METHODS OF DETERMINING CERULOPLASMIN ACTIVITY AND APPLICATIONS THEREOF
20250369031 ยท 2025-12-04
Assignee
Inventors
Cpc classification
International classification
Abstract
Disclosed is a method of determining ceruloplasmin activity using a mass spectrometry technique. The method comprises reacting a biological sample containing ceruloplasmin with a compound of formula I to produce an oxidized product of the compound, measuring a level of the oxidized product by a mass spectrometry technique, and determining a level of ceruloplasmin activity based on the level (amount) of the oxidized product.
Claims
1. A method of determining ceruloplasmin activity, comprising: (i) contacting a biological sample containing ceruloplasmin with a compound of formula I under a condition such that the compound reacts with the ceruloplasmin to produce an oxidized product of the compound ##STR00028## wherein R.sub.1 is hydrogen, an alkyl group, an alcohol group, an alkoxy group or an alkyl ether group containing one or more oxyethylene units; R.sub.2 is hydrogen or hydroxyl; and R.sub.3 and R.sub.4, the same or different, are independently hydrogen or an alkylamine group, or R.sub.3 and R.sub.4 together form a benzene ring fused to the nitrogen-containing bicyclic heteroring, wherein the benzene ring is substituted by NH.sub.2; (ii) measuring a level of the oxidized product by a mass spectrometry technique; and (iii) determining a level of ceruloplasmin activity based on the level of the oxidized product, wherein a decreased level of the oxidized product is indicative of a decreased level of ceruloplasmin activity, and an increased level of the oxidized product is indicative of an increased level of ceruloplasmin activity.
2. The method of claim 1, wherein R.sub.1 is hydrogen, an alkyl group, an alcohol group, an alkoxy group or an alkyl ether group containing one or more oxyethylene units; R.sub.2 is hydrogen or hydroxyl; and R.sub.3 and R.sub.4, the same or different, are independently hydrogen or an alkylamine group.
3. The method of claim 2, wherein R.sub.1 is hydrogen; R.sub.2 is hydroxyl; R.sub.3 is an alkylamine group; R.sub.4 is hydrogen.
4. The method of claim 3, wherein the compound is represented by formula Ia ##STR00029##
5. The method of claim 1, wherein R.sub.1 is hydrogen, an alkyl group, an alcohol group, an alkoxy group or an alkyl ether group containing one or more oxyethylene units; R.sub.2 is hydrogen or hydroxyl; and R.sub.3 and R.sub.4 together form a benzene ring fused to the nitrogen-containing bicyclic heteroring, wherein the benzene ring is substituted by NH.sub.2.
6. The method of claim 5, wherein R.sub.1 is an alkyl group, an alcohol group or an alkyl ether group containing one or more oxyethylene units; R.sub.2 is hydrogen; and R.sub.3 and R.sub.4 together form a benzene ring fused to the nitrogen-containing bicyclic heteroring, wherein the benzene ring is substituted by NH.sub.2.
7. The method of claim 6, wherein the compound is represented by formula Ib ##STR00030## wherein R.sub.1 is CH.sub.3, CH.sub.2CH.sub.3, CH.sub.2CH.sub.2CH.sub.2CH.sub.3, CH.sub.2CH.sub.2OH, CH.sub.2CH.sub.2CH.sub.2OH or CH.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.4OCH.sub.3.
8. The method of claim 7, wherein the compound is selected from the group consisting of ##STR00031##
9. The method of claim 1, wherein the spectrometry technique comprises a tandem mass spectrometry (MS/MS) technique.
10. The method of claim 9, wherein the spectrometry technique comprises an LC-MS/MS technique.
11. The method of claim 1, wherein the biological sample is a liquid biological sample.
12. The method of claim 11, wherein the liquid biological sample is a serum sample or a urine sample.
13. The method of claim 1, wherein biological sample has a volume of 0.01 L to 10 L.
14. The method of claim 13, wherein the biological sample has a volume of 0.05 L to 5 L.
15. The method of claim 13, wherein the biological sample has a volume of 0.05 L to 2 L.
16. A method for determining ceruloplasmin activity and predicting the risk of a disease related to ceruloplasmin activity in a subject, comprising (i) providing a biological sample which is obtained from the subject; (ii) contacting the biological sample with a compound of formula I under a condition such that the compound is oxidized to produce an oxidized product of the compound, ##STR00032## wherein R.sub.1 is hydrogen, an alkyl group, an alcohol group, an alkoxy group or an alkyl ether group containing one or more oxyethylene units: R.sub.2 is hydrogen or hydroxyl; and R.sub.3 and R.sub.4, the same or different, are independently hydrogen or an alkylamine group, or R.sub.3 and R.sub.4 together form a benzene ring fused to the nitrogen-containing bicyclic heteroring, wherein the benzene ring is substituted by NH.sub.2; (iii) measuring a level of the oxidized product by a mass spectrometry technique; and (iv) determining a level of ceruloplasmin activity and the likelihood of having a disease or condition related to ceruloplasmin activity of said subject based on the level of the oxidized product, wherein a decreased level of the oxidized product below a reference level is indicative of deficiency of ceruloplasmin activity and an increased likelihood of having a disease or condition related to deficiency of ceruloplasmin activity; and/or an increased level of the oxidized product above a reference level is indicative of excess of ceruloplasmin activity and an increased likelihood of having a disease or condition related to excess of ceruloplasmin activity.
17. The method of claim 16, wherein the disease or condition related to deficiency of ceruloplasmin activity is selected from the group consisting of aceruloplasminemia, Wilson's disease, Menke's disease, Alzheimer's disease and Parkinson's disease (PD).
