Patent Application
20120165227
The present disclosure is in the field of analysis of carboxylic acids in samples, in particular, using differential isotope labeling coupled with mass spectrometry.
One of the early reports of using differential isotope labeling (DIL) for metabolite analysis was the use of the iTRAQ reagent, commonly known as the labeling reagent for peptides for quantitative proteomics, to label amino acids for quantitative analysis of these small molecules in urine and blood samples.1 Fukusaki et al reported the use of 13C- and 12C-methylation to introduce differential isotope tags to flavonoids for relative quantification.2 Yang et al described a LC/MS method for amino acid analysis involving derivatization with an N-hydroxysuccinimide ester of N-alkylnicotinic acid where the alkyl chain can contain deuterium, instead of hydrogen, to provide a differential isotope tag.3 Shortreed, et al reported the use of heavy and light isotopic forms of methyl acetimidate for the relative metabolome quantifications of amine-containing species.4 Guo et al used the reductive amination reaction to label amine-containing metabolites with 13C- and 12C-formaldehyde for relative metabolome quantifications.5 Ji et al reported the use of acetaldehyde d(4) to label and quantify the monoamine neurotranmitters in rat brain microdialysates.6 Abello et al developed isotope tagged pentafluorophenyl-activated esters of poly(ethylene glycol) to label amine-containing metabolites with multiplexing capability.7 13C4 labeled succinic anhydride and deuterated (D9) butanol have been used for labeling metabolites for relative metabolome analysis.8 While LC/MS is commonly used for detecting the differential isotope labeled metabolites, GC/MS has also been combined with chemical derivatization with isotope-coded reagents for metabolome analysis.9 It should be noted that a related method using isotope enriched media for cell culturing has been used for quantitative metabolomics.10-16 More recently, Guo and Li reported the use of dansyl chloride as a labeling reagent for analyzing amine- and phenol-containing metabolites.17
Phenacyl bromide has been used to form derivatives of carboxylic acids for analytical applications such as improving the performance of HPLC and UV detection.18,19 The synthesis of an isotope form of phenacyl bromide (5 hydrogen atoms in the benzene ring were replaced by 5 deuterium atoms) has been reported.20 This reagent was used to label peptides for MS analysis.20,21
Novel stable-isotope forms of p-dimethylaminophenacyl bromide (DmPA) have been prepared and shown to be useful as reagents for binary and multiplex quantification of carboxylic acids.
Accordingly, the present disclosure includes a compound of Formula I:
wherein
Ar is phenyl or naphthyl;
R1 is a suitable leaving group;
R2 is C1-10alkyl in which one, two or three of the carbon atoms, with the exception of the carbon atom attached to the nitrogen, is optionally replaced with O and/or NR4 and one or more of the carbon atoms in R2 is present as a carbon-13 isotope in amounts greater than the natural abundance of carbon-13 and/or one or more of the oxygen atoms, if present, in R2 is present as an oxygen-18 isotope in amounts greater than the natural abundance of oxygen-18;
R3 is selected from C1-6alkoxy and N(C1-6alkyl)2;
R4 is selected from H and C1-6alkyl; and
n is 0, 1, 2, 3 or 4, and
salts and solvates thereof.
The present application also includes carboxylic acids derivatized with a compound of Formula I as defined above. It will be appreciated by those skilled in the art that any compound comprising at least one carboxylic acid moiety is capable of reacting with the compounds of Formula I to form the corresponding ester. It is an embodiment of the disclosure that the carboxylic acid is a metabolite found in a biological sample.
Also included in the present disclosure in a method of preparing an ester of a carboxylic acid comprising reacting a compound comprising at least one carboxylic acid with a compound of Formula I as defined above in the presence of a suitable base under conditions to form the ester of the one or more carboxylic acids.
The present application also includes a library comprising, consisting essentially of or consisting of two or more esters of a carboxylic acid, wherein the esters are formed by the reaction of a compound of Formula I as defined above with the carboxylic acid in the presence of a base.
The disclosure also includes a binary method of quantifying one or more carboxylic acids in first and second samples comprising:
In an embodiment of the disclosure, one of the first or second sample comprises one or more standard carboxylic acids with known concentrations and the method provides an absolute quantification of the one or more carboxylic acids in the other of the first or second sample.
The present disclosure also includes a multiplex method of quantifying one or more carboxylic acids in three or more samples comprising:
In an embodiment of the disclosure, where the standard sample comprises one or more standard carboxylic acids with known concentrations, the method provides an absolute quantification of the one or more carboxylic acids in the two or more samples.
