TUNED SYNTHETIC DENDRIMER CALIBRANTS FOR MASS SPECTROMETRY
20200090917 ยท 2020-03-19
Inventors
Cpc classification
Y10T436/10
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
H01J49/004
ELECTRICITY
International classification
Abstract
Provided are synthetic dendrimer calibrants for mass spectrometry. The calibrants are distinguished by their relative case and rapidity of synthesis, comparatively low cost, long shelf life, high purity, and amenability to batch synthesis as mixtures. The latter characteristic enables parallel preparation of higher molecular weight compounds displaying useful distributions of discrete molecular weights, thereby providing multi-point mass spectrometry calibration standards. Methods of making, tuning and using said calibrants are provided.
Claims
1. A composition comprising: a first dendrimer comprising a first core molecule, wherein said first core molecule is selected from the group consisting of: a molecule comprising between 1 and 8 alcohol functionalities, a molecule comprising between 1 and 8 amine functionalities, and a molecule comprising at least one amine functionality and at least one alcohol functionality wherein the combined number of amine and alcohol functionalities of said first core molecule is at least 2 but no greater than 8; a second dendrimer comprising a second core molecule, wherein said second core molecule is selected from the group consisting of: a molecule comprising between 1 and 8 alcohol functionalities, a molecule comprising between 1 and 8 amine functionalities, and a molecule comprising at least one amine functionality and at least one alcohol functionality wherein the combined number of amine and alcohol functionalities of said second core molecule is at least 2 but no greater than 8; and wherein said first core molecule has a different number of total alcohol functionalities and amine functionalities than said second core molecule.
2.-16. (canceled)
17. A method of manufacturing, comprising the steps of: providing a composition comprising a first core molecule wherein said first core molecule is selected from the group consisting of: a molecule comprising between 1 and 8 alcohol functionalities, a molecule comprising between 1 and 8 amine functionalities, and a molecule comprising at least one amine functionality and at least one alcohol functionality wherein the combined number of amine and alcohol functionalities of said first core molecule is at least 2 but no greater than 8; a second core molecule wherein said second core molecule is selected from the group consisting of: a molecule comprising between 1 and 8 alcohol functionalities, a molecule comprising between 1 and 8 amine functionalities, a molecule comprising at least one amine functionality and at least one alcohol functionality wherein the combined number of amine and alcohol functionalities of said second core molecule is at least 2 but no greater than 8; and wherein said first core molecule has a different number of total alcohol functionalities and amine functionalities than said second core molecule; and subjecting said first core molecule and said second core molecule to a round of dendronization.
4.-28. (canceled)
29. A method of manufacturing, comprising the steps of: providing a composition comprising a first dendrimer comprising a first core molecule, wherein said first core molecule is selected from the group consisting of: a molecule comprising between 1 and 8 alcohol functionalities, a molecule comprising between 1 and 8 amine functionalities, and a molecule comprising at least one amine functionality and at least one alcohol functionality wherein the combined number of amine and alcohol functionalities of said first core molecule is at least 2 but no greater than 8; and a second dendrimer comprising a second core molecule, wherein said second core molecule comprises a subsequent generation dendrimer of said first core molecule; and subjecting said first core molecule and said second core molecule to a round of dendronization.
30.-40. (canceled)
41. A method of determining physical properties of a sample, the method comprising: providing a composition comprising a first dendrimer comprising a first core molecule, wherein said first core molecule is selected from the group consisting of: a molecule comprising between 1 and 8 alcohol functionalities, a molecule comprising between 1 and 8 amine functionalities, and a molecule comprising at least one amine functionality and at least one alcohol functionality wherein the combined number of amine and alcohol functionalities of said first core molecule is at least 2 but no greater than 8; a second dendrimer comprising a second core molecule, wherein said second core molecule is selected from the group consisting of: a molecule comprising between 1 and 8 alcohol functionalities, a molecule comprising between 1 and 8 amine functionalities, and a molecule comprising at least one amine functionality and at least one alcohol functionality wherein the combined number of amine and alcohol functionalities of said second core molecule is at least 2 but no greater than 8; wherein said first core molecule has a different number of total alcohol functionalities and amine functionalities than said second core molecule; and wherein said composition has physical properties; ionizing at least a portion of said composition; providing an analyte sample wherein said analyte sample has physical properties; ionizing at least a portion of said analyte; collecting data from said ionized portion of said composition and said ionized portion of said analyte sample; and relating said data to said physical properties of said portion of said composition, thereby determining said physical properties of said analyte sample.
42.-48. (canceled)
49. A method of determining physical properties of a sample, the method comprising: providing a composition comprising a first dendrimer comprising a first core molecule, wherein said first core molecule is selected from the group consisting of: a molecule comprising between 1 and 8 alcohol functionalities, a molecule comprising between 1 and 8 amine functionalities, and a molecule comprising at least one amine functionality and at least one alcohol functionality wherein the combined number of amine and alcohol functionalities of said first core molecule is at least 2 but no greater than 8; a second dendrimer comprising a second core molecule, wherein said second core molecule comprises a subsequent generation dendrimer of said first core molecule; and wherein said composition has physical properties; ionizing at least a portion of said composition; providing an analyte sample wherein said analyte sample has physical properties; ionizing at least a portion of said analyte; collecting data from said ionized portion of said composition and said ionized portion of said analyte sample; and relating said data to said physical properties of said portion of said composition, thereby determining said physical properties of said analyte sample.