18. The method of claim 16, wherein the disease or condition related to excess of ceruloplasmin activity includes Type II diabetes mellitus, valvular heart disease, coronary heart disease, bladder cancer, chronic obstructive pulmonary disease and kidney diseases.
19. The method of claim 16, wherein the subject is a fetus, a neonate, a child, an adolescent or an adult.
20. The method of claim 16, wherein the biological sample is a liquid biological sample.
21. The method of claim 20, wherein the liquid biological sample is a serum sample or a urine sample.
22. A kit, which comprises a compound of formula I, ##STR00033## wherein R.sub.1 is hydrogen, an alkyl group, an alcohol group, an alkoxy group or an alkyl ether group containing one or more oxyethylene units; R.sub.2 is hydrogen or hydroxyl; and R.sub.3 and R.sub.4, the same or different, are independently hydrogen or an alkylamine group, or R.sub.3 and R.sub.4 together form a benzene ring fused to the nitrogen-containing bicyclic heteroring, wherein the benzene ring is substituted by NH.sub.2, a reaction buffer, a quenching agent and instructions for using the kit for performing the method of determining ceruloplasmin activity as defined in claim 1.
23. (canceled)
24. (canceled)
25. (canceled)
26. A kit, which comprises a compound of formula I, ##STR00034## wherein R.sub.1 is hydrogen, an alkyl group, an alcohol group, an alkoxy group or an alkyl ether group containing one or more oxyethylene units; R.sub.2 is hydrogen or hydroxyl; and R.sub.3 and R.sub.4, the same or different, are independently hydrogen or an alkylamine group, or R.sub.3 and R.sub.4 together form a benzene ring fused to the nitrogen-containing bicyclic heteroring, wherein the benzene ring is substituted by NH.sub.2, a reaction buffer, a quenching agent and instructions for using the kit for performing the method of predicting the risk of a disease related to ceruloplasmin activity in a subject as defined in claim 16.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0042] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.
[0043] In the drawings:
[0044]
[0045]
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[0049]
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DETAILED DESCRIPTION OF THE INVENTION
[0055] The following description is merely intended to illustrate various embodiments of the invention. As such, specific embodiments or modifications discussed herein are not to be construed as limitations to the scope of the invention. It will be apparent to one skilled in the art that various changes or equivalents may be made without departing from the scope of the invention.
[0056] In order to provide a clear and ready understanding of the present invention, certain terms are first defined. Additional definitions are set forth throughout the detailed description. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as is commonly understood by one of skill in the art to which this invention belongs.
[0057] As used herein, the singular forms a, an, and the include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a component includes a plurality of such components and equivalents thereof known to those skilled in the art.
[0058] As used herein, the term comprise or comprising is generally used in the sense of include/including which means permitting the presence of one or more features, ingredients or components. The term comprise or comprising encompasses the term consists or consisting of.
[0059] As used herein, the term about or approximately refers to a degree of acceptable deviation that will be understood by persons of ordinary skill in the art, which may vary to some extent depending on the context in which it is used. In general, about or approximately may mean a numeric value having a range of 10% around the cited value.
[0060] As used herein, the terms subject, individual and patient refer to any mammalian subject for whom diagnosis, prognosis, treatment, or therapy is desired, particularly humans. Other subjects may include cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and so on.
[0061] As used herein, the term a normal individual may be used to refer to an individual who is basically in a healthy condition without particular diseases (e.g., Wilson's disease, Alzheimer's disease and Parkinson's disease), and may refer to a single normal/healthy individual or a group of normal/healthy individuals.
[0062] As used herein, the term ceruloplasmin or the abbreviation Cp refers to a ferroxidase enzyme which in humans is encoded by the CP gene. Ceruloplasmin is made in the liver and then secreted into the blood. It contains multiple copper atoms and oxidizes ferrous ion (Fe.sup.2+) into ferric iron (Fe.sup.3+) to allow the transport of iron. A blood test can determine ceruloplasmin's amount or activity in the blood. For example, the normal values (reference range) for ceruloplasmin's amount in the blood have been investigated and are in general between 20 and 50 mg/dL which can vary with different populations. Measurement of ceruloplasmin in the blood can be used to evaluate and manage of copper/iron-related diseases/conditions. A low amount or low activity of ceruloplasmin can possibility indicate that the person suffers from, for example, aceruloplasminemia characterized by iron accumulation in the brain and other organs which is caused by lack of ceruloplasmin ferroxidase activity due to mutation of the corresponding CP gene; Wilson's disease in which excessive amounts of copper accumulate in the body; and Menke's disease in which copper absorption is decreased from the intestine, causing systemic copper and ceruloplasmin deficiencies. A low amount or low activity of ceruloplasmin is also correlated with some neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease (PD). On the other hand, increased levels of ceruloplasmin may indicate acute inflammation, rheumatoid arthritis or infection.
[0063] According to the present invention, a compound of formula I is used as an enzymatic substrate of ceruloplasmin in a mass spectrometry technique. The compound of formula I is of the structure as follows.
##STR00005## [0064] wherein [0065] R.sub.1 is hydrogen, an alkyl group, an alcohol group, an alkoxy group or an alkyl ether group containing one or more oxyethylene units; [0066] R.sub.2 is hydrogen or hydroxyl; and [0067] R.sub.3 and R.sub.4, are independently hydrogen or an alkylamine group, or [0068] R.sub.3 and R.sub.4 together form a benzene ring fused to the nitrogen-containing bicyclic heteroring, wherein the benzene ring is substituted by NH.sub.2.