Advantages of the compounds of Formula I as differential isotope labeling reagents, include, for example,
Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the disclosure are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.
The present disclosure will now be described in greater detail with reference to the drawings in which:
The term “alkyl” as used herein refers to straight and branched chain alkyl groups having 1, 2, 3, 4, 5 or 6 carbon atoms.
The term “salt” means an acid addition salt or basic addition salt.
The term “acid addition salt” as used herein means any organic or inorganic salt of any base compound of the disclosure, or any of its intermediates. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable salts include mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, oxalic acid cinnamic and salicylic acids, as well as sulfonic acids such as p-toluene sulfonic and methanesulfonic acids. Either the mono or di-acid salts can be formed, and such salts may exist in either a hydrated, solvated or substantially anhydrous form. In general, the acid addition salts of the compounds of the invention are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms. The selection of the appropriate salt will be known to one skilled in the art.
The term “basic addition salt” as used herein means any organic or inorganic base addition salt of any acid compound of the disclosure, or any of its intermediates. Illustrative inorganic bases which form suitable salts include lithium, sodium, potassium, calcium, magnesium or barium hydroxide. Illustrative organic bases which form suitable salts include aliphatic, alicyclic or aromatic organic amines such as methylamine, trimethylamine and picoline or ammonia. The selection of the appropriate salt will be known to a person skilled in the art.
The formation of a desired compound salt is achieved using standard techniques. For example, the neutral compound is treated with an acid or base in a suitable solvent and the formed salt is isolated by filtration, extraction or any other suitable method.
The term “solvate” as used herein means a compound or a salt of a compound, wherein molecules of a suitable solvent are incorporated in the crystal lattice. Examples of suitable solvents are ethanol, water and the like. When water is the solvent, the molecule is referred to as a “hydrate”. The formation of solvates will vary depending on the compound and the solvate. In general, solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent. The solvate is typically dried or azeotroped under ambient conditions.
“Stable isotopes” of elements as used herein means an isotope of an element having identical numbers of protons and electrons, but having an additional neutron, which increases the molecular weight of the element by one mass unit.
In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The term “consisting essentially of”, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and/or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and/or steps.
Terms of degree such as “substantially”, “about” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies.
The present disclosure includes a compound of Formula I:
wherein
Ar is phenyl or naphthyl;
R1 is a suitable leaving group;
R2 is C1-10alkyl in which one, two or three of the carbon atoms, with the exception of the carbon atom attached to the nitrogen, is optionally replaced with 0 and/or NR4 and one or more of the carbon atoms in R2 is present as a carbon-13 isotope in amounts greater than the natural abundance of carbon-13 and/or one or more of the oxygen atoms, if present, in R2 is present as an oxygen-18 isotope in amounts greater than the natural abundance of oxygen-18;
R3 is selected from C1-6alkoxy and N(C1-6alkyl)2;
R4 is selected from H and C1-6alkyl; and
n is 0, 1, 2, 3 or 4, and salts and solvates thereof.
In an embodiment of the disclosure Ar is phenyl.
In an embodiment of the disclosure, R1 is a halogen selected from Br, I, Cl and F or R1 is OTosyl. In a further embodiment R1 is Br.
In a further embodiment R2 is 13CH3, 13CH3CH2, CH313CH2, 13CH313CH2, (CH3)213CH, (13CH3)2CH, (13CH3)213CH, 13CH3OCH2CH2, CH3O13CH213CH2, 13CH3O13CH213CH2, CH318OCH2CH2, or CH318O13CH213CH2. In another embodiment, R2 is 13CH3.
In another embodiment, R3 is CH3O or CH3CH2O and n is 1 or 2. In a further embodiment n is 0.
In another embodiment, R4 is H, CH3 or CH2CH3. In a further embodiment R4 is H or CH3.
In another embodiment of the disclosure, when Ar is phenyl, the group N(R2)2 is attached to the phenyl ring at the position that is para to the C(O)CH2R1 group. In another embodiment, when Ar is naphthyl, the group N(R2)2 is attached to the naphthyl ring at the position that is para to the C(O)CH2R1 group.