50.-56. (canceled)
57. A method of calibrating a mass spectrometer, the method comprising: providing a composition comprising a first dendrimer comprising a first core molecule, wherein said first core molecule is selected from the group consisting of: a molecule comprising between 1 and 8 alcohol functionalities, a molecule comprising between 1 and 8 amine functionalities, and a molecule comprising at least one amine functionality and at least one alcohol functionality wherein the combined number of amine and alcohol functionalities of said first core molecule is at least 2 but no greater than 8; and a second dendrimer comprising a second core molecule, wherein said second core molecule is selected from the group consisting of: a molecule comprising between 1 and 8 alcohol functionalities, a molecule comprising between 1 and 8 amine functionalities, and a molecule comprising at least one amine functionality and at least one alcohol functionality wherein the combined number of amine and alcohol functionalities of said second core molecule is at least 2 but no greater than 8; wherein said first core molecule has a different number of total alcohol functionalities and amine functionalities than said second core molecule; wherein at least one of properties (A) and (B) are satisfied: (A) the first dendrimer and the second dendrimer each have a mass defect falling within a scarcine valley of an average amino acid, and (B) at least one of the first core molecule and the second core molecule comprises a tertiary amine functionality; and wherein said composition has physical properties; ionizing at least a portion of said composition; collecting data from said ionized portion of said composition; and relating said data to said physical properties.
58. The method of calibrating of claim 57 wherein said first core molecule further comprises a halogen atom.
59. The method of calibrating of claim 57 wherein said first core molecule comprises three iodine atoms.
60. The method of calibrating of claim 57 wherein said first core molecule comprises 2,4,6-triiodolphenol.
61. The method of calibrating of claim 57 wherein at least one of the first core molecule and the second core molecule comprises a tertiary amine functionality.
62. The method of calibrating of claim 57 wherein said first core molecule comprises triethanolamine.
63. The method of calibrating of claim 57 wherein said first core molecule comprises N,N,NN-tetrakis(2-hydroxyethyl)ethylenediamine.
64. The method of calibrating of claim 57 wherein said first core molecule comprises bis-tris.
65. A method of calibrating a mass spectrometer, the method comprising: providing a composition comprising a first dendrimer comprising a first core molecule, wherein said first core molecule is selected from the group consisting of: a molecule comprising between 1 and 8 alcohol functionalities, a molecule comprising between 1 and 8 amine functionalities, and a molecule comprising at least one amine functionality and at least one alcohol functionality wherein the combined number of amine and alcohol functionalities of said first core molecule is at least 2 but no greater than 8; a second dendrimer comprising a second core molecule, wherein said second core molecule comprises a subsequent generation dendrimer of said first core molecule; and wherein said composition has physical properties; ionizing at least a portion of said composition; collecting data from said ionized portion of said composition; and relating said data to said physical properties.
66. The method of calibrating of claim 65 wherein said first core molecule further comprises a halogen atom.
67. The method of calibrating of claim 65 wherein said first core molecule comprises three iodine atoms.
68. The method of calibrating of claim 65 wherein said first core molecule comprises 2,4,6-triiodolphenol.
69. The method of calibrating of claim 65 wherein said first core molecule a tertiary amine functionalities.
70. The method of calibrating of claim 65 wherein said first core molecule comprises triethanolamine.
71. The method of calibrating of claim 65 wherein said first core molecule comprises N,N,NN-tetrakis(2-hydroxyethyl)ethylenediamine.
72. The method of calibrating of claim 65 wherein said first core molecule comprises bis-tris.
73. The method of calibrating of claim 57 wherein the first dendrimer and the second dendrimer each have a mass defect falling within a scarcine valley of an average amino acid.
74. The method of calibrating of claim 73 wherein the first core molecule and the second core molecule each comprise a mass defect marker.
75. The method of calibrating of claim 74 wherein the mass defect marker for the first core molecule and for the second core molecule is selected from the group consisting of fluorine, phosphorous, iodine, chlorine, bromine, and combinations thereof.
76. The method of calibrating of claim 73 wherein the first dendrimer and the second dendrimer each have a mass defect distinct from an averagine ridge of an average amino acid.
77. The method of calibrating of claim 76 wherein the average amino acid has an average chemical formula of C.sub.4.9384H.sub.7.7583N.sub.1.3577O.sub.1.4773S.sub.0.0417 with a molecular weight of 111.05431.
78. The method of calibrating of claim 76 wherein the first dendrimer and the second dendrimer each have a mass difference of about 0.2 amu about 0.5 amu relative to the averagine ridge.
79. The method of calibrating of claim 76 wherein the first dendrimer and the second dendrimer each have a mass difference of about 0.3 amu to about 0.5 amu relative to the averagine ridge.
80. The method of calibrating of claim 76 wherein the first dendrimer and the second dendrimer each have a mass difference of about 0.4 amu to about 0.5 amu relative to the averagine ridge.
81. The method of calibrating of claim 76 wherein the first dendrimer and the second dendrimer each have a mass difference of about 0.5 amu relative to the averagine ridge.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] For a further understanding of the nature, objects, and advantages of the present disclosure, reference should be had to the following detailed description, read in conjunction with the following drawings, wherein like reference numerals denote like elements.
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DETAILED DESCRIPTION
[0064] Before the subject disclosure is further described, it is to be understood that the disclosure is not limited to the particular embodiments of the disclosure described below, as variations of the particular embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present disclosure will be established by the appended claims.
