Selective detection of amino group containing thiols

Patent metadata

Field	Value
Publication number	US-9506929-B2
Application number	US-201314380704-A
Country	US
Kind code	B2
Filing date	Feb 22, 2013
Priority date	Feb 23, 2012
Publication date	Nov 29, 2016
Grant date	Nov 29, 2016

Abstract

Probes for selectively detecting compounds comprising a thiol group and an amino group (“thiols”) are disclosed. Exemplary thiols include cysteine, homocysteine, and glutathione. Embodiments of the disclosed probes produce a detectable change in absorbance spectrum and/or emission spectrum when reacted with one or more thiols in solution. Methods and kits for performing the detection also are disclosed. The probes have a general formula

where each bond depicted as “ ” is a single or double bond; R 1 , R 3 -R 6 and R 8 independently are hydrogen, hydroxyl, thiol, lower alkyl, carboxyalkyl, amino, alkoxy, or halogen; R 2 is an α,β-unsaturated aliphatic ester; R 7 is oxygen, sulfur, hydrogen, hydroxyl, thiol, lower alkyl, carboxyalkyl, amino, alkoxy, or halogen, or R 7 and R 8 together form a cycloalkyl or aryl ring; X 1 is CH 2 , S, NH, O, Se, Si(CH 3 ) 2 , Ge(CH 3 ) 2 , Sn(CH 3 ) 2 , or C(CH 3 ) 2 ; and X 2 is CH, CH 2 , N, NH, or CR 9 where R 9 is aryl.

First claim

We claim:

1. A compound having a chemical structure according to general formula I:

where each bond depicted as “ ” is a single or double bond as needed to satisfy valence requirements;
R 1 , R 3 -R 6 and R 8 independently are H, hydroxyl, thiol, lower alkyl, carboxyalkyl, amino, alkoxy, or halogen;
R 2 is an α,β-unsaturated aliphatic ester;
R 7 is O, S, H, hydroxyl, thiol, lower alkyl, carboxyalkyl, amino, alkoxy, or halogen, or R 7 and R 8 together form a cycloalkyl or aryl ring;
X 1 is CH 2 , S, NH, O, Se, Si(CH 3 ) 2 , Ge(CH 3 ) 2 , Sn(CH 3 ) 2 , or C(CH 3 ) 2 ; and
X 2 is CH, CH 2 , N, NH, or CR 9 where R 9 is aryl, wherein
when X 1 is O, then R 2 is an acrylate ester, X 2 is CH or N, R 7 is O, and R 1 , R 3 -R 6 , and R 8 independently are H or lower alkyl, or
when X 1 is O or S, then R 7 and R 8 together form an aryl ring.

2. The compound of claim 1 where X 1 is O or S, and R 1 and R 3 -R 6 independently are H or lower alkyl.

3. The compound of claim 1 where X 1 is O, R 7 and R 8 together form an aryl ring, X 2 is N or CR 9 , R 1 is lower alkyl or H, R 2 is an acrylate ester, and R 3 -R 6 are H.

4. The compound of claim 1 having the structure:

5. A kit for detecting at least one compound comprising a thiol group and an amino group, comprising at least one probe according to claim 1 .

6. The kit of claim 5 where the probe is

or a combination thereof.

7. A compound having a chemical structure according to general formula II:

where “ ” indicates a single or double bond as needed to satisfy valence requirements and “------” indicates an optional single bond;
R 1 , R 3 -R 6 and R 8 independently are H, hydroxyl, thiol, lower alkyl, carboxyalkyl, amino, alkoxy, or halogen;
X 1 is CH 2 , S, NH, O, Se, Si(CH 3 ) 2 , Ge(CH 3 ) 2 , Sn(CH 3 ) 2 , or C(CH 3 ) 2 ;
R 2 and R 11 independently are an α,β-unsaturated aliphatic ester;
R 10 , R 12 , and R 13 independently are H, hydroxyl, thiol, lower alkyl, carboxyalkyl, amino, alkoxy, or halogen;
R 14 -R 17 independently are H, alkyl, acyl, carboxyl, nitro, amino, alkyl amino, or —SO 3 H; and
R 18 is H, hydroxyl, lower alkyl, lower alkoxy, —SO 3 H or —COOR 19 where R 19 is H or lower alkyl and the bond depicted as “ ” in ring B is a double bond, or R 18 is one or more atoms forming a ring system with rings B and E and the bond depicted as “ ” in ring B is a single bond.