An exemplary preparation of the compounds of Formula I is shown in Scheme 1. Generally, aminoacetophenones of Formula II, wherein Ar, R3 and n are as defined in Formula I (which are commercially available or are prepared using methods known in the art), are alkylated, for example with a reagent of Formula III, wherein R2 is as defined in Formula I (available from Sigma Aldrich), in the presence of a base under conditions to form a compound of the Formula IV, wherein Ar, R2, R3 and n are as defined in Formula I. In an embodiment of the disclosure, the alkylation is performed in two steps, with the second alkylation being performed in the presence of a stronger base. In an embodiment, the compound of the Formula IV is reacted with bromine in the presence of an acid, under conditions to prepare the compound of the Formula V, wherein Ar, R2, R3 and n are as defined in Formula I, which is then mono-debrominated, for example using diethyl phosphite in the presence of a base, under conditions to form the compound of the Formula I, wherein R1 is Br and Ar, R2, R3 and n are is as defined in Formula I. The compounds of Formula I, wherein R1 is Br are converted to other compounds of Formula I, wherein R1 is an alternative suitable leaving group, such as I and Otosyl, using methods known in the art.
The present application also includes the carboxylic acids derivatized with a compound of Formula I as defined above. It will be appreciated by those skilled in the art that any compound comprising at least one carboxylic acid moiety is capable of reacting with the compounds of Formula I to form the corresponding ester. It is an embodiment of the disclosure that the carboxylic acid is a metabolite found in a biological sample or an aqueous sample such as an agricultural or environmental sample. In a further embodiment of the disclosure, the biological sample is blood, plasma, serum or urine.
Accordingly, also included in the present disclosure in a method of preparing an ester of a carboxylic acid comprising reacting a compound comprising at least one carboxylic acid with a compound of Formula I as defined above in the presence of a suitable base under conditions to form the ester of the one or more carboxylic acids.
The formation of the esters VI, wherein Ar, R2, R3 and n are as defined in Formula I and R is any residue of a carboxylic acid, using the compound of Formula I is shown generally in Scheme 2:
In an embodiment of the disclosure, the base is a non-nucleophilic organic amine base, for example a trialkylamine, such as triethylamine. In a further embodiment the formation of the compounds of Formula VI is performed in a suitable reaction solvent, such as a buffer at a pH of about 7 to about 10, at a temperature of about 60° C. to about 130° C., suitably about 80° C. to about 95° C., for about 5 minutes to about 60 minutes, suitably about 10 minutes to about 30 minutes.
In a further embodiment, the present disclosure also includes a library comprising, consisting essentially of or consisting of two or more compounds of Formula VI as defined above wherein each of the two or more compounds of Formula VI contains a different residue “R” corresponding to a known carboxylic acid. In a further embodiment, the amount of each compound of Formula VI is known so that the library represents a standard or control sample that is used to quantitatively determine an amount of one or more of the carboxylic acids in a test sample.
The natural abundance of various isotopes in nature has been approximated, for example, in the CRC Handbook of Chemistry and Physics, (D. R. Lide, Ed. 89th Edition, 2008-2009, CRC Press Inc. U.S.). The most abundantly occurring form of carbon, the carbon-12 (12C) isotope, is approximately 98.90% abundant in nature. The stable carbon-13 (13C) isotope, by contrast, is only approximately 1.10% naturally abundant. The most abundantly occurring form of oxygen, the carbon-16 (16O) isotope, is approximately 99.765% abundant in nature. The stable oxygen-18 (18O) isotope, by contrast, is only approximately 0.1995% naturally abundant. Accordingly, standard molecules known in the art will generally have incorporated therein various isotopes in these respective percentages of natural abundance. The present disclosure, however, relates to analogs of standard compounds, for example, compounds of Formula I and VI, in which the less naturally abundant stable isotope is selectively incorporated into the structure at desired positions thereof, such that a given analog will have a characteristic molecular weight different from the molecular weight of its corresponding standard compound.
Isotopically labeled carboxylic acid esters, for example the compounds of Formula VI, according to the present disclosure suitably differ from their corresponding standard carboxylic acid ester by a molecular weight of between 2 and 16 atomic mass units (amu's). In particular, it is desirable that isotopes be incorporated in such a manner, and the mass difference be sufficient such that, the mass spectrometric molecular ion peaks of the isotopically-labeled derivative and standard carboxylic acid are distinguishable.
A benefit offered by all isotopically labeled analog internal standards reported herein is that their chemical properties are essentially identical to the target analyte. This means that during sample extraction and workup there can be no or very little differential loss of internal standard versus the target analyte due to differing chemical properties, as may be the case with a chemical analog with differing chemical properties. Again, this translates to an inherently more accurate analytical method when using the compounds of Formula I as defined herein as isotope mass-coded derivatives.
