Enzymatic substrates for multiple detection systems
View Patent ↗An inventive substrate is provided which includes a substrate compound of formula A-B 1 -B 2 -B 3 -B 4 : wherein A is a sugar moiety; B 1 is a linker moiety allowing the conjugation of moiety A and the remaining structure of the substrate; B 2 is a linker moiety with a free reactive amino group so as to be available for reaction with carboxylic acids or detectable tags; B 3 contains a permanently charged element such as a quaternary ammonium group so as to increase sensitivity for mass spectrometry analysis; and B 4 of various carbon length conferring specificity amongst individual substrates in detection methods. Also provided is a molecule of the formula B 1 -B 2 -B 3 -B 4 , with similar structural characteristics to an enzymatic product produced by the action of a target enzyme on an inventive substrate. Further provided are methods for using inventive substrates for detecting enzymatic activity.
1. A molecule comprising the formula:
B 1 -B 2 -B 3 -B 4 (I)
where B 1 is a methylene or 4-aminophenol; B 2 is a diaminohexanoyl or an amidylaminoacyl said B 2 covalently bound to B 1 by an amide bond and to B 3 by an amide bond;
B 3 includes a quaternary ammonium and is a carnitinyl;
and B 4 is an alkyl chain with 10-20 carbons linked to B 3 via an ester bond.
2. The molecule of claim 1 wherein B 1 is a 4-aminophenol.
3. The molecule of claim 1 where B 2 is a lysine.
4. The molecule of claim 1 wherein B 2 is derivatized by the addition of a fluorophore.
5. The molecule of claim 1 wherein (B 1 -B 2 -B 3 -B 4 ) includes a stable secondary prevalence isotope of an element.
6. The molecule of claim 5 wherein said stable secondary prevalence isotope in each occurrence is selected from the group consisting of 2 D, 13 C, 15 N, 17 O, 18 O, 31 P and 34 S.
7. A method for detecting enzymatic activity, comprising: contacting a sample containing a target enzyme with a substrate having the formula:
A-B 1 -B 2 -B 3 -B 4
where A is a monosaccharide or a disaccharide, said A covalently bound to said B 1 , and wherein said B 1 -B 2 -B 3 -B 4 is the molecule of claim 1 , under conditions wherein the target enzyme is capable of acting on the substrate to produce an enzymatic product; and
detecting the amount of the enzymatic product.
8. The method of claim 7 wherein the B 2 of said enzymatic product is derivatized by the addition of a fluorophore.
9. The method of claim 7 wherein said substrate is linked to a solid support.
10. The method of claim 7 wherein said detecting is by detecting the amount of substrate remaining on the solid support.
11. The method of claim 7 wherein said B 1 -B 2 -B 3 -B 4 includes a stable secondary prevalence isotope of an element.
12. The method of claim 11 wherein said stable secondary prevalence isotope in each occurrence is selected from the group consisting of 2 D, 13 C, 15 N, 17 O, 18 O, 31 P and 34 S.