[0069] As used herein, the term alkyl refers to an aliphatic hydrocarbon chain and includes straight and branched chains. An alkyl group may be a C.sub.1-C.sub.12 alkyl such as a C.sub.1-C.sub.10 alkyl, a C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.4 alkyl and C.sub.1-C.sub.3 alkyl. Examples of alkyl, include but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, n-pentyl, and isobutyl, and the like.
[0070] As used herein, an alcohol group refers to a hydrocarbon group that includes at least one hydroxy group. An alcohol group may be represented by ROH wherein R is a C.sub.1-C.sub.12 alkylene such as a C.sub.1-C.sub.10 alkylene, a C.sub.1-C.sub.8 alkylene, C.sub.1-C.sub.6 alkylene, C.sub.1-C.sub.4 alkylene and C.sub.1-C.sub.3 alkylene. Examples of alkylene include, but are not limited to, methylene, ethylene, n-propylene, n-butylene, and the like.
[0071] As used herein, the term alkoxyl refers to an alkyl group, as defined above, having an oxygen radical attached thereto. Examples of alkoxyl include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, iso-butoxy, sec-butoxy, and tert-butoxy.
[0072] As used herein, the term alkyl ether refers to an alkyl group having at least one oxygen incorporated into the alkyl chain.
[0073] As used herein, the term oxyethylene units can be represented by (OCH.sub.2CH.sub.2).sub.n wherein n can be from 1 to 8, e.g., 1 to 7, 1 to 6, 1 to 5, or 1 to 4.
[0074] As used herein, the term an alkyl ether group containing one or more oxyethylene units may be represented by formula of (CH.sub.2).sub.m(OCH.sub.2CH.sub.2).sub.n OCH.sub.3, wherein m and n, the same or different, are independently an integer from 1 to 5, e.g., 1, 2, 3, 4 or 5. In some embodiments, m is 2 and n is 4.
[0075] As used herein, the term alkylamine refers to an alkyl-NH.sub.2 group.
[0076] In some embodiments, R.sub.1 is hydrogen, an C.sub.1-C.sub.12 alkyl group, an C.sub.1-C.sub.12 alcohol group, an C.sub.1-C.sub.12 alkoxy group or an alkyl ether group containing one or more oxyethylene units represented by formula of (CH.sub.2).sub.m(OCH.sub.2CH.sub.2).sub.n OCH.sub.3 wherein m and n, the same or different, are independently an integer from 1 to 5, e.g., 1, 2, 3, 4 or 5; R.sub.2 is hydrogen or hydroxyl; and R.sub.3 and R.sub.4, the same or different, are independently hydrogen or an C.sub.1-C.sub.12 alkylamine group.
[0077] In some embodiments, R.sub.1 is hydrogen, R.sub.2 is hydroxyl, R.sub.3 is an C.sub.1-C.sub.12 alkylamine group and R.sub.4 is hydrogen.
[0078] In some particular embodiments, the compound of the present invention is represented by formula Ia
##STR00006##
[0079] In some embodiments, R.sub.1 is hydrogen, an C.sub.1-C.sub.12 alkyl group, an C.sub.1-C.sub.12 alcohol group, an C.sub.1-C.sub.12 alkoxy group or an alkyl ether group containing one or more oxyethylene units represented by formula of (CH.sub.2).sub.m(OCH.sub.2CH.sub.2).sub.n OCH.sub.3 wherein m and n, the same or different, are independently an integer from 1 to 5, e.g., 1, 2, 3, 4 or 5; R.sub.2 is hydrogen or hydroxyl; and R.sub.3 and R.sub.4 together form a benzene ring fused to the nitrogen-containing bicyclic heteroring, wherein the benzene ring is substituted by NH.sub.2.
[0080] In some embodiments, R.sub.1 is an C.sub.1-C.sub.12 alkyl group, an C.sub.1-C.sub.12 alcohol group or an alkyl ether group containing one or more oxyethylene units represented by formula of (CH.sub.2).sub.m(OCH.sub.2CH.sub.2).sub.n OCH.sub.3 wherein m and n, the same or different, are independently an integer from 1 to 5, e.g., 1, 2, 3, 4 or 5; Rz is hydrogen; and R.sub.3 and R.sub.4 together form a benzene ring fused to the nitrogen-containing bicyclic heteroring, wherein the benzene ring is substituted by NH.sub.2.
[0081] In some certain embodiments, R.sub.1 is selected from the group consisting of CH.sub.3, CH.sub.2CH.sub.3, CH.sub.2CH.sub.2CH.sub.2CH.sub.3, CH.sub.2CH.sub.2OH, CH.sub.2CH.sub.2CH.sub.2OH and CH.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.4OCH.sub.3.
[0082] In some particular embodiments, the compound of the present invention is represented by formula Ib
##STR00007##
[0083] Some examples of the compound of formula I are provided in Table 1 below.
TABLE-US-00001 TABLE 1 Examples of commpound of formula I Name Structure ACK-1000
[0084] According to the present invention, the compound of the present invention can be oxidized by ceruloplasmin to an oxidized product. In some embodiments, the oxidized compound reacts with the original (unoxidized) compounds and forms a polymeric (e.g. dimeric or trimeric) structure. In some embodiments, the resultant oxidized product in a polymeric structure is of formula II
##STR00015##
wherein R.sup.1 is as defined as R.sub.1 as described herein. Certain examples of the resultant oxidized product in a polymeric structure are provided as follows.
TABLE-US-00002 TABLE 2 Examples of resultant oxidized product Name Structure Oxi- dized ACK- 1001 Formula II
[0085] In step (i) of the method of the invention, the biological sample is reacted with the compound of formula I to obtain a reaction mixture comprising the oxidized product of the compound.