[0065] Furthermore, this application incorporates by reference, in their entireties, U.S. Non-Provisional application Ser. No. 11/290,998, which is the National Stage of International Application No. PCT/US10/23087 filed on 3 Feb. 2012, U.S. Provisional Patent Application No. 61/149,506, filed 3 Feb. 2009, U.S. Provisional Patent Application No. 61/167,708, filed on 8 Apr. 2009, and U.S. Provisional Patent Application No. 61/185,665, filed on 10 Jun. 2009.
[0066] In this specification and the appended claims, the singular forms a, an, and the include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs.
[0067] As used herein, the term [M+Ag]+ indicates that one silver cation is attached per molecule, during ionization of samples, as the counterion. Other counterions may include, for example and without limitation, H, Na, and K, as will be readily appreciated by those persons having ordinary skill in the relevant art. As used herein, the term m/z denotes the mass-to-charge ratio. As used herein, MW means molecular weight.
[0068] The recently developed divergent aliphatic poly(ester) synthesis appears to offer the advantages of both techniques, while minimizing the shortcomings of both. A divergent dendritic synthesis is an iterative process that involves a well-defined (though exponential) increase of mass with each repetition of two synthetic steps: the coupling step, and the deprotection step. In
[0069] The synthetic dendrimer calibrants of the present disclosure offer a number of distinct advantages over other calibrants. Peptides and proteins have been used as commercial standards for calibration because, traditionally, these were the only monodisperse polymers which could be prepared and purified with sufficiently high molecular weight. While peptide and protein calibrants provide a viable standard, they suffer from short shelf-life (because of the prevalence of peptidases) and high cost (because their synthesis and purification is typically carried out on a milligram scale). A representative example of these calibrants is provided in TABLE 1.
TABLE-US-00001 TABLE 1 Prior Art Peptide and Protein Calibrants Molecu Price per Calibrant Weight (USD) Bradykinin Fragment 756 38,300 Angiotensin II 1,046 6,580 P.sub.14R 1,533 8,733,300 ACTH Fragment 18- 2,464 220,500 Insulin Chain B 3,496 8,160 Insulin 5,730 2,652,900 Cytochrome c 12,362 1,181,000 Apomyoglobin 16,952 861,200 Aldolase 39,211 372,300 Albumin 66,429 219,800
[0070] Source: Sigma-Aldrich, Inc.
[0071] Synthetic calibrants offer a number of potential advantages, including increased shelf-life, but until recently the only products that could be produced at a competitive price were polydisperse polymers (i.e., they exhibit a broad range of mass characteristics). The presence of multiple species (and the prevalence of different counterions in MS, including MALDI-TOF, ESI, APCI, and FAB) has prevented these from becoming an attractive alternative to peptides and proteins. Monodisperse synthetic calibrants, such as P.sub.14R, are at least 3 times as expensive as the next-cheapest peptide calibrant (Insulin), and more than 1,000 times more expensive than the cheapest peptide calibrant (Insulin Chain B).
[0072] The synthetic dendrimer calibrants of the present disclosure, in contrast, are less expensive to produce. Because of this rapid synthetic access to cost-efficient, yet highly pure dendritic compounds, the dendrimer calibrants of the present disclosure offer a competitive solution to the calibration of mass spectrometers, particularly when using MALDI-TOF, ESI, APCI, or FAB methods. In addition, they can be synthesized as mixtures, thus reducing preparation, purification, and packaging costs. While presently-available peptide and protein calibrants are widely used and accepted, the reduced cost of the dendrimer calibrants of the present disclosure, as well as their improved shelf-life and solvent compatibility, should result in their ready acceptance.
[0073] The dendrimers are given a standard nomenclature to denote their architecture. For example, in the names CX-([G-n]Ph.sub.p).sub.z, and CX-([G-n]OH.sub.q).sub.z, the CX term refers to the number of alcohol functionalities on the corethe core numberwhere X is an integer. Thus, C3 refers to 1,1,1-trishydroxyethylmethane (a triol) as the core, C4 refers to pentaerythritol (a tetraol) as the core, C5 refers to xylitol (a pentaol) as the core, and C6 refers to dipentaerythritol (a hexaol) as die core. The G-n term refers to the generation number, which denotes the number of layers of branching points which have been added, and which also refers to the number of coupling-and-deprotection iterations that have taken place. For example, [G-1] denotes generation one, and indicates that one round of coupling has occurred (see, e.g., dendrimer 1 of
Example 1
[0074] General Synthetic Procedure
[0075] The general procedure for the preparation of the dendritic calibrants follows generally those published by Grayson et al. (Grayson, S. M.; Frchet, J. M. J. Macromolecules, 2001; 34:6542-6544) and by Ihre et al. (Ihre, H.; Padilla de Jesus, O. L.; Frchet, J. M. J J. Am. Chem. Soc. 2001; 123:5908-5917), each of which are hereby incorporated by reference in their entireties.
[0076] As shown in
Example 2
[0077] Preparation of Benzylidene Protected Bis-MPA Anhydride Monomer
[0078] The benzylidene protected bis-MPA anhydride monomer was prepared according to the synthesis reported previously by Ihre, H.; Padilla de Jesus, O. L.; Frchet, J. M. J J. Am. Chem. Soc. 2001, 123, 5908-5917, which is hereby incorporated by reference in its entirety.