The compounds of Formula I as defined herein are useful for quantitative analysis of carboxylic acids in samples, for example, biological samples. In an embodiment, the quantitative analysis is performed using differential isotope labeling methods. In general, this method involves reacting a first sample comprising one or more carboxylic acids with a compound of Formula I as defined above. A second comparative (or standard) sample comprising one or more carboxylic acids is reacted with a compound having the same structure as that of the compound of Formula I, but that includes an amount of carbon-13 and oxygen-18, if present, that corresponds to its natural abundance. The first and second reacted samples are then analyzed. In one embodiment, the first and second reacted samples are combined and then analyzed by mass spectrometry. The mass spectral analysis of the first and second reacted samples provides quantitative information relating to the amount of carboxylic acids in the first and second samples. This is done by analyzing the peak intensity ratio of the isotope-labeled samples and can be done as a relative quantification of the carboxylic acids in two comparative samples or absolute quantification of the carboxylic acids in a sample if the other sample is a standard compound with known concentration.
Accordingly, the present disclosure also includes a method of quantifying one or more carboxylic acids:
In an embodiment of the disclosure, one of the first or second sample comprises one or more standard carboxylic acids with known concentrations and the method provides an absolute quantification of the one or more carboxylic acids in the other of the first or second sample. In a further embodiment, the first and second samples are comparative samples, such as urine, plasma, serum or blood samples, from diseased and healthy individuals.
The present disclosure also includes a multiplex method of quantifying one or more carboxylic acids in three or more samples comprising:
In an embodiment of the disclosure, where the standard sample comprises one or more standard carboxylic acids with known concentrations, the method provides an absolute quantification of the one or more carboxylic acids in the two or more samples.
In an embodiment of the disclosure, in the multiplex method, the two or more samples are samples from diseased individuals and the standard sample is as sample from healthy individuals. In another embodiment, the sample is from urine, plasma, serum or blood.
It is an embodiment of the binary and multiplex methods of the present disclosure that the mass spectrometry analysis is liquid chromatography/mass spectrometry (LC/MS), flow injection mass spectrometry, or direct sample introduction mass spectrometry.
In an embodiment of the disclosure, the LC comprises the use of reversed phase liquid chromatography, although a person skilled in the art would understand that the specific form of LC will vary depending on the identity of the carboxylic acids(s). In a further embodiment the mass spectrometry (MS) comprises the use of electrospray ionization (ESI) mass spectrometry.
The stable isotope-labeled compounds of the present disclosure are demonstrably useful for improving the efficiency of methodologies for analysis of biological samples for the presence of carboxylic acids and for determining the concentrations of carboxylic acids. In particular, the carbon-13 and/or oxygen-18 labeled compounds of Formula I of the present disclosure are especially useful compounds in the analysis of carboxylic acids in samples, particularly biological samples, for example, for metabolite analysis, for metabolome analysis, in pharmacokinetic and pharmacodynamic studies or for quantitative proteomics.
One major targeted area of application of the present disclosure is for metabolomics which involves a large scale analysis of metabolome (all metabolites) in biological samples. In particular the present disclosure is directed to the use of compounds of Formula I as defined herein in generating quantitative information on metabolome changes in comparative samples, such as urine, plasma, serum or blood samples, from diseased and healthy individuals. In this embodiment, a chemical reaction is used to introduce an isotope tag to an analyte(s) in one sample and another mass-different isotope tag is introduced via a separate reaction to the same analyte(s) in a comparative sample (or standard), followed by mixing the two labeled samples for mass spectrometric analysis. The peak intensity ratio of the isotope labeled analyte pair provides the basis of relative quantification of the analyte(s) in the two comparative samples or absolute quantification of the analyte(s) in a sample if the one sample is a standard with a known concentration of analyte(s).
A Bruker apex-Qe™ 9.4-T FT-ICR-MS was employed. A Waters Acquity™ BEH C18 column (2.1×50 mm, 1.7 m) was used for fast reverse phase (RP) separation.
Synthesis of 13C2-DmPA was based on a three-step procedure as shown in Scheme 3. The first reaction involved a dimethylation reaction using conditions as described in J. Physical Org. Chem. 1996, 9, 35-40 and J. Org. Chem. 1968, 33, 318-322. The second and third reactions involved bromination and debromination reactions using conditions as described in Tetrahedron Lett. 1998, 39, 4987-4990. The three-step procedure was optimized for the preparation of ˜1 gram of labeling reagent which ensured a good supply of this reagent. In most LC/MS work, only ˜100 μg of the labeling reagent are needed for one sample. Two semi-preparative reversed-phase (RP) separations and normal phase flash chromatography were used to produce high purity reagents. The purity of the labeling reagent was >99.5% by HPLC, UV, MS and NMR analysis.