[0086] In some embodiments, said compound is allowed to react with the biological sample for about 15 to about 60 minutes at about 35 C. to about 40 C. Preferably, the reaction is allowed for about 20 to about 30 minutes at about 35 C. to about 40 C. More preferably, the reaction is allowed for about 30 minutes at about 35 C. to about 40 C.
[0087] In some embodiments, the method of the present invention may further comprise before step (ii) the following step: (i-a) quenching the reaction mixture. Preferably, the reaction mixture is quenched by adding a quenching agent. A particular example of the quenching agent is dimethyl sulfoxide (DMSO).
[0088] In some embodiments, the method of the present invention may further comprise before step (ii) the following step: (i-b) collecting from the reaction mixture a solution comprising the oxidized product of the compound. In certain embodiments, the solution is collected by a method comprising: subjecting the quenched reaction mixture to centrifugation, and then collecting the supernatant liquid as the solution comprising the oxidized product.
[0089] In step (ii) of the method of the invention, the oxidized product as produced is measured by a mass spectrometry technique. A mass spectrometry technique is known and available in this art which is an analytical technique that measures the mass-to-charge ratio of charged particles. In some embodiments, the mass spectrometry technique is a tandem mass spectrometry (MS/MS) technique, such as an LC-MS/MS technique. In general, in a MS analysis, the area under the peak is proportional to the amount of analyte injected onto the column. A peak's area can be determined by integration which is usually handled by the instrument's computer. If the peak is clear, the determination of the area under the peak is straightforward. As shown in the examples below, unique and distinct peaks with strong signal intensity are observed for the oxidized products of the compounds of the present invention.
[0090] In step (iii) of the method of the invention, a level of ceruloplasmin activity is determined based on the level (amount) of the oxidized product as measured through the mass spectrometry technique. According to the present invention, the level (amount) of the oxidized product as measured is positively correlated with the level of ceruloplasmin activity. Therefore, a decreased level (amount) of the oxidized product is indicative of a decreased level of ceruloplasmin activity and/or an increased level (amount) of the oxidized product is indicative of an increased level of ceruloplasmin activity. Particularly, the level (amount) of the oxidized product as measured can be further divided by the volume of the biological sample to represent a specific enzymatic activity unit. As shown in the examples below, the specific ceruloplasmin activity unit can be calculated by the formula: Specific Cp activity unit=Peak area of oxidized product MS2 ion/amount of serum (L).
[0091] As used herein, a decreased level or increased level can refer to a level that is decreased or increased compared with a reference level. For example, a decreased level can be lower than a reference level by more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%; and an increased level can be higher than a reference level by more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In some embodiments, a reference level can be a standard (or a threshold) level in a control group. For example, a standard level can be set based on an average or median level obtained from a cohort of normal subjects. In particular embodiments, the cohort of subjects can be a population of normal human.
[0092] In some embodiment, the biological sample used in the present invention is a liquid biological sample, which includes but is not limited to a serum sample or a urine sample.
[0093] The method of the present invention provides excellent reliability and sensitivity. More specifically, the method of the present invention requires trace amounts of a biological sample to be tested and achieves desired reliability and sensitivity. In some embodiments, the method of the present invention requires the biological sample having a volume of 0.01 L to 10 L, for example, 0.05 L to 5 L, such as 0.05 L to 2 L, 0.05 L to 1 L or 0.05 L to 0.5 L.
[0094] Since ceruloplasmin activity is known to be associated with a number of diseases/conditions, the method of the present invention of determining ceruloplasmin activity is applicable in clinical applications for predicting the risk of a disease related to ceruloplasmin activity in a subject.
[0095] Therefore, the present invention also provides a method for determining ceruloplasmin activity and predicting the risk of a disease related to ceruloplasmin activity in a subject, comprising [0096] (i) providing a biological sample which is obtained from the subject; [0097] (ii) contacting the biological sample with a compound of formula I as described herein under a condition such that the compound is oxidized to produce an oxidized product of the compound; [0098] (iii) measuring a level of the oxidized product by a mass spectrometry technique; and [0099] (iv) determining a level of ceruloplasmin activity and the likelihood of having a disease or condition related to ceruloplasmin activity of said subject based on the level of the oxidized product, wherein [0100] a decreased level of the oxidized product below a reference level is indicative of deficiency of ceruloplasmin activity and an increased likelihood of having a disease or condition related to deficiency of ceruloplasmin activity; and [0101] an increased level of the oxidized product above a reference level is indicative of excess of ceruloplasmin activity and an increased likelihood of having a disease or condition related to excess of ceruloplasmin activity.
[0102] Some diseases or conditions are known to be corelated with deficiency of ceruloplasmin activity. For example, aceruloplasminemia exhibits deficiency of ceruloplasmin activity which is directly caused by Cp protein gene.sup.20. Some neurodegenerative diseases, including Alzheimer's disease.sup.26 and Parkinson's disease.sup.27-28 are known to exhibit deficiency of ceruloplasmin activity. Additional disease or condition related to deficiency of ceruloplasmin activity include Wilson's disease.sup.21 and Menke's disease.
[0103] Some diseases or conditions are known to be corelated with excess of ceruloplasmin activity. Examples of such diseases or conditions include Type II diabetes mellitus.sup.22, cardiovascular diseases, such as valvular heart disease.sup.24 and coronary heart disease.sup.25, bladder cancer,.sup.29 chronic obstructive pulmonary disease.sup.30 and kidney diseases..sup.23
[0104] In some embodiments, the method of the present invention is applicable for a fetus, a neonate, a child, an adolescent or an adult. In particular, the method of the present invention is applicable for a fetus, a neonate or a child where blood is difficult to draw.
