Electrochemical methods and compounds for the detection of enzymes
Disclosed are compositions and methods for the electrochemical detection of enzymes, such as enzymes that are indicative of disease, disorders, or pathogens, such as viruses, bacteria, and fungi, or other disorders. These methods can be used in point-of-care diagnostic assays for the detection of disease, disorder, or pathogen (e.g., to identify the strain of pathogen infecting a patient in a healthcare setting). The electrochemical methods described herein can also be used to assess the susceptibility of a pathogen to an antipathogen drug. Also provided are probes suitable for use in conjunction with the methods described herein.
1. A compound defined by Formula I below
wherein
A is —OH, —OMe, —OC(═O)CH 3 , —NH 2 , —NHC(═O)CH 3 , —NHCH 2 OH, or —NHC(═NH)NH 2 ;
R 1 is, individually for each occurrence, —H, —Me, or —C(═O)CH 3 ;
R 2 is —H or —Me;
R 3 is —OH, —OMe, —OC(═O)CH 3 , —NH 2 , —NHC(═O)CH 3 , or —NHCH 2 OH;
n is an integer from 1 to 16;
L is selected from a polyvalent linking moiety or a bivalent linking moiety comprising
wherein R 5 and R 6 are independently selected from methyl, ethyl, isopropyl, and tertbutyl; Z is C or S; and m is 0, 1, 2 or 3; and
E is, individually for each occurrence, an electrochemically active moiety defined by one of the general formulae shown below
wherein
R 4 is a C 1 -C 6 alkyl group, and
X is, individually for each occurrence, —OH, —OMe, —OC(═O)CH 3 , —F, —CF 3 , —CH 2 F, —CHF 2 , —Cl, —Br, —I, H or sulfonate.
2. The compound of claim 1 , wherein E is
3. The compound of claim 1 , wherein E comprises a moiety defined by one of the general formulae below
wherein X is, individually for each occurrence, —OH, —OMe, —OC(═O)CH 3 , —F, —CF 3 , —CH 2 F, —CHF 2 , —Cl, —Br, —I, H or sulfonate.
4. The compound of claim 3 , wherein E comprises a moiety defined the general formula below
wherein X is, individually for each occurrence, —OH, —OMe, —OC(═O)CH 3 , —F, —CF 3 , —CH 2 F, or —CHF 2 .
5. The compound of claim 3 , wherein X is, in each occurrence —OH.
6. The compound of claim 1 , wherein R 1 and R 2 are, in each occurrence, —H.
7. The compound of claim 1 , wherein A is —OH.
8. The compound of claim 1 , wherein R 3 is —NHC(═O)CH 3 .
9. The compound of claim 1 , wherein L is chosen from one of the following
10. The compound of claim 1 , wherein L is a polyvalent linking moiety.
11. The compound of claim 1 , wherein L is chosen from one of the following
12. The compound of claim 1 , wherein n is 1.
13. The compound of claim 1 , wherein n is from 2 to 8.
14. A method of electrochemically detecting an enzyme in a sample comprising:
(a) providing a probe comprising a compound defined by Formula I below, having a substrate for the enzyme covalently linked to an electrochemically active moiety via a bond that is cleavable by the enzyme;
wherein
A is —OH, —OMe, —OC(═O)CH 3 , —NH 2 , —NHC(═O)CH 3 , —NHCH 2 OH, or —NHC(═NH)NH 2 ;
R 1 is, individually for each occurrence, —H, -Me, or —C(═O)CH 3 ;
R 2 is —H or -Me;
R 3 is —OH, —OMe, —OC(═O)CH 3 , —NH 2 , —NHC(═O)CH 3 , or —NHCH 2 OH;
n is an integer from 1 to 16;
L is absent or is a linking moiety, wherein when L is absent, 01 is also absent and n is 1; and
E is, individually for each occurrence, an electrochemically active moiety defined by one of the general formulae shown below
wherein
R 4 is a C 1 -C 6 alkyl group, and
X is, individually for each occurrence, —OH, —OMe, —OC(═O)CH 3 , —F, —CF 3 , —CH 2 F, —CHF 2 , —Cl, —Br, —I, H or sulfonate,
(b) contacting the sample with the probe under conditions effective for enzymatic cleavage of the bond by the enzyme;
(c) electrochemically detecting the electrochemically active moiety enzymatically cleaved by the enzyme.
15. The method of claim 14 , wherein the enzyme is a pathogen-specific enzyme selected from a viral neuraminidase, a reverse transcriptase or protease, an isoform of β-lactamase sulfatase, a β-glucuronidase, a carbapenamase, a lysyl aminopeptidase, or an α mannoside.
16. The method of claim 14 , wherein the enzyme is a pathogen-specific enzyme selected from a peptidase, sulfatase, phosphatase, esterase, or combination thereof.
17. A kit comprising:
(i) a device for collecting a sample comprising an enzyme of interest; and
(ii) a probe comprising a compound defined by Formula I below, having a substrate for the enzyme of interest covalently linked to an electrochemically active moiety via a bond that is cleavable by the enzyme,
wherein
A is —OH, —OMe, —OC(═O)CH 3 , —NH 2 , —NHC(═O)CH 3 , —NHCH 2 OH, or —NHC(═NH)NH 2 ;
R 1 is, individually for each occurrence, —H, -Me, or —C(═O)CH 3 ;
R 2 is —H or -Me;
R 3 is —OH, —OMe, —OC(═O)CH 3 , —NH 2 , —NHC(═O)CH 3 , or —NHCH 2 OH;
n is an integer from 1 to 16;
L is absent or is a linking moiety, wherein when L is absent, 01 is also absent and n is 1; and
E is, individually for each occurrence, an electrochemically active moiety defined by one of the general formulae shown below
wherein
R 4 is a C 1 -C 6 alkyl group, and
X is, individually for each occurrence, —OH, —OMe, —OC(═O)CH 3 , —F, —CF 3 , —CH 2 F, —CHF 2 , —Cl, —Br, —I, H or sulfonate.