST-3">3</A> Jelinek R. Drug Dev. Res.  2000,  50:  497 

<A NAME="RU08509ST-4">4</A> Reichert A. Nagy JO. Spevak W. Charych D. J. Am. Chem. Soc.  1995,  117:  829 

<A NAME="RU08509ST-5">5</A> Pan JJ. Charych D. Langmuir  1997,  13:  1365 

<A NAME="RU08509ST-6">6</A> Ma Z. Li J. Liu M. Cao J. Zou Z. Tu J. Jiang L.
J. Am. Chem. Soc.  1998,  120:  12678 

<A NAME="RU08509ST-7">7</A> Wang C. Ma Z. Anal. Bioanal. Chem.  2005,  382:  1708 

<A NAME="RU08509ST-8">8</A> Gill I. Ballesteros A. Angew. Chem. Int. Ed.  2003,  42:  3264 

<A NAME="RU08509ST-9">9</A> Kolusheva S. Kafri R. Katz M. Jelinek R. J. Am. Chem. Soc.  2001,  123:  417 

<A NAME="RU08509ST-10">10</A> Lee SW. Kang CD. Yang DH. Lee JS. Kim JM. Ahn DJ. Sim S. J. Adv. Funct. Mater.  2007,  17:  2038 

<A NAME="RU08509ST-11">11</A> Schlossbauer A. Schaffert D. Kecht J. Wagner E. Bein T. J. Am. Chem. Soc.  2008,  130:  12558 

<A NAME="RU08509ST-12">12</A> Kaiser E. Colescott RL. Bossinger CD. Cook PI. Anal. Biochem.  1970,  34:  595 

<A NAME="RU08509ST-13A">13a</A> Shim HY. Lee SH. Ahn DJ. Kim JM. Mater. Sci. Eng. C  2004,  24:  157 

<A NAME="RU08509ST-13B">13b</A> Kim JM. Ji EK. Woo SM. Lee H. Ahn DJ. Adv. Mater.  2003,  15:  1118 

<A NAME="RU08509ST-13C">13c</A> Jonas U. Shah K. Norvez S. Charych DH. J. Am. Chem. Soc.  1999,  121:  4580 

<A NAME="RU08509ST-14">14</A> Kim J.-M. Lee J.-S. Choi H. Sohn D. Ahn DJ. Macromolecules  2005,  38:  9366 

Preparation of an Alkyne-Terminated PCDA Linker

<A NAME="RU08509ST-15A">15a</A>

To 1 g of pretreated 2-CTC resins (1.3 mmol/g of Cl, 100-200 mesh) with 10 mL of CH₂Cl₂, A (1.9 mL, 10 equiv based on the amount of Cl) and Et₃N (3.6 mL, 20 equiv based on the amount of Cl) were added. The reaction mixture was shaken for 3 h at r.t. After that, the resins were filtered and washed with CH₂Cl₂ and MeOH (5 times each), and dried in a vacuum for 12 h. The loading level of amino groups was determined to be 0.76 mmol/g by Fmoc titration.

<A NAME="RU08509ST-15B">15b</A>

Then, 300 mg of B (0.76 mmol/g of amino groups) in DMF (2 mL, for HBTU) and CH₂Cl₂ (2 mL, for PCDA), HBTU (260 mg, 3 equiv), DIEA (93 µL, 3 equiv), and PCDA (256 mg, 3 equiv) were added one by one; the equivalent ratios were based on the amount of amino groups on B. The reaction mixture was shaken for 4 h at r.t. and washed with CH₂Cl₂ and MeOH (5 times each). The resulting products were cleaved from the beads with 2% of TFA in CH₂Cl₂ (2 min, r.t.) several times. The combined CH₂Cl₂ solution was washed with deionized H₂O, and the solvent was removed, affording the product C in 50% yield (94% LC purity). The product was characterized by ^¹H NMR.

<A NAME="RU08509ST-15C">15c</A>

To 20 mL of an CH₂Cl₂-CHCl₃ (4:1) solution of C (210 mg, 0.42 mmol), pentynoic acid (150 mg, 0.84 mmol) and EDCI˙HCl (160 mg, 0.46 mmol) were added, and the reaction mixture was stirred for 3 h at r.t. After adding 20 mL of deionized H₂O to the reaction solution, the product was extracted with an excess of EtOAc. The organic layer was dried with MgSO₄, and the solvent was removed to give the final product D in quantitative yield. It was identified by ^¹H NMR and IR spectroscopy (characteristic IR peak of alkyne C-H stretching at 3291 cm^-¹).

<A NAME="RU08509ST-16">16</A>

Preparation of PDA Liposomes in PBS Solution
An alkyne-terminated PCDA linker solution and PCDA solution were prepared by dissolving D (5.8 mg, 0.01 mmol) in 10 mL of CHCl₃ and PCDA (3.7 mg, 0.01 mmol) in 10 mL of CHCl₃, respectively. The linker solution (1 mL) and 9 mL of PCDA solution were taken and mixed. After removing the solvent by a rotary evaporator, 10 mL of PBS solution (pH 7.4) was added to give a total PCDA lipid concentration of 1.0 mM. Then, the sample was homogenized for 10 min, and the resulting solution was filtered by a polytetrafluoro-ethylene (PTFE) valve (Libra tube). The filtrate was cooled to 4 ˚C and kept at this temperature for 12 h to stabilize the PDA vesicles formed in the PBS solution.

<A NAME="RU08509ST-17">17</A>

Reaction of PDA Liposomes Having Terminal Alkynes with Benzyl Azide
Benzyl azide (5.32 mg, 40 µmol) was dissolved in 1.5 mL
of t-BuOH-H₂O (1:2) and 150 µL of the solution (4 equiv based on the amount of terminal triple bonds) was taken
and added to 10 mL of the previously prepared liposome solution. Ascorbic acid (2 mg) was added to a 1 mM aqueous solution of CuSO₄˙5H₂O (10 mL), and the resulting mixture was stirred for 10 min at r.t. Subsequently, 2 mL of copper-containing solution was added to the reaction mixture. The resulting mixture was shaken for 3 h at r.t.