[0105] The present invention also provides a kit for performing the method as described herein, which comprises the compound of formula I as the enzymatic substrate, a reaction buffer for oxidation, a quenching agent for stopping oxidation, and instructions for using the kit to determine ceruloplasmin activity and/or predicting the risk of a disease related to ceruloplasmin activity in a subject by using a mass spectrometry technique. The components including the compound of formula I, the reaction buffer and the quenching agent can be packaged in the form of a kit. For example, the components can respectively be packaged in separate containers or some of the components packed in one container and the other packed in another container.
[0106] The present invention is further illustrated by the following examples, which are provided for the purpose of demonstration rather than limitation. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Examples
[0107] A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of serum oxidase activity. Through the combinatorial library screening of in-house compounds, including natural products, synthetic compounds, and heterocyclic molecules, hit substrates which provided unique and selective oxidized products in ESI-MS analysis were selected. To optimized the detection method, the modification of hit substrates and the alternation of assay conditions were applied. Furthermore, the lead substrates provided good quantitative measurement of serum oxidases activity with less amount of serum samples (<5 L) in LC-MS/MS. At last, spike-in ceruloplasmin showed good linearity (R.sup.2=0.9971), representing that serum oxidases had positive correlation with ceruloplasmin as well as the modified substrates could be oxidized by both ceruloplasmin and serum oxidases. This method can be widely applied to the detection of disease related to serum oxidase activity.
1. Material and Methods
1.1 General Information
[0108] All reagents and solvents were purchased from commercial suppliers and used without further purification. All non-aqueous reactions were performed in oven-dried glassware under a slight positive pressure of argon unless otherwise noted. NMR spectra were recorded on dilute solutions in CDCl.sub.3 and CD.sub.3OD on Bruker AVANCE 600 spectrometer at ambient temperature. Chemical shifts are given in values and coupling constants J are given in Hz. The splitting patterns are reported as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and dd (double of doublets). Reactions were magnetically stirred and monitored by thin-layer chromatography on silica gel. Flash column chromatography was performed on silica gel of 40-63 m particle size. Preparative TLC chromatography was performed on silica gel F254 preparative TLC plates (2020 cm, 0.5 mm layer and fluorescence at 254 nm, Merck). Rotary evaporation was used to concentrate the reaction mixture.
1.2 Screening for Candidate Chemicals
[0109] The chemical library dissolved in DMSO individually to give stock concentrations of 1 mg/mL. Each chemical was placed in individual well and incubated with serum sample in acetate buffer (0.1M, pH 5.0) at 37 C. for 1 h. Additional DMSO was provided to inhibit oxidase activity. High-resolution ESI mass spectra (Bruker Daltonics spectrometer) was utilized to monitor the oxidized products.
1.3 Synthesis of Compounds as CP Substrates
1.3.1 Serotonin (or 5-Hydroxytryptamine) (ACK-1000)
[0110] Serotonin (or 5-Hydroxytryptamine), named as ACK-1000 herein, was purchased from Cayman Chemical Company (Ann Arbor, MI, U.S.A.).
1.3.2 Amino Carbazoles (ACK-1001 to ACK-1006)
1.3.2.1 General Procedure for 9-Alkyl-9H-Carbazole (Compound 2)
[0111] Carbazole (Compound 1) (500 mg, 3 mmol) was dissolved in dried DMF (4 mL), and sodium hydride (60%) (300 mg, 7.4 mmol) was added proportionally at 0 C. After vigorously stirring for 30 min, each alkyl compound (6 mmol) as an alkylating reagent was prepared in DMF (1 mL) and added dropwise to the reaction at 0 C. The mixture was stirred for 4 hr then the reaction was quenched by water (10 mL). The precipitate was dissolved by ethyl acetate (30 mL) and washed with brine. The organic layers were collected, dried with MgSO.sub.4, and concentrated. The crude product was purified by CC and characterized by NMR and mass spectrometry.
(i) 9-buyl-9H-carbazole
[0112] 1-Bromobutane (652 L, 6 mmol) was used as the alkyl compound. The crude product was purified by CC (10% EA in hexanes, silica gel) to give N-butyl carbazole (647 mg, 2.9 mmol, 98% yield) as a white solid. .sup.1H NMR (600 MHz, CDCl.sub.3) 3.89 (t, J=4.8 Hz, 3H), 7.25-7.29 (m, 2H), 7.42-7.45 (m, 2H), 7.49-7.53 (m, 2H), 8.12-8.15 (m, 2H).
(ii) 9-methyl-9H-carbazole
[0113] Iodomethane (374 L, 6 mmol) was used as the alkyl compound. The crude product was purified by CC (20% EA in hexanes, silica gel) to give N-methyl carbazole (436 mg, 2.4 mmol, 80% yield) as a white solid..sup.1H NMR (600 MHz, CDCl.sub.3) 3.89 (t, 3H, J=4.1 Hz), 7.25-7.29 (m, 2H), 7.42-7.45 (m, 2H), 7.49-7.53 (m, 2H), 8.12-8.15 (m, 2H).
