Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

Summary

The synthesis of asymmetric species of ferrocene is challenging using solution techniques. This report focuses on the methods carried out to produce a ferrocene-biotin bioconjugate using facile and clean reactions accomplished via solid-phase synthesis. Incorporation of a thiolate moiety is shown to impart the ability for immobilization on gold surfaces.

Abstract

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. The most common techniques utilized are imaging modalities such as Magnetic Resonance Imaging (MRI), Positron Emission Topography (PET), and Computed Topography (CT) and are optimal for understanding the physical structure of the disease but can only be performed once every four to six weeks due to the use of imaging agents and overall cost. With this in mind, the development of “point of care” techniques, such as biosensors, which evaluate the stage of disease and/or efficacy of treatment in the clinician’s office and do so in a timely manner, would revolutionize treatment protocols.¹ As a means to exploring ferrocene based biosensors for the detection of biologically relevant molecules², methods were developed to produce ferrocene-biotin bio-conjugates described herein. This report will focus on a biotin-ferrocene-cysteine system that can be immobilized on a gold surface.

Introduction

Biosensors are small devices that employ biomolecular recognition technology as the platform for selective analysis and are utilized for their specificity, speed, and low-cost. Electrochemical biosensors for the detection of biomolecules are at the forefront of this field due to their simplicity, cost effectiveness, and high sensitivity.^1,3 The general anatomy of these sensors is an electrode equipped with a recognition molecule specific for the biological marker of interest. Binding of the biomarker by the recognition molecule results in a local change of potential or current that can be detected by simple measurement. To date the recognition moiety can ra....

Protocol

1. Synthesis of Biotin-Fc-cysteine (1)

1. Solid phase methods to produce resin-bound 1.
   1. Place biotin loaded resin (250 mg, 0.145 mmol) into a fritted syringe and swell the resin by drawing up dimethylformamide (5 ml) and shaking the syringe on a lab shaker for 20 min. Expel the solution and repeat dimethylformamide swelling one more time.
   2. Remove the Fmoc protecting group by adding 4-6 ml of 20% piperidine in dimethylformamide to the syringe followed by 10-15 min of shaking. Repeat the deprotection process with another 4-6 ml of piperidine. Wash the resin with a sequence of 3x dimethylformamide, 3x dimethylformamide:meth....

Results

The resin bound form of 1 is shown in Figure 2. The covalent attachment of the ferrocene component gives rise to an orange tint to the resin beads that is persistent with continuous washing and indicative of an immobilized iron containing complex as opposed to iron absorption by the PEG component of the resin bead. The resin-free form of 1 is identical in color to the resin beads. Following removal of the compound from the resin-beads, the purity and yield (68%) resultin.......

Discussion

The synthesis of asymmetric ferrocene derivatives is challenging in solution. For example, attempts to produce 1 in solution resulted in low yields of the desired product (less than 20%). Likewise, reactions utilizing 1’-amino-ferrocene carboxylic acid (sans Fmoc) and resin bound biotin resulted in insoluble product consistent with the polymerized product reported by Baristic et al. and minimal product.⁴⁷ This is further complicated by ferrocene and its derivatives bein.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Biotin Novatag Resin	NovaBiochem	8550510001
TORVIQ 10 ml Luer Lock Fritted Syringe	Fisher	NC9299151
piperidine	Acros	P/3520/PB05
ninhydrin test	Sigma-Aldrich	60017-1ea
1’-Fmoc-amino-ferrocene-1-carboxylic acid	Omm Scientific	Special Order
N,N′-Diisopropylcarbodiimide	Sigma-Aldrich	D125407-5G
Fmoc-Cys(Trt)-OH	Novabiochem	8520080025
trifluoroacetic acid	Sigma-Aldrich	T5408
1,2-ethanedithiol	Sigma-Aldrich	2930
triisopropyl silane	Sigma-Aldrich	233781
Eppendorf tubes (20 ml)	any source
methanol	any source		dry with molecular sieves prior to use & store in 100 ml media bottle for easy usage
dichloromethane	any source		dry with molecular sieves prior to use & store in 100 ml media bottle for easy usage
dimethylformamide	any source		dry with molecular sieves prior to use & store in 100 ml media bottle for easy usage
centrifuge	any source