# US 8,771,977 B2 · Method of microbial profiling of a sample using phenanthridium derivatives

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- Number: US 8,771,977 B2
- Document ID: US8771977B2
- Status: Granted
- Kind: B2
- Application number: 13/470,716
- Filed: 2012-05-14
- Published: 2012-11-29
- Granted: 2014-07-08
- Earliest claimed: 2006-02-28
- Assignees: Montana State University
- Applicants: Andreas Nocker; Anne K. Camper
- Inventors: Andreas Nocker; Anne K. Camper
- Web page: https://patentgrep.com/patent/US8771977B2

## CPC

- C12Q 1/18: Testing for antimicrobial activity of a material
- C07D 221/12: Phenanthridines
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- G01N 1/30: Staining; Impregnating {; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis}
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The present disclosure provides for a method of microbial profiling of a sample using phenanthridium derivatives with a 2+ charge or higher.

## Claims

### Claim 1

A method of microbial profiling of a sample, comprising:
a. collecting a sample;
b. exposing said sample to a phenanthridium derivative of formula (II):
wherein
R1 is an alkyl having 1 to 6 carbon atoms, perfluoroalkyl having 1 to 6 carbon atoms, or tetra-alkyl ammonium;
R2 is a substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted cycloalkyl having 3 to 10 carbon atoms;
R3 is amino, N-alkylamino, N,N-dialkylamino, alkoxy, or carboxyalkyl; and
R4 and R5 are independently selected from the group consisting of: hydrogen, halide, hydroxyl, amino, N-alkylamino, N,N-dialkylamino, alkoxy, carboxyalkyl, alkyl having 1 to 6 carbon atoms, perfluoroalkyl having 1 to 6 carbon atoms, azide, and tetra-alkyl ammonium;
or a mono or bis halide salt thereof,
wherein the phenanthridium derivative has an overall charge of 2+ or higher;
c. exposing said phenanthridium derivative containing sample to a light source;
d. optionally isolating genomic DNA from said sample; and
e. performing PCR on said isolated DNA.

### Claim 2 (depends on 1)

The method of claim 1, wherein said sample is selected from the group consisting of: soil, water, sewage, food, agricultural, pharmaceuticals, and cosmetics.

### Claim 3 (depends on 1)

The method of claim 1, wherein said phenanthridium derivative is PMA.

### Claim 4

A method of microbial profiling of a sample comprising:
a. collecting a sample;
b. exposing said sample to a phenanthridium derivative of formula (II):
wherein
R1 is an alkyl having 1 to 6 carbon atoms, perfluoroalkyl having 1 to 6 carbon atoms, or tetra-alkyl ammonium;
R2 is a substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted cycloalkyl having 3 to 10 carbon atoms;
R3 is amino, N-alkylamino, N,N-dialkylamino, alkoxy, or carboxyalkyl; and
R4 and R5 are independently selected from the group consisting of: hydrogen, halide, hydroxyl, amino, N-alkylamino, N,N-dialkylamino, alkoxy, carboxyalkyl, alkyl having 1 to 6 carbon atoms, perfluoroalkyl having 1 to 6 carbon atoms, azide, and tetra-alkyl ammonium;
or a mono or bis halide salt thereof,
wherein the phenanthridium derivative has an overall charge of 2+ or higher;
c. exposing said phenanthridium derivative containing sample to a light source;
d. optionally isolating RNA from said sample; and
e. performing RT-PCR on said isolated RNA.

### Claim 5 (depends on 4)

The method of claim 4, wherein said sample is selected from the group consisting of: soil, water, sewage, food, agricultural, pharmaceuticals, and cosmetics.

### Claim 6 (depends on 4)

The method of claim 4, wherein said phenanthridium derivative is PMA.

### Claim 7 (depends on 1)

The method of claim 1, wherein said phenanthridium derivative has a structure of formula (III):
wherein
R1 is an alkyl having 1 to 6 carbon atoms, perfluoroalkyl having 1 to 6 carbon atoms, or tetra-alkyl ammonium;
R2 is a substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted cycloalkyl having 3 to 10 carbon atoms;
R3a and R3b are independently hydrogen or alkyl having 1 to 6 carbon atoms; and
R4 and R5 are independently selected from the group consisting of hydrogen, halide, hydroxyl, amino, N-alkylamino, N,N-dialkylamino, alkoxy, carboxyalkyl, alkyl having 1 to 6 carbon atoms, perfluoroalkyl having 1 to 6 carbon atoms, azide, and tetra-alkyl ammonium;
or a mono or bis halide salt thereof,
wherein the phenanthridium derivative has an overall charge of 2+ or higher.

### Claim 8 (depends on 7)

The method of claim 7, wherein said phenanthridium derivative is selected from the group consisting of: 3-amino-8-azido-5-(3-(diethylmethylammonio)propyl)-6-phenylphenanthridium diiodide, 3-amino-8-azido-5-(3-(diethylmethylammonio)propyl)-6-phenylphenanthridium dibromide, and 3-amino-8-azido-5-(3-(diethylmethylammonio)propyl)-6-phenylphenanthridium dichloride.

### Claim 9 (depends on 4)

The method of claim 4, wherein said phenanthridium derivative has a structure of formula (III):
wherein
R1 is an alkyl having 1 to 6 carbon atoms, perfluoroalkyl having 1 to 6 carbon atoms, or tetra-alkyl ammonium;
R2 is a substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted cycloalkyl having 3 to 10 carbon atoms;
R3a and R3b are independently hydrogen or alkyl having 1 to 6 carbon atoms; and
R4 and R5 are independently selected from the group consisting of hydrogen, halide, hydroxyl, amino, N-alkylamino, N,N-dialkylamino, alkoxy, carboxyalkyl, alkyl having 1 to 6 carbon atoms, perfluoroalkyl having 1 to 6 carbon atoms, azide, and tetra-alkyl ammonium;
or a mono or bis halide salt thereof,
wherein the phenanthridium derivative has an overall charge of 2+ or higher.

### Claim 10 (depends on 9)

The method of claim 9, wherein said phenanthridium derivative is selected from the group consisting of: 3-amino-8-azido-5-(3-(diethylmethylammonio)propyl)-6-phenylphenanthridium diiodide, 3-amino-8-azido-5-(3-(diethylmethylammonio)propyl)-6-phenylphenanthridium dibromide, and 3-amino-8-azido-5-(3-(diethylmethylammonio)propyl)-6-phenylphenanthridium dichloride.