(iii) 2-(9H-carbazol-9-yl)ethan-1-ol
[0114] 3-Bromoethanol (440 L, 6 mmol) was used as the alkyl compound. The crude product was purified by CC (40% EA in hexanes, silica gel) to give N-(2-hydroxyl)ethyl carbazole (506 mg, 2.4 mmol, 40% yield) as a white solid. .sup.1H NMR (600 MHz, CDCl.sub.3) 4.06 (dt, J=5.3, 5.5 Hz, 2H), 4.48 (t, J=5.5 Hz, 2H), 7.23-7.25 (m, 2H), 7.46-7.47 (m, 4H), 8.09-8.10 (m, 2H)
(iv) 3-(9H-carbazol-9-yl)propan-1-ol
[0115] 3-Bromopropanol (545 L, 6 mmol) was used as the alkyl compound. Furthermore, the reaction mixture was microwaved for 1 hr at 135 C. The crude product was purified by CC (40% EA in hexanes, silica gel) to give N-(3-hydroxyl)propyl carbazole (715 mg, 3.2 mmol, 53% yield) as a white solid. .sup.1H NMR (600 MHz, CDCl.sub.3) 2.13 (quin, J=6.0 Hz, 2H), 3.62 (dt, J=5.6, 3.9 Hz, 2H), 4.48 (t, J=6.7 Hz, 2H), 7.22-7.25 (m, 2H), 7.46-7.47 (m, 2H), 8.10-8.11 (m, 2H).
(v) 9-(2,5,8,11-tetraoxatridecan-13-yl)-9H-carbazole
[0116] Tetraethylene glycol (288 mg, 1.48 mmol) and potassium hydroxide (99 mg, 1.77 mmol) were added to the mixed solvent of water (1 mL) and 1,4-dioxane (4 mL). After stirring for 2 min, dimethylsulfate (50 L, 0.47 mmol) was added dropwise to the reaction at room temperature. Subsequently, the mixture was heated by microwave at 135 C. for 1 hour. To the end of the reaction, the solvent was evaporated and water (30 mL) was added in. The solution was extracted by DCM and washed with brine. The combination of organic layers were dried with MgSO.sub.4 and concentrated. The crude product (100 mg, 0.48 mmol) and triethylamine (88 L, 0.62 mmol) were added to dried DCM under ice bath. Tosylchloride (110 mg, 0.57 mmol) prepared in DCM (1 mL) was added dropwise to the solution. After 2 hours of stirring, the solvent was evaporated and re-dissolved with EA. The organic layers were washed with brine, dried with MgSO.sub.4, and concentrated. The crude product was purified by CC (DCM: methanol=19:1) to give 2,5,8,11-tetraoxatridecan-13-yl 4-methylbenzenesulfonate (133 mg, 0.4 mmol, 85% yield).
[0117] 2,5,8,11-Tetraoxatridecan-13-yl 4-methylbenzenesulfonate (323 mg, 0.9 mmol) was used as the alkyl compound. The crude product was purified by CC (5% methanol in DCM, silica gel) to give the N-alkylated carbazole (80 mg, 0.22 mmol, 37% yield) as a colorless oil. .sup.1H NMR (600 MHz, CDCl.sub.3) 3.15 (s, 3H), 3.28-3.36 (m, 14H), 3.64 (t, J=6.0 Hz, 2H), 4.27 (t, J=6.1 Hz, 2H), 7.01-7.03 (m, 2H), 7.24 (m, 4H), 7.86-7.88 (m, 2H).
(vi) 9-ethyl-9H-carbazole
[0118] 1-Bromoethane (445 L, 6 mmol) was used as the alkyl compound. The crude product was purified by CC (10% EA in hexanes, silica gel) to give N-butyl carbazole (556 mg, 2.9 mmol, 95% yield) as a white solid. .sup.1H NMR (600 MHz, CDCl.sub.3) 1.40 (t, 3H, J=7.2 Hz), 4.31 (q, 2H, J=7.2 Hz), 7.25-7.29 (m, 2H), 7.42-7.45 (m, 2H), 7.49-7.53 (m, 2H), 8.12-8.15 (m, 2H).
1.3.2.2 General Procedure for 9-alkyl-9H-carbazol-3-amine (Compound 3)
[0119] Compound 2 (100 mg, 0.45 mmol) was dissolved in acetic acid (3 mL), and stirred for 5 min under room temperature. A dissolved Cu(NO.sub.3).sub.2 solution (containing Cu(NO.sub.3).sub.2.Math.3H.sub.2O (140 mg, 0.60 mmol), acetic anhydride (0.2 mL) and acetic acid (1 mL)) was added dropwise to the carbazole solution. After stirring for 1 hr, water was added to the solution and the pH value was adjusted to 7-8 with 10% NaOH.sub.(aq). Subsequently, the mixture was extracted by EA (30 mL, three times) and washed with brine. The combined organic layers were dried with MgSO.sub.4 and concentrated. The crude product was purified by CC to afford nitrated compounds. The nitrated compounds (100 mg, 0.37 mmol) was dissolved in THF/methanol (2 mL, 2 mL) then Pd/C (1 mg) was added to the solution. The mixture was stirred at 60 C. for 2 min and the solution of hydrazine monohydrate (600 L in 1 mL methanol) was added dropwise to the mixture. After the reaction was checked by TLC, the solvent of the reaction was evaporated and the residue was dissolved by EA (45 mL). The organic layers were washed with brine, dried with MgSO.sub.4 and concentrated. The crude product was purified by CC and characterized by NMR and mass spectrometry.