Example 3
[0079] General Dendronization Procedure for Preparation of CX-([G-n]pH.sub.p).sub.z
[0080] The procedure of this EXAMPLE is shown schematically as step i of
Example 4
[0081] General Deprotection Procedure for Preparation of CX-([G-n]OH.sub.q).sub.z
[0082] The procedure of this EXAMPLE is shown schematically as step ii of
Example 5
[0083] Synthesis of Tri-Functional C-3 Calibrants
[0084] The tri-functional dendrimer species of this EXAMPLE 5 are shown in
[0085] Synthesis of C3-([G-1]Ph).sub.3, dendrimer 1 of
[0086] Synthesis of C3-([G-1]OH.sub.2).sub.3, dendrimer 2 of
[0087] Synthesis of C3-([G-2]Ph.sub.2).sub.3, dendrimer 3 of
[0088] Synthesis of C3-([G-2]OH.sub.4).sub.3, dendrimer 4 of
[0089] Synthesis of C3-([G-3]Ph.sub.4).sub.3, dendrimer 5 of
[0090] Synthesis of C3-([G-3]OH.sub.8).sub.3, dendrimer 6 of
[0091] Synthesis of C3-([G-4]Ph.sub.8).sub.3, dendrimer 7 of
[0092] Synthesis of C3-([G-4]OH.sub.16).sub.3, dendrimer 8 of
[0093] Synthesis of C3-([G-5]Ph.sub.16).sub.3, dendrimer 9 of
[0094] Synthesis of C3-([G-5]OH.sub.32).sub.3, dendrimer 10 of
Example 6
[0095] Synthesis of Tetra-Functional C-4 Calibrants
[0096] The tetra-functional dendrimer species of this EXAMPLE 6 are shown in
[0097] Synthesis of C4-([G-1]Ph).sub.4, dendrimer 11 of
[0098] Synthesis of C4-([G-1]OH.sub.2).sub.4, dendrimer 12 of
[0099] Synthesis of C4-([G-2]Ph.sub.2).sub.4, dendrimer 13 of
[0100] Synthesis of C4-([G-2]OH.sub.4).sub.4, dendrimer 14 of
[0101] Synthesis of C4-([G-3]Ph.sub.4).sub.4, dendrimer 15 of
[0102] Synthesis of C4-([G-3]OH.sub.8).sub.4, dendrimer 16 of
[0103] Synthesis of C4-([G-4]Ph.sub.8).sub.4, dendrimer 17 of
[0104] Synthesis of C4-([G-4]OH.sub.16).sub.4, dendrimer 18 of
[0105] Synthesis of C4-([G-5]Ph.sub.16).sub.4, dendrimer 19 of
[0106] Synthesis of C4-([G-5]OH.sub.32).sub.4, dendrimer 20 of
Example 7
[0107] Synthesis of Penta-Functional C-5 Calibrants
[0108] The penta-functional dendrimer species of this EXAMPLE 7 are shown in
[0109] Synthesis of C5-([G-1]Ph).sub.5, dendrimer 21 of
[0110] Synthesis of C5-([G-1]OH.sub.2).sub.5, dendrimer 22 of
[0111] Synthesis of C5-([G-2]Ph.sub.2).sub.5, dendrimer 23 of
[0112] Synthesis of C5-([G-2]OH.sub.4).sub.5, dendrimer 24 of
[0113] Synthesis of C5-([G-3]Ph.sub.4).sub.5, dendrimer 25 of
[0114] Synthesis of C5-([G-3]OH), dendrimer 26 of
[0115] Synthesis of C5-([G-4]Ph.sub.8).sub.5, dendrimer 27 of
[0116] Synthesis of C5-([G-4]OH.sub.16).sub.5, dendrimer 28 of
[0117] Synthesis of C5-([G-5]Ph.sub.16).sub.5, dendrimer 29 of
[0118] Synthesis of C5-([G-5]OH.sub.32).sub.5, dendrimer 30 of
Example 8
[0119] Synthesis of Hexa-Functional C-6 Calibrants
[0120] The hexa-functional dendrimer species of this EXAMPLE 8 are shown in
[0121] Synthesis of C6-([G-1]Ph).sub.6, dendrimer 31 of
[0122] Synthesis of C6-([G-1]OH.sub.2).sub.6, dendrimer 32 of
[0123] Synthesis of C6-([G-2]Ph.sub.2).sub.6, dendrimer 33 of
[0124] Synthesis of C6-([G-2]OH.sub.4).sub.6, dendrimer 34 of
[0125] Synthesis of C6-([G-3]Ph.sub.4).sub.6, dendrimer 35 of
[0126] Synthesis of C6-([G-3]OH.sub.8).sub.6, dendrimer 36 of
[0127] Synthesis of C6-([G-4]Ph.sub.8).sub.6, dendrimer 37 of
[0128] Synthesis of C6-([G-4]OH.sub.16).sub.6, dendrimer 38 of
[0129] Synthesis of C6-([G-5]Ph.sub.16).sub.6, dendrimer 39 of
[0130] Synthesis of C6-([G-5]OH.sub.32).sub.6, dendrimer 40 of
Example 9
[0131] Parallel Synthesis of Dendrimers 1, 11, 21, and 31
[0132] In the prior art, a broad range calibrant is made by mixing appropriate quantities of individual peptides, which have been prepared and purified separately, to yield a calibrant cocktail. The synthetic methodology described herein and shown schematically in
[0133] By serial repetitions of steps i and ii as detailed in EXAMPLES 3 and 4 (and as shown, for example, in
[0134] A particularly efficient way to make a calibrant mixture is to carry out the dendronization process using a mixture of cores in a single batch (e.g., equimolar mixtures of the C-3, C-4, C-5, and/or the C-6 cores). For example, and as shown in
[0135] Because the most desirable calibrant would be a mixture of numerous, well-defined monodisperse compounds (e.g., as shown in the reaction scheme of
[0136] Synthesis of CX-([G-1]Ph).sub.z, an equimolar mixture of dendrimers 1, 11, 21, and 31 (see, e.g., reaction scheme of
Example 10
[0137] Parallel Synthesis of Dendrimers 2, 12, 22, and 32
[0138] Synthesis of CX-([G-1]OH.sub.2).sub.z, an equimolar mixture of dendrimers 2, 12, 22, and 32 (not shown) (see, e.g., reaction scheme of
Example 11
[0139] Parallel Synthesis of Dendrimers 3, 13, 23, and 33
[0140] Synthesis of CX-([G-2]Ph.sub.2).sub.z, an equimolar mixture of dendrimers 3, 13, 23, and 33 (see, e.g., reaction scheme of
Example 12
[0141] Parallel Synthesis of Dendrimers 4, 14, 24, and 34
[0142] Synthesis of CX-([G-2]OH.sub.4).sub.z, an equimolar mixture of dendrimers 4, 14, 24, and 34 (see, e.g., reaction scheme of
Example 13
[0143] Parallel Synthesis of Dendrimers 5, 15, 25, and 35
