Chromogenic and fluorogenic compounds and their use for biological detection
The disclosure provides chromogenic and fluorogenic compounds comprising amino acid or peptide appendages that are capable of detecting an activity in a bioassay. Also provided herein are methods for detecting a biological activity in a sample with the subject chromogenic and fluorogenic compounds. In one embodiment, provided herein is a method for detecting endotoxin in a sample. Further provided herein are kits including the subject chromogenic and fluorogenic compounds for detecting a biological activity in a sample.
1 . A chromogenic and/or fluorogenic compound having the structure of Formula 1:
or a salt thereof, wherein:
each A is an amino acid or a peptide fragment connected to a masked chromophore and/or fluorophore moiety via an enzyme-cleavable amide bond;
R 1 to R 8 are each independently selected from hydrogen, halogen, a carboxy, an alkyl, a substituted alkyl, an aryl, a substituted aryl, a heteroaryl group, and a substituted heteroaryl group; and
Z is C(R 10 R 11 ), wherein R 10 and R 11 are independently selected from hydrogen, an alkyl, a substituted alkyl, an aryl, a substituted aryl, a heteroaryl group and a substituted heteroaryl group; and
wherein the compound is capable of releasing a detectable chromophore and/or fluorophore in the presence of a target enzyme or analyte that provides for cleavage of the enzyme-cleavable amide bonds.
2 . The compound of claim 1 , wherein:
R 1 to R 8 are independently selected from H, F, Cl, a methyl, and a carboxy; and
R 10 and R 11 are each alkyl.
3 . The compound of claim 1 , wherein:
each A is a peptide fragment comprising one or more amino acids selected from Arg, Ala, Pro, D-Ala, Beta-Ala and Asp;
R 1 to R 8 are each H; and
R 10 and R 11 are each methyl.
4 . The compound of claim 1 , wherein:
each A is selected from Ac-Ala-Asn-Trp, Cbz-Asp-Glu-Val-Asp, Ac-Asp-Glu-Val-Asp, succinimidyl-Leu-Leu-Val-Tyr, Ac-Trp-Leu-Ala, Ac-Lys-Gln-Leu, Cbz-Leu-Leu-Glu, Ac-Pro-Ala-Leu, Ac-Ile-Thr-Asp, Ac-Arg-Arg-Arg, Boc-Leu-Leu-Val-Tyr, Ac-Leu-Glu-His-Asp, Gly-Pro, Cbz-Leu-Arg-Gly-Gly, Cbz-Lys-Lys-Ala-Gly, and Cbz-Lys-Ala-Gly-Gly;
R 1 to R 8 are each H; and
R 10 and R 11 are each methyl.
5 . The compound of claim 1 , wherein the compound is of the formula (IA):
wherein:
A 1 -A 4 and A 1′ -A 4′ are each independently selected from an amino acid;
Y 1 and Y 1′ are each an optional terminal group;
R 10 and R 11 are independently selected from H, alkyl and substituted alkyl; and
n-o and n′-o′ are each independently selected from 0 or 1.
6 . The compound of claim 5 , wherein A 1 -A 4 and A 1′ -A 4′ are each independently an amino acid selected from Alanine (Ala), isoleucine (Ile), leucine (Leu), valine (Val), tryptophan (Trp), asparagine (Asn), glutamine (Gln), proline (Pro), glycine (Gly), lysine (Lys), aspartic acid (Asp), glutamic acid (Glu) and arginine (Arg), wherein the amino acid is of L-configuration or D-configuration.
7 . The compound of claim 5 , wherein A 1 -A 4 and A 1′ -A 4′ are each selected from Asp-Glu-Val-Asp and Arg-Ala-Glu-Ile.
8 . The compound of claim 1 , wherein R 10 and R 11 are independently selected from H, alkyl and substituted alkyl.
9 . The compound of claim 1 , wherein the compound is selected from:
Cpd #
Structure
4
5
6
20
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26
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34
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37
10 . A method of detecting a biological activity in a sample, the method comprising:
a) contacting the sample with a detection reagent comprising a compound according to claim 1 under conditions in which said detection reagent releases the detectable chromophore and/or fluorophore in the presence of a target analyte, if present in the sample, to produce an optical signal; and
b) assessing whether the target analyte is present in the sample by detecting the optical signal.
11 . The method of claim 10 , wherein the target analyte is a protease.
12 . The method of claim 10 , further comprising assessing a biological activity in the sample.
13 . The method of claim 12 , wherein the biological activity is an apoptosis or cell viability.
14 . A method of determining an endotoxin level in a sample, comprising:
a) contacting said sample with a detection reagent comprising a compound according to claim 1 under conditions in which said compound releases the detectable chromophore and/or fluorophore in the presence of endotoxin in the sample to produce an optical signal; and
b) detecting the endotoxin level in the sample by detecting the optical signal.
15 . A kit for detecting a biological activity in a sample, the kit comprising:
a) one or more compounds according to claim 1 ; and
b) one or more components selected from a buffer, an organic solvent, one or more detection reagents, luminescence standards, an enzyme, an enzyme indicator and an instruction sheet concerning the use of the kit for detecting a biological activity in a sample.