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 substrate for analysis of an enzyme having formula:
A-(B 1 -B 2 -B 3 -B 4 ) (I)
where A is a monosaccharide or a disaccharide and B 1 is a C 1 -C 20 alkyl, a substituted C 6 -C 20 aryl, C 6 -C 20 heterocyclic containing a heteroatom of N, O or S, said B 1 covalently associated with A;
B 2 is an amino acid; 2,6-diaminohexanoic acid; or
where R 1 ′ is a C 1 -C 20 alkyl, C 1 -C 20 alkyl having a substituent of N, O, or S, heteroatom containing C 6 -C 20 aryl, C 2 -C 20 carbonyl, C 1 -C 20 amidyl, C 1 -C 20 ether, C 6 -C 20 aryl, C 6 - C 20 heterocyclic containing a heteroatom of N, O, or S, said R1′ covalently bound to both B 1 and B 3 ; X is a nullity, oxygen, sulfur, or nitrogen; R 2 ′ is a C 1 -C 20 alkyl, C 4 -C 20 ether, C 1 -C 20 alkyl having a substituent of N, O, or S, C 1 -C 20 ester, C 1 -C 20 alcohol, C 1 -C 20 alkenyl, heteroatom containing C 6 -C 20 aryl, or a C 6 -C 20 heterocyclic containing a heteroatom of N, O or S; Y is a nitrogen, oxygen, or sulfur nucleophilic group; and n is an integer between 1 and 30;
B 3 includes a quaternary ammonium and is
where R 1 is a C 1 -C 20 alkyl, C 1 -C 20 alkyl having a substituent of N, O, or S, a heteroatom containing C 6 -C 20 aryl, C 2 -C 20 carbonyl, C 1 -C 20 amidyl, C 1 -C 20 ether, C 6 -C 20 aryl, or a C 6 -C 20 heterocyclic containing a heteroatom of N, O, or S, said R 1 covalently associated with B 2 , said R 1 covalently associated with said B 4 directly or through an intermediate (XR 3 )n-R 4 ; R 2 is independently in each occurrence a C 1 -C 20 alkyl, a C 2 -C 20 alkyl having a substituent of C 1 -C 20 alkyl; X is independently in each occurrence a nullity, oxygen, sulfur, or nitrogen; R 3 is independently in each occurrence a nullity, C 1 -C 20 alkyl, C 1 -C 20 having a substituted C 6 -C 20 aryl, C 6 -C 20 heterocyclic containing a heteroatom of N, O or S; n is an integer between 0 and 30, inclusive; R 4 is independently in each occurrence a nullity, C 1 -C 20 alkyl, C 1 -C 20 having a substituted C 6- C 20 aryl, C 1 -C 20 carbonyl, C 1 -C 20 amidyl, C 1 -C 20 ether, C 6 -C 20 aryl, C 6 -C 20 heterocyclic containing a heteroatom of N, O, or S; and
B 4 is a C 2 -C 20 alkyl, C 4 -C 20 ether, C 2 -C 20 alkyl having a substituent of N, O, or S, C 1 -C 20 ester, C 1 -C 20 alcohol, C 1 -C 20 alkenyl, heteroatom containing C 6 -C 20 aryl, or a C 6 -C 20 heterocyclic containing a heteroatom of N, O or S.
2. The substrate of claim 1 wherein A and (B 1 -B 2 -B 3 -B 4 ) are separable by an action of an enzyme.
3. The substrate of claim 1 wherein A is an aldohexose or a ketohexose.
4. The substrate of claim 1 wherein A is a D-glucose or a D-galactose.
5. The substrate of claim 1 wherein B 1 is C 6 aryl having a substituent extending from the ring.
6. The substrate of claim 1 where B 2 is a lysine.
7. The substrate of claim 1 wherein B 2 is derivatized by the addition of a fluorophore.
8. The substrate of claim 1 wherein B 3 is a carnitinyl.
9. The substrate of claim 8 wherein B 4 is C 2 -C 20 alkyl having a substituent of O present as a carbonyl.
10. The substrate of claim 1 wherein A and (B 1 -B 2 -B 3 -B 4 ) portions each include a stable secondary prevalence isotope of an element.
11. The substrate of claim 10 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.
12. A method for detecting enzymatic activity, comprising: contacting a sample containing a target enzyme with a substrate of claim 1 , wherein said target enzyme is selected from the group consisting of acid α-galactosidase A, acid β-glucocerebrosidase, galactocerebroside α-galactosidase and acid α-glucosidase; and said substrate is linked to a solid support, under conditions wherein the target enzyme is capable of acting on the substrate to produce an enzymatic product; and detecting the amount of substrate remaining on the solid support.