[0144] Synthesis of CX-([G-3]Ph.sub.4).sub.z, an equimolar mixture of dendrimers 5, 15, 25, and 35 (see, e.g., reaction scheme of
Example 14
[0145] Parallel Synthesis of Dendrimers 6, 16, 26, and 36
[0146] Synthesis of CX-([G-3]OH.sub.8).sub.z, an equimolar mixture of dendrimers 6, 16, 26, 36 (see, e.g., reaction scheme of
Example 15
[0147] Parallel Synthesis of Dendrimers 7, 17, 27, and 37
[0148] Synthesis of CX-([G-4]Ph.sub.8).sub.z, an equimolar mixture of dendrimers 7, 17, 27, and 37 (see, e.g., reaction scheme of
Example 16
[0149] Parallel Synthesis of Dendrimers 8, 18, 28, and 38
[0150] Synthesis of CX-([G-4]OH.sub.16).sub.z, an equimolar mixture of dendrimers 8, 18, 28, and 38: The mixture of benzylidene protected dendrimers 7, 17, 27, and 37 from EXAMPLE 15 was deprotected using 5% Pd(OH).sub.9/C and hydrogen gas following the General Deprotection Procedure of EXAMPLE 4, to afford the CX-([G-4]OH.sub.16).sub.z, mixture of dendrimers 8, 18, 28, and 38. MALDI-TOF MS: Theo. Avg. MW: [M+Na].sup.+ m/z=5,365.2; 7,121.9; 8,878.6; 10,721.4. Observed MW: [M+Na].sup.+ m/z=5,366.619; 7,123.504; 8,880.111; 10,722.572. As can be appreciated from
Example 17
[0151] Parallel Synthesis of Dendrimers 9, 19, 29, and 39
[0152] Synthesis of CX-([G-5]Ph.sub.16).sub.z, an equimolar mixture of dendrimers 9, 19, 29, and 39: The mixture of hydroxyl functionalized dendrimers 8, 18, 28, and 38 from EXAMPLE 16 would be esterified following the General Dendronization Procedure of EXAMPLE 3, using the benzylidene-protected Bis-MPA anhydride of EXAMPLE 3 and DMAP to afford the CX-([G-5]Ph.sub.16).sub.z mixture of dendrimers 9, 19, 29, and 39. MALDI-TOF MS: Theo. Avg. MW: [M+Ag].sup.+ m/z=15,244.9; 20,266.9; 25,288.8; 30,396.9. Observed MW: [M+Ag].sup.+ m/z=to be determined.
Example 18
[0153] Parallel Synthesis of Dendrimers 10, 20, 30, and 40
[0154] Synthesis of CX-([G-5]OH.sub.32).sub.z, an equimolar mixture of dendrimers 10, 20, 30, and 40: The mixture of benzylidene protected dendrimers, 9, 19, 29, and 39 from EXAMPLE 17 would be deprotected using 5% Pd(OH).sub.2/C and hydrogen gas following the General Deprotection Procedure of EXAMPLE 4, to afford the CX-([G-5]OH.sub.32).sub.z mixture of dendrimers 10, 20, 30, and 40. MALDI-TOF MS: Theo. Avg. MW: [M+Na].sup.+ m/z=10,935.5; 14,548.9; 18,162.4; 21,861.9. Observed MW: [M+Na].sup.+ m/z=to be determined
Example 19
[0155] Calibrant TestsDendronized Cavitand
[0156] To verify the utility of the calibrants of the present disclosure in acquiring accurate MALDI-TOF data with high mass resolution, a dendronized cavitand (a monodisperse synthetic molecule) was examined, and the results are shown in
Example 20
[0157] Calibrant TestPoly(ethylene) Glycol, PEG 1970
[0158] To further verify the utility of the calibrants of the present disclosure in acquiring accurate MALDI-TOF data with high mass resolution, synthetic polymer PEG 1970 (a polydisperse polymer of three different oligomers: a 33-mer, a 43-mer, and a 53 mer), was examined. The number average molecular weight (M.sub.n) of PEG 1970 is 1970, and its polydispersity index (PDI) is 1.05. The spectrometric results are shown in
[0159] The PEG 1970 33-mer has the molecular formula C.sub.66H.sub.134O.sub.34. As shown in
[0160] The PEG 1970 43-mer has the molecular formula C.sub.86H.sub.174O.sub.44. As shown in
[0161] The PEG 1970 53-mer has the molecular formula C.sub.106H.sub.214O.sub.54. As shown in
Example 21
[0162] Calibrant TestProprietary Peptide JF-1485
[0163] To further verify the utility of the calibrants of the present disclosure in acquiring accurate MALDI-TOF data with high mass resolution, peptide JF-1485 having the formula C.sub.88H.sub.118N.sub.16O.sub.22S.sub.5 (and having a proprietary structure) was examined. As shown in
[0164] Alternative Hydroxyl-Terminated Cores
[0165] As will be appreciated by those having ordinary skill in the art, dendrimers of various functionalities other than the ones described above may be synthesized via the General Dendronization Procedure of EXAMPLE 3 followed (optionally) by the General Deprotection Procedure of EXAMPLE 4. This could be accomplished, for example, and without intending to be limited, simply by choosing a hydroxyl-terminated core different from the ones disclosed above (e.g., a core other than 1,1,1-tris(hydroxymethyl)ethane, pentaerythritol, xylitol, or dipentaerythritol) for the General Dendronization Procedure of EXAMPLE 3. Exemplary alternative hydroxyl-terminated cores include, without intending to be limited: tripentaerythritol (eight hydroxyl termini) and tetrapentaerythritol (ten hydroxyl termini). Those having ordinary skill in the art will also understand from the foregoing description that each dendrimer created via the General Dendronization Procedure of EXAMPLE 3 may also function as an alternative hydroxyl-terminated core. For example, the dendrimer denoted C3-([G-2]OH.sub.4).sub.3dendrimer 4 of