(i) 9-buyl-9H-carbazol-3-amine (ACK-1001)
[0120] The crude product was purified by preparative TLC (50% DCM and 1% NH.sub.4OH in hexanes) to give ACK-1001 (22 mg, 0.09 mmol, 25% yield) as a dark red solid. .sup.1H NMR (600 MHz, CDC.sub.3), =0.93 (t, J=7.3 Hz, 3H), 1.38 (sext, J=7.7 Hz, 2H), 1.82 (quint, J=7.5 Hz), 4.24 (t, J=7.2 Hz), 6.91 (dd, J=1.7, 8.6 Hz, 1H), 7.14 (t, J=7.5 Hz, 1H), 7.21 (d, J=8.4 Hz, 1H), 7.33 (d, J=8.1 Hz, 1H), 7.39-7.42 (m, 2H), 7.99 (d, J=7.6 Hz, 1H).
##STR00022##
(ii) 9-methyl-9H-carbazol-3-amine (ACK-1002)
[0121] The crude product was purified by CC (50% DCM and 1% NH.sub.4OH in hexanes) to give ACK-1002 (36 mg, 0.18 mmol, 40% yield) as a dark red oil. .sup.1H NMR (600 MHz, CDCl.sub.3), =3.79 (s, 3H), 6.92 (dd, J=1.5, 8.3 Hz, 1H), 7.15 (t, J=7.4 Hz, 1H), 7.21 (d, J=8.2 Hz, 1H), 7.33 (d, J=8.2 Hz, 1H), 7.41-7.44 (m, 2H), 7.99 (d, J=7.7 Hz, 1H).
##STR00023##
(iii) 2-(3-amino-9H-carbazol-9-yl)ethan-1-ol (ACK-1003)
[0122] The crude product was purified by CC (5% methanol and 1% NH.sub.4OH in DCM) to give ACK-1003 (7 mg, 0.03 mmol, 8% yield) as a dark red oil. .sup.1H NMR (600 MHz, CDCl.sub.3), =4.03 (t, J=5.3 Hz, 2H), 4.42 (t, J=5.3 Hz, 2H), 6.90 (dd, J=2.2, 8.5 Hz, 1H), 7.17 (m, 1H), 7.27 (m, 1H), 7.40-7.41 (m, 3H), 7.89 (dd, J=3.3, 7.5 Hz, 1H).
##STR00024##
(iv) 3-(3-amino-9H-carbazol-9-yl)propan-1-ol (ACK-1004)
[0123] The crude product was purified by CC (5% methanol and 1% NH.sub.4OH in DCM) to give ACK-1004 (13 mg, 0.05 mmol, 27% yield) as a dark red solid. .sup.1H NMR (600 MHz, CDCl.sub.3), =2.09 (quint, J=6.0 Hz, 2H), 3.61 (t, J=6.5 Hz, 2H), 4.40 (dt, J=6.4, 6.6 Hz, 2H), 6.97 (dd, J=7.7, 7.7 Hz, 1H), 7.08 (dd, J=7.7, 7.7 Hz, 1H), 7.20 (dd, J=6.8, 7.4 Hz, 1H), 0.7.27 (s, 1H), 7.40-7.44 (m, 2H), 8.23 (t, J=7.44 Hz, 1H).
##STR00025##
(v) 9-(2,5,8,11-tetraoxatridecan-13-yl)-9H-carbazol-3-amine (ACK-1005)
[0124] The crude product was purified by CC (5% methanol and 1% NH.sub.4OH in DCM) to give ACK-1005 (5 mg, 0.01 mmol, 26% yield) as a dark red oil. .sup.1H NMR (600 MHz, CDCl.sub.3), =3.35 (s, 3H), 3.50-3.55 (m, 10H), 3.58-3.59 (m, 2H), 3.83 (t, J=6.5 Hz, 2H), 4.43 (t, J=6.4 Hz, 2H), 6.89 (dd, J=2.4, 8.6 Hz, 1H), 7.14 (ddd, J=2.1, 5.8, 7.4 Hz, 1H), 7.27 (d, J=8.5 Hz, 1H), 7.39-7.40 (m, 3H), 7.96 (m, 1H).
##STR00026##
(vi) 9-ethyl-9H-carbazol-3-amine (ACK-1006)
[0125] The crude product was purified by CC (50% DCM and 1% NH.sub.4OH in hexanes) to give ACK-1006 (70 mg, 0.33 mmol, 90% yield) as a dark red oil. .sup.1H NMR (600 MHz, CDCl.sub.3), =1.40 (t, 3H, J=7.2 Hz), 4.31 (q, 2H, J=7.2 Hz), 6.91 (dd, J=2.1, 6.42 Hz, 1H), 7.15 (t, J=7.4 Hz, 1H), 7.21 (d, J=8.5 Hz, 1H), 7.33 (d, J=8.1 Hz, 1H), 7.41-7.44 (m, 2H), 7.99 (d, J=7.7 Hz, 1H).
##STR00027##
1.4 LC-MS/MS Based Serum Oxidase Activity Assay
[0126] Samples were detected by LC-ESI-MS on a Velos Pro dual-pressure linear ion trap mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with an Ultimate 3000 RSLC system from Dionex (Dionex Corporation, Sunnyvale, CA), using a Xbridge C18 (HPLC column 1 mm15 cm i.d., 3.5 m particle size, 130 pore size). Briefly, the gradient employed was 50% buffer B at 5 min to 98% buffer B at 23 min with a flow rate of 50 L/min, where buffer A was 0.1% formic acid/H.sub.2O and buffer B was 0.1% formic acid/acetonitrile. All spectra were acquired in the mass range m/z 200-2000. Electrospray voltage was maintained at 4.0 kV and capillary temperature was set at 275 C.
[0127] The stock solution of modified amino carbazoles (125 M, 20 L) were incubated with 5 L of serum (estimate<10 nM of serum oxidases) in sodium acetate buffer (0.1M, pH 5.0, 5 L) at 37 C. for 30 min. The serum oxidase oxidation was quenched by DMSO (90 L), followed detection of LC-MS/MS.
