Monday, August 2, 2010

Synthesis of cyclic heptapeptide, cyclo(Gly-Tyr-Val-Pro-Leu-Trp-Pro)

To synthesize cyclo(Gly-Tyr-Val-Pro-Leu-Trp-Pro) (14), 9.44 gm of linear heptapeptide unit(10 mmol) (13) was deprotected at carboxyl end using 0.36 gm of LiOH (15 mmol) to get Boc-Tyr-Val-Pro-Leu-Trp-Pro-Gly-OH (13a) following the same procedure as adopted for the synthesis of compounds 8a and 9a from compounds 8 and 9 respectively.

The deprotected heptapeptide unit 13a (4.65 gm, 5 mmol) was dissolved in 50 ml of CHCl3 at 0ºC. To the above solution, 0.94 gm of pnp (6.7 mmol) was added and stirred at room temperature for 12 hrs. The reaction mixture was filtered and the filtrate was washed with 10% NaHCO3 solution (3 x 15 ml) until excess of pnp was removed and finally washed with 5% HCl (2 x 10 ml) to get the corresponding p-nitrophenyl ester Boc-Tyr-Val-Pro-Leu-Trp-Pro-Gly-O-pnp (13b). To compound 13b (4.20 gm, 4 mmol) dissolved in 35 ml of CHCl3, 0.91 gm of TFA (8 mmol) was added, stirred at room temperature for 1 hr and washed with 10% NaHCO3 solution (2 x 25 ml).

Tyr-Val-Pro-Leu-Trp-Pro-Gly-O-pnp (13c), which was dissolved in 25 ml of CHCl3 and 2.3 ml of NMM (21 mmol) was added. Then all the contents were kept at 0ºC for 7 days. The reaction mixture was washed with 10% NaHCO3 until the byproduct p-nitrophenol was removed completely and finally washed with 5% HCl (3 x 15 ml). The organic layer was dried over anhydrous sodium sulphate. Finally, chloroform was distilled off and the crude cyclized product was crystallized from CHCl3 and n-hexane to get the pure compound 14.
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Bioelectronic DNA detection involves forming an electronic circuit mediated by nucleic acid hybridization and it serves as the basis for a DNA detection system called eSensor™ [1-4]. This system uses low-density DNA chips containing electrodes coated with DNA capture probes. Target DNA present in the sample hybridizes specifically both to capture probes and ferrocene labeled signal probes in solution thereby generating an electric current. Currente Sensor DNA chips contain as many as 36 electrodes for simultaneous detection of multiple pathogens from a single sample.

Many pathogens cause both acute and chronic disease at relatively low copy number and may be difficult or impossible to propagate in culture. Thus, most pathogen detection systems rely on nucleic acid amplification by using polymerase chain reaction (PCR). One highly effective amplification strategy targets conserved sequences among the family of organisms of interest. Such broad-range PCR strategies have been used to identify and characterize several known and previously uncharacterized bacteria [5,6] and viruses [7,8]. In order to maximize the utility of these effective pathogen nucleic acid amplification systems, amplification needs to be coupled with rapid, sensitive, and specific detection. Bioelectronic DNA detection by use of the eSensor chip might fulfill this need.

Human papillomaviruses (HPV) serve as an ideal model system for determining the efficiency and feasibility of eSensor DNA detection technology since there are at least 30 distinct genital HPV types that can be effectively amplified with broad-range consensus PCR primers. We designed two eSensor chips, each containing 14 probes specific for the conserved L1 region of the HPV genome. We evaluated clinical cervical cytology samples known to contain one or more HPV types. The eSensor DNA detection platform successfully detected the correct HPV type in most of these clinical samples, demonstrating that the system provides a rapid, sensitive, specific, and economical approach for multiple-pathogen detection and identification from a single sample.Background We used human papillomaviruses (HPV) as a model system to evaluate the utility of a nucleic acid, hybridization-based bioelectronic DNA detection platform (eSensor) in identifying multiple pathogens.