[0166] In addition, the coupling acylation chemistry used to covert alcohols to the corresponding esters during the coupling or dendronization step as described in EXAMPLE 3 is equally amenable to the acylation reaction, using the same reagents, that converts amines to amides. As a result, polyamine core molecules can also be used (as core molecules), including commercially available families of dendritic polyamine such as the poly(amidoamine) (PAMAM) and poly(propylene amine) (PPI) dendrimers.
[0167] Trismonomer
[0168] The benzylidene protected bis-MPA monomer described above may be modified by substituting a hydroxymethyl group for the pendent methyl group, to produce a protected trismonomer, as shown in Formula 1 below:
##STR00001##
[0169] By substituting a hydroxymethyl group for the pendent methyl group of the benzylidene protected bis-MPA anhydride monomer ((bis(5-methyl-2-phenyl-1,3-dioxane-5-carboxylic) acid anhydride monomer), each dendrimer layer could contain three branches, rather than the two branches shown in
##STR00002##
[0170] The OH groups of Formula 2 may be protected using methylidene orthoesters to carry out subsequent dendronization and deprotection steps.
[0171] Tuning the Dendrimers
[0172] Because the dendrimers described originate almost exclusively from the bis(hydroxymethyl)propanoic acid monomer, the composition of the overall structure can be easily tuned by subtle changes in the monomer structure. Such tuning could include modification of a pendant methyl group and/or synthesis of dendrimers using .sup.12C isotopically-enriched monomer.
[0173] The exact atomic masses of all atoms are close to, but not exactly, whole numbers. Because larger molecular weight (MW) compounds are comprised of multiple atoms, they have a significant mass defectan offset from the nominal mass (the value of the nearest integer approximation of the most abundant isotope for each atomic mass). Simply put, the mass defect is the difference between the whole number approximate nominal mass and the actually-observed monoisotopic mass. The mass defect can be used to identify classes of compounds, and can be used to distinguish natural biomolecules from unnaturally modified ones. By tuning the elemental composition of the dendrimer backbone, the mass defect can be adjusted to ensure that they do not overlap withand can be easily differentiated fromnatural compounds. Such tuning can also facilitate automated data analysis by simplifying the distinction between analyte and calibrant. Because the disclosed dendrimers are made predominantly by multiple layers of the same monomer, tuning the elemental composition of that monomer allows the mass defect of all of the disclosed dendrimers to be tuned. For example, an average peptide will exhibit the averagine mass defect of +0.506 daltons (Da) per 1000 Da of molecular weight. Averagine is the theoretical average amino acid in regards to its elemental composition (with the non integer molecular formula: C.sub.4.9384H.sub.7.7583N.sub.1.3577O.sub.1.4773S.sub.0.0417), and can be used to calculate the expected elemental composition and mass defect of peptides and proteins across a range of molecular weights. The hydroxyl-functionalized dendrons (see, e.g., dendrimers 2, 4, 6, 8, etc.) exhibit a mass defect of +0.420.02 Da per 1000 Da of molecular weight, while the benzylidene functionalized dendrons (see, e.g., dendrimers 1, 3, 5, 7, 9, etc.) exhibit a mass defect of 0.390.02 Da per 1000 Da of molecular weight. In order to differentiate this mass defect further, the pendant methyl of the hydroxyl-functionalized dendrons can be modified or functionalized with a variety of longer alkyl chains or with halogenated alkyl chains, without any significant effect on the synthetic procedure. This may be accomplished by modifying the benzylidene protected bis-MPA anhydride monomer (bis(5-methyl-2-phenyl-1,3-dioxane-5-carboxylic) acid anhydride monomer at the 5-methyl position as shown in Formula 3 below:
##STR00003##
[0174] In Formula 3, X may be: alkyl (e.g., CH.sub.3, CH.sub.2CH.sub.3, CH.sub.2CH.sub.2CH.sub.3, or (CH.sub.2).sub.nCH.sub.3, where n is an integer from 0 to 16); CH.sub.2OCH.sub.2Ph, where Ph represents phenyl; CQ.sub.3, where Q represents a halogen, preferably fluorine (F) or chlorine (Cl) (e.g., CF.sub.3, CCl.sub.3, etc.); or (CQ.sub.2).sub.nCQ.sub.3, where Q represents a halogen, preferably fluorine (F) or chlorine (Cl), and where n is an integer from 1 to 16. For example, a rather significant shift in MW can be demonstrated by replacing the methyl group with a trifluoromethyl group, resulting in a shift in the mass defect to +0.110.02 Da per 1000 Da of MW. The molecular mass defect can also be modified by a simple functionalization of the periphery with a substituent with the desired mass defect. Despite modification at X, dendrimer synthesis using the benzylidene protected monomer of Formula 3 may proceed via serial iterations of the General Dendronization Procedure of EXAMPLE 3 and the General Deprotection Procedure of EXAMPLE 4.