The synthesis of other isotope reagents, such as p-diethylaminophenacyl bromide with varying numbers of carbon-13 (13Cn-DePA) and p-diisopropylaminophenacyl bromide containing varying numbers of carbon-13 (13Cn-DipPA) can be readily performed using a reaction scheme similar to that shown in Scheme 3. In these cases, dimethyl sulfate-13C2 is replaced with diethyl sulfate-13C2 and diisopropyl sulfate-13C2, respectively. It is noted that dimethyl sulfate-13C2 is commercially available, for example from Sigma Aldrich, however diethyl sulfate-13C2 and diisopropyl sulfate-13C2 is made by reacting ethanol or isopropanol with sulfuric acid or SO2Cl2 as described in J. Amer. Chem. Soc. 1924, 46, 999 and Compt. Rend. 1929, 188, 261. Isotope-containing ethanol is available, for example from Sigma Aldrich, in various forms, such as 12CH3CH2OH or 13CH313CH2OH. Isotope-containing isopropanol is also available, for example from Sigma Aldrich, in forms such as isopropanol-2-13C, isopropanol-1,2-13C2 and isopropanol-13C3. Scheme 3 only illustrates one route of preparing DmPA. Alternative routes can also be used to prepare DmPA, DePA or DipPA.
Scheme 4 shows the labeling reaction using 13C2-DmPA and a carboxylic acid. The labeling procedure was fast (˜15-20 min at 85-90° C. in a water bath), simple and robust. Triethylamine (TEA) was used in Scheme 4 as base. Other examples of bases include triethanolamine (TEOA) and N-methyldiethanolamine.
Relative quantification of carboxylic acids in two comparative metabolome samples can be done by making 12C-DmPA derivatives from one sample and 13C-DmPA derivatives from the other sample, followed by mixing the two labeled samples and injecting the mixture into LC/MS for analysis. The intensities of the mass spectral peak pairs are compared to generate information on the relative quantity differences of the metabolites in the two samples.
With the availability of a DmPA compound library, it is possible to determine the absolute concentration of each metabolite in a biological sample, as long as the carboxylic acid analyte standard is present in the library. A strategy of measuring absolute metabolite concentrations of individual samples is explored using a pooled sample as an internal standard. A pooled sample is prepared by taking aliquots from individual samples and then combining them to form a composite sample. This sample is labeled with 12C-DmPA. The 13C-DmPA metabolite standards are spiked to an aliquot of the pooled sample, followed by running the mixture in LC/MS. The metabolites present in the pooled sample can be identified based on the retention time match and accurate molecular mass measurement. The metabolite concentration can be determined based on the measured peak abundance ratios of 13C-/12C-DmPA derivatives and the amount of the 13C-standards spiked to the sample. To determine the concentrations of metabolites in the individual samples, each sample is labeled by 13C-DmPA and then mixed with an aliquot of the 12C-DmPA pooled sample. Based on individual metabolites already measured in the pooled sample, the absolute concentration of each metabolite in the individual samples can be determined.
To demonstrate the utility of this method of using a pooled sample as an internal standard, human urine samples are collected over five consecutive mornings from the same healthy individual. A pooled urine sample is then prepared by mixing equal volume aliquots of “Day-1” to “Day-5” urine samples. The carboxylic acid-12C-DmPA standards are grouped into mixtures to minimize the complexity of the samples and reduce the possibility of ion suppression in LC/MS (i.e., the spiked standards may suppress the analyte signals in the urine sample). Note that, depending on the type of biological samples analyzed, the concentrations of individual standards in the mixture may be adjusted so that the 13C-/12C-DmPA peaks do not fall off the linear dynamic range of relative quantification. Each group of mixture is esterified with 13C-DmPA and then spiked into the 12C-DmPA pooled urine for absolute quantification.
While the present disclosure has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the disclosure is not limited to the disclosed examples. To the contrary, the disclosure is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.
All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Where a term in the present application is found to be defined differently in a document incorporated herein by reference, the definition provided herein is to serve as the definition for the term.
This application is a PCT application that claims the benefit of priority of co-pending U.S. Provisional Patent Application 61/224,500 filed Jul. 9, 2010, which is herein incorporated in its entirety by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CA2010/001064 | 7/9/2010 | WO | 00 | 3/8/2012 |
| Number | Date | Country | |
|---|---|---|---|
| 61224500 | Jul 2009 | US |
