SNP Discrimination by Tolane-Modified Peptide Nucleic Acids: Application for the Detection of Drug Resistance in Pathogens

During the treatment of viral or bacterial infections, it is important to evaluate any resistance to the therapeutic agents used. An amino acid substitution arising from a single base mutation in a particular gene often causes drug resistance in pathogens. Therefore, molecular tools that discriminate a single base mismatch in the target sequence are required for achieving therapeutic success. Here, we synthesized peptide nucleic acids (PNAs) derivatized with tolane via an amide linkage at the N-terminus and succeeded in improving the sequence specificity, even with a mismatched base pair located near the terminal region of the duplex. We assessed the sequence specificities of the tolane-PNAs for single-strand DNA and RNA by UV-melting temperature analysis, thermodynamic analysis, an in silico conformational search, and a gel mobility shift assay. As a result, all of the PNA-tolane derivatives stabilized duplex formation to the matched target sequence without inducing mismatch target binding. Among the different PNA-tolane derivatives, PNA that was modified with a naphthyl-type tolane could efficiently discriminate a mismatched base pair and be utilized for the detection of resistance to neuraminidase inhibitors of the influenza A/H1N1 virus. Therefore, our molecular tool can be used to discriminate single nucleotide polymorphisms that are related to drug resistance in pathogens.


2-(4-(Phenylethynyl)phenyl)acetic acid (2) [1]
2-(4-(Phenylethynyl)phenyl)acetate (2b) (1.40 g, 5.58 mmol) was dissolved in 25ml of THF, and lithium hydroxide monohydrate (470 mg, 11.2 mmol) dissolved in 10 ml of H2O was added to the solution and stirred at room temperature until all the starting material was consumed, as was monitored by TLC. Water was added to the solution and the pH was adjusted to pH < 1 with 3% HCl solution. The solution was extracted three times with ethyl acetate, washed with brine, and then dried over magnesium sulfate. The

Methyl 5-(4-iodophenyl)pentanoate (4b)
Acetyl chloride (0.3 mL, 4.00 mmol) was dropped into 5 ml of methanol at 0 °C and stirred for Supporting information S-10 ten minutes under Ar. 5-(4-iodophenyl)pentanoic acid (4a) (405 mg, 1.33 mmol) was added to the solution, and stirred at room temperature for two hours. The solution was neutralized with potassium carbonate and evaporated under reduced pressure. The residue was dissolved in ethyl acetate, washed with water and brine, and the organic layer was dried over magnesium sulfate.
The solvent was evaporated under reduced pressure to yield the product (445 mg, quant) and used to next step without further purification; 1

6-(4-(Phenylethynyl)phenyl)hexanoic acid (5)
Product 5 was synthesized from 6-phenylhexanoic acid (1.0g, 5.20 mmol) employing the same procedure used for product 4. However, its iodo-derivative at the first step could not be recrystallized because of its oily nature. Therefore, crude products was used in each step and finally, the crude product at the last step was recrystallized from hexane-chloroform to yield product 5 (226mg, 15% over 4 steps); 1

2-(4-(Phenylethynyl)phenethoxy)acetic acid (6)
Ethyl 2-(4-(phenylethynyl)phenethoxy)acetate (6b) (676 mg, 2.19 mmol) was dissolved in 8 mL of THF, and lithium hydroxide monohydrate (185 mg, 4.38 mmol) dissolved in 4 ml of H2O was added to the solution and stirred at room temperature until all the starting material was consumed, as was monitored by TLC. Water was added to the solution and the pH was adjusted to pH < 1 with 3% HCl solution. The combined organic layers were extracted three times with ethyl acetate, washed with brine, and then dried over magnesium sulfate. The solvent was evaporated under reduced pressure to yield the product 6 (583 mg, 95%); 1

S-14
Methyl 3-((4-(phenylethynyl)benzyl)oxy)propanoate (7b) (978 mg, 3.32 mmol) was dissolved in 15 mL of THF, and lithium hydroxide monohydrate (280 mg, 6.65 mmol) dissolved in 7 ml of H2O was added to the solution and stirred at room temperature until all the starting material was consumed, as was monitored by TLC. Water was added to the solution and the pH was adjusted to pH < 1 with 3% HCl solution. The combined organic layers were extracted three times with ethyl acetate, washed with brine, and then dried over magnesium sulfate. The solvent was evaporated under reduced pressure to yield the product 7 (700 mg, 75%); 1

S-15
Ethyl 2-(4-(naphthalen-2-ylethynyl)phenethoxy)acetate (9a) (72 mg, 0.20 mmol) was dissolved in 1.5 mL of THF, and lithium hydroxide monohydrate (17 mg, 0.40 mmol) dissolved in 0.5 ml H2O was added to the solution and stirred at room temperature until all the starting material was consumed, as was monitored by TLC. Water was added to the solution and the pH was adjusted to

2-(4-(Pyren-1-ylethynyl)phenethoxy)acetic acid (10)
consumed, as was monitored by TLC. Water was added to the solution and the pH was adjusted to pH < 1 with 3% HCl solution. The combined organic layers were extracted three times with ethyl acetate, washed with brine, and then dried over magnesium sulfate. The solvent was evaporated under reduced pressure to yield the product 10 (64 mg, 98%); 1