[0175] As the molecular weight of carbon-containing molecules increases, the natural prevalence of .sup.13C (natural abundance=1.109%) in the molecules leads to a broadening of the molecular isotopic distribution in their mass spectra. Above about 8,000 Da, the signal corresponding to the monoisotopic species (having only .sup.12C) is so small, relative to polyisotopic species, that exact mass determination is difficult because the monoisotopic species' peak is difficult to identify amongst the peaks from polyisotopic species. Consequently, the presence of polyisotopic species greatly reduces the resolution of molecular weight calculations. Take, for example, Formula 4:
##STR00004##
[0176] which can be represented by the formula C.sub.5H.sub.12O.sub.4. Because greater than 1% of C is .sup.13C, the MS of any carbon-containing compound will exhibit higher molecular weight signals corresponding to these .sup.13C isotopes. As the number of carbons in a compound increases, the likelihood that .sup.13C is present in the compound increases. This is seen in the isotopic distribution of the monomer of Formula 4, which has an exact mass of 136.07356, exhibits a monoisotopic signal at 136.07356 (m/z; 100.0% relative signal intensity), and a higher molecular weight species at 137.07691 (m/z; 5.4% relative signal intensity).
[0177] With increasing carbon content (e.g., without intending to be limited, 500 carbon atoms per molecule) the statistical distribution of molecular weights from different polyisotopic species becomes so broad that the single monoisotopic peak can become difficult to resolve. The native abundance of .sup.12C is 98.89%, of .sup.13C is 1.109%, of .sup.1H is 99.99%, of .sup.2H is 0.01%, of .sup.16O is 99.76%, of .sup.18O is 0.20%, and of .sup.17O is 0.04%. The .sup.13C isotope is the most common higher isotope in most organic compounds. Thus, the simplest way to narrow the isotopic distribution at high molecular weights is to start with building materials in which .sup.13C has been depletedfor example, starting materials in which all carbon is .sup.12C.
[0178] Because the dendrimers described originate almost exclusively from the bis(hydroxymethyl)propanoic acid monomer, if the synthesis is carried out with .sup.12C isotopically enriched monomer then the mass spectral peak broadening will be reduced substantially, and high accuracy calibration above 10,000 Da can be achieved easily. While isotopic broadening due to .sup.18O is much less pronounced (because 180 represents only 0.201% of all O species) .sup.16O isotopic enrichment can also be carried out to improve the accuracy even further. These isotopic enrichments contemplated here are not expected to have any effect on the synthetic parameters, beyond subtly altering the molecular weights of the reactants and the dendrimer products.
[0179] As shown in the General Dendronization Procedure for Preparation of CX-([G-n]Ph.sub.p).sub.z described in EXAMPLE 3, the alcohol functionalities of the monomer must be protected in order to control the iterative dendrimer growth that yields exact monodisperse structures. Two alcohols can be protected simultaneously with benzylidene (described in EXAMPLE 3 and shown below at Formula 5), and those of ordinary skill in the art will also recognize that they may be protected with acetonide (Formula 6), or other acetal or ketal protecting group (see, e.g., Formulae 7 & 8, where R.sup.3 is H or CHA, R.sup.4 is Ph, CH.sub.3, C.sub.6H.sub.4OCH.sub.3, or C.sub.6H.sub.4NO.sub.2, R.sub.5 is CH.sub.2Ph, Si(CH.sub.3).sub.3, C.sub.6H.sub.5NO.sub.2, CH.sub.2OCH.sub.3, C.sub.5H.sub.9O (Tetrahydropyranyl ether), or SiPh.sub.2t-Bu, and where Ph is phenyl).
##STR00005##
[0180] Further examples of protecting groups may be found in Protective Groups in Organic Synthesis by P. G. M. Wuts and T. W. Greene (4th edition, 2007, John Wiley and Sons Inc. Hoboken, N.J.), which is incorporated by reference herein in its entirety. In addition, a number of labile ether linkages, including benzyl ethers, substituted benzyl ethers, and silyl ethers, can be also be used instead of, or in addition to, to enable the synthesis of structurally pure dendrimers. Such modifications to the dendronization procedure lie within the scope of the present disclosure.
[0181] Tuning the Dendrimers Via the Core Molecule
[0182] Another method of tuning is to modify the core molecule of the dendrimer. In one embodiment, the dendrimer is tuned so as to incorporate a specific element or elements not commonly found in biomolecules into the core molecule in order to create a dendritic calibrant with a mass defect marker distinct from common, natural biomolecules. By tuning the elemental composition of the dendrimer core molecule, the mass defect can be adjusted to ensure that the observed masses of the dendrimers do not overlap withand therefore can be easily differentiated fromthe more common natural compounds during mass spectrometry.