8. The compound of claim 7 where each of R 2 and R 11 is an acrylate ester, and R 18 is —COOH or R 18 is —C(O)O— and forms a ring system with rings B and E.

9. The compound of claim 8 where R 1 , R 3 -R 6 , R 10 and R 12 -R 17 independently are H or lower alkyl.

10. The compound of claim 9 where X 1 is O.

11. The compound of claim 7 having the structure:

12. A method for selectively detecting a compound comprising a thiol group and an amino group, comprising:
combining a sample potentially comprising at least one compound comprising a thiol group and an amino group with a solution comprising a probe having a structure according to claim 1 to produce a reaction mixture;
allowing a reaction between the sample and the probe to proceed for an effective period of time to produce a detectable change in the reaction mixture's absorbance spectrum, emission spectrum, or both, where the change indicates that the compound is present; and
detecting the change.

13. The method of claim 12 where the sample comprises blood, a blood product, a blood component, urine, a urine product, or a urine component.

14. The method of claim 13 , further comprising:
obtaining plasma;
reducing the plasma by addition of a reducing agent;
precipitating proteins in the plasma;
separating precipitated proteins from the plasma to form the sample; and
subsequently combining the sample with the solution comprising the probe.

15. The method of claim 13 , further comprising:
obtaining a dried blood spot prepared by spotting whole blood onto an absorbent material and allowing the blood to dry;
extracting the dried blood spot with a solvent to produce an extract;
reducing the extract by addition of a reducing agent to form a reduced extract;
fractionating the reduced extract to obtain the sample; and
subsequently combining the sample with the solution comprising the probe.

16. The method of claim 12 where detecting the change comprises (i) comparing a color of the reaction mixture before the reaction to a color of the reaction mixture after the reaction, (ii) detecting a change in absorbance of the reaction mixture at one or more wavelengths after the reaction has proceeded for the effective period of time, (iii) comparing an absorbance spectrum of the reaction mixture at a first time after combining the sample and the probe to an absorbance spectrum of the reaction mixture after the reaction has proceeded for the effective period of time, (iv) detecting a change in emission of the reaction mixture at one or more wavelengths after the reaction has proceeded for the effective period of time, (v) comparing an emission spectrum of the reaction mixture at a first time after combining the sample and the probe to an emission spectrum of the solution after the reaction has proceeded for the effective period of time, or (vi) any combination thereof.

17. The method of claim 12 where the probe is

18. The method of claim 17 where the at least one compound is glutathione, and the reaction mixture further comprises a cationic surfactant.

19. The method of claim 12 where the at least one compound is cysteine, and the probe is

20. The method of claim 19 where the reaction mixture further comprises a cationic surfactant.

Assignees

• Oregon State Board Of Higher Education On Behalf Of Portland State Univ
• Univ Portland State

Inventors

• Strongin Robert M
• Guo Yixing
• Hakuna Lovemore
• Lowry Mark Allen
• Escobedo Córdova Jorge Omar

Classifications

• G01N33/6848

  Methods of protein analysis involving mass spectrometry · CPC title

• G01N33/582

  with fluorescent label · CPC title

• G01N33/52

  Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper {and including single- and multilayer analytical elements (immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72)} · CPC title

• C07D265/38

  [b, e]-condensed with two six-membered rings · CPC title

• G01N33/6815 Primary

  containing sulfur, e.g. cysteine, cystine, methionine, homocysteine · CPC title