[0128] The amount of the oxidized product was determined according to the area under the peak by integration. The specific Cp activity unit was calculated by the formula: Peak area of oxidized product MS2 ion/serum volume (L).
2. Results
2.1 Compounds as Substrates for Serum (ACK-1000 and ACK-1001 to ACK-1006)
[0129] To screen for suitable compounds as Cp substrates for MS analysis, an approach of combinatorial library screening in primary MS analysis was used to find out oxidized products and their relationship with initial substrates. Briefly, a platform for rapidly and conveniently screening various small molecules was established. In the chemical library collection as obtained, including natural products, synthetic compounds and heterocyclic molecules, each member was parallelly incubated with a certain small amount of serum for a desired reaction time. After quenching each enzyme reaction, analysis of the reaction products with ESI-MS was performed. Hits with reliable, major, and distinct oxidized product peaks in ESI-MS analysis were selected for further studies (
[0130] Through screening, certain heterocyclic compounds, ACK-1000 and ACK-1001 (
2.2 LC-MS/MS Detection of Serum Oxidase Activity
[0131] Detection of serum oxidase activity with the candidate compounds was performed by LC-MS/MS. Unique and distinct peaks with strong signal intensity were observed, indicating that the oxidized products of these candidate compounds (ACK-1001 to ACK-1006) are reliable bio-markers for detection of serum oxidases (
2.3 Substrates with Specific Reactivity for CP
[0132] To understand the relation between ceruloplasmin and serum oxidases and the reactivity of modified substrates and ceruloplasmin, spike-in experiment was performed. Different amounts of recombinant ceruloplasmin (0, 0.5, and 2.5 L) were added in the normal serum sample (5 L) and reacted with ACK-1006. The assay was incubated at 37 C. for 30 mins, and quenching the reaction by DMSO. The final products were analyzed by LC-MS/MS. By comparing the LC peak area, the increasing amount of ceruloplasmin contributed the growing quantity of oxidized product and showed good linearity (R.sup.2=0.9971) (
[0133] Another way to confirm that the ACK-1006 substrate exclusively reacts with ceruloplasmin, protein depletion test was applied. The Cp depletion serum was obtained by size selection method. Briefly, the centrifuge filter was applied in normal serum to eliminate those proteins larger than 100 kDa, including Cp (130 kDa). ACK-1006 was incubated with the Cp depleted serum and trace amount of oxidized product were detected by LCMS. Comparison with serum and plasma from normal adults representing p value smaller than 0.001 indicating that ACK-1006 is affected by Cp activity (
2.4 Establishment of LC-MS/MS Based Measurement of Serum Cp Oxidase Activity
[0134] Accordingly, the method of LC-MS/MS based assay based on the determination of the oxidized products was established (
[0135] To obtain the accurate and precise analysis, assay conditions have been further optimized. Substrate ACK-1006 was used in the test and detection was taken at different reaction times. The time-dependent assay exhibited that oxidized product reached highest amount after 30 min reaction, indicating that serum Cp oxidase is active within 30 min incubation. Thus, the assay time has been adjusted to accomplish within 30 mins (
2.5 Limitations of Detection
[0136] The limitations of current methods and available commercial kits were described. In current method, the colorimetric methods using p-phenylenediamine (PPD) and o-dianisidine dihydrochloride (ODD) as substrates are used for quantification of ceruloplasmin oxidative activity. Those substrates oxidized by ceruloplasmin and resulted in obvious color changes. The activity of ceruloplasmin were positively correlated to absorbance. However, the correlation of absorbance and serum volume small than 10 L was poor (
[0137] Two commercial kits were available (BioVision kit and Invitrogen kit) for quantification of serum Cp activity. There was a good correlation between absorbance and serum volume from 0.25 L to 10 L. (
[0138] To detect the limitation of ACK1006-LC/MS method, the ACK-1006 and serum oxidation mixture were diluted to 10-1,000 folds and analyzed by LC-MS/MS. The calibration curve exhibited a good correlation between the observed peak area of MS2 ion (y) and the serum volume (x) (R.sup.2=0.9987) (
2.6 Detection of Cp Activity in Newborn Serum
[0139] This established Cp-activity assay by novel substrate ACK-1006 in LC-MS was also reliable even in the newborn plasma which contains extreme lower ceruloplasmin than normal adults. Substrate ACK-1006 were oxidized by new born plasma and showed a distinctive signal (>10.sup.5) by LCMS (
3. CONCLUSIONS
[0140] Through our efforts, ACK-1000 was initially found out via diverse library screening with the LC-MS analysis. After structural modifications, a series of analogues (ACK1001-1006) were designed and synthesized for further investigation. Fortunately, qualified new Cp substrates were identified with an assistance of LC-MS/MS detection. The relative standard deviation (<12%) exhibited excellent stability of MS spectrometry, and a squares regression R.sup.2=0.9968 displayed that the LC-MS/MS based assay provided decent linearity and sensitivity. In addition, spike-in recombinant ceruloplasmin showed that the modified substrate ACK-1006 as an example had a positive correlation with ceruloplasmin amounts. In contrast, the incubation of Cp-depletion serum and ACK-1006 produced trace amount of an oxidized signal from the oxidation of substrate in comparison with normal serum (P value=0.001). Both Cp spike-in and depletion experiment showed that ACK-1006 is specifically affected by Cp activity. Thus, the ACK1006-LC-MS/MS micro-analytical method is suitable for clinical applications of those diseases with abnormal amounts or oxidative activities of Cp in newborns.
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