[0183] Fluorine, phosphorus and iodine are all speculated to be desirable elements for incorporation into the core molecule because they are believed to result in a comparatively stable dendrimer calibrant that further results in a mass spectral peak of a narrower width. Specifically, because halogens (e.g., fluorine, chlorine, bromine, iodine) are capable of relatively easily substituting for hydrogen atoms, and as such will bond with the carbons of the core molecule, they should result in relatively stable dendrimers. Furthermore, because fluorine, phosphorous and iodine are monoisotopic, their incorporation into the dendrimers should further result in a desirable relatively narrow mass spectral peak.
[0184] Additionally, it is preferable to incorporate an element with a larger negative mass defect into the core molecule as it results in a more substantial shift in the peak of the mass spectra. The mass defects for a sampling of various elements are provided in TABLE 2.
TABLE-US-00002 TABLE 2 Atomic Mass % Isotopic Mass defect Element Isotope Mass (u) Defect comp. per 1000 u Hydrogen .sup.1H 1.00783 0.00783 99.9885 7.7692 .sup.2H 2.01410 0.01410 0.0115 7.0065 Carbon .sup.12C 12.00000 0.00000 98.93 0.0000 .sup.13C 13.00335 0.00335 1.07 0.2576 Nitrogen .sup.14N 14.00307 0.00307 99.632 0.2192 .sup.15N 15.00011 0.00011 0.368 0.0073 Oxygen .sup.16O 15.99491 0.00509 99.757 0.3182 .sup.17O 16.99913 0.00087 0.038 0.0512 .sup.18O 17.99916 0.00084 0.205 0.4667 Fluorine .sup.19F 18.99840 0.00160 100 0.0842 Phosphorus .sup.31P 30.97377 0.02623 100 0.8468 Sulfur .sup.32S 31.97207 0.02793 94.93 0.8736 .sup.33S 32.97146 0.02854 0.76 0.8656 .sup.34S 33.96787 0.03213 4.29 0.9459 Chlorine .sup.35Cl 34.96885 0.03115 75.78 0.8908 .sup.37Cl 36.96885 0.03419 24.22 0.9248 Bromine .sup.79Br 78.91834 0.08166 50.69 1.0347 .sup.81Br 80.90585 0.08371 49.31 1.0347 Iodine .sup.127I 126.93032 0.09553 100 0.7526
[0185] Knowing the mass defect for each element, one skilled in the art can create a graphical representation of the total population of all possible peptides (composed of the 20 standard amino acids) per 0.01u of mass defect for each nominal molecular weight. Such a graphical representation is shown, in various views, in
[0186] As can be seen in
[0187] Thus, in use as a calibrant, it is desirable to have a dendrimer tuned so that it has a mass defect that falls within the scarcine valley. In one embodiment, this can be accomplished by using a dendrimer calibrant with 2,4,6-triiodalphenol as the core molecule, as shown in Formula 9.
##STR00006##
[0188] In alternative embodiments, various other tuned core molecules can be utilized. Such core molecules include, but are not limited to, hydroxyl-functional cores such as pentose sugars (linear and furanose forms), hexaose sugars (linear, pyranose and furanose forms), oligomers of pentose sugars, oligomers of hexose sugars, and cyclodextrins.
[0189] Other core molecules may include, but are not limited to, amino-functional cores such as diethylenetriamine[N.sup.1-(2-aminoethyl)ethane-1,2-diamine], N,N-Bis(3-aminopropyl)ethylenediamine[N.sup.1,N.sup.1 (ethane 1,2 diyl)bis(ethane 1,2 diamine)], bis(hexamethylene)triamine [N.sup.1-(6-aminohexyl)hexane-1,6-diamine], spermidine [N.sup.1-(3-aminopropyl)butane-1,4-diamine], tetraethylenepentamine [N.sup.1,N.sup.1-(ethane-1,2-diyl)bis(N.sup.2-(2-aminoethyl)ethane-1,2-diamine)], spermine [N.sup.1,N.sup.1-(butane-1,4-diyl)bis(propane-1,3-diamine)], N,N-bis(2-aminoethyl) 1,3-propanediamine [N.sup.1,N.sup.1-(propane-1,3-diyl)bis(ethane-1,2-diamine)], and pentaethylenehexamine [N.sup.1-(2-aminoethyl)-N.sup.2-(2-((2-((2-((2-aminoethyl)amino)ethyl)amino) ethyl) amino)ethyl) ethane-1,2-diamine].
[0190] Yet other core molecules may include, but are not limited to, hydroxyl-functional tertiary amine cores such as N-methyldiethanolamine[2,2-(methylazadiyl)diethanol], N-ethyldiethanolamine [2,2-(ethylazadiyl)diethanol], N-propyldiethanolamine [2,2-(propylazadiyl)diethanol], N-butyldiethanolamine [2,2-(propylazadiyl)diethanol], N,N-Bis(2-hydroxyethyl)-p-toluidine [2,2-(p-tolylazadiyl)diethanol], N,N-bis(2-hydroxyethl)-m-toluidine [2,2-(m-tolylazadiyl)diethanol], N-phenyldiethanolamine [2,2-(phenylazadiyl) diethanol], triethanolamine [2,2,2 nitrilotriethanol], 1 (N,N-bis(2-hydroxyethyl)amino) 2 propanol [2,2 ((2 hydroxypropyl)azanediyl)diethanol], triisopropanolamine [1,1,1-nitrilotris(propan-2-ol)], 3-(dimethylamino)-1,2-propanediol, 3-(diethylamino)-1,2-propanediol, 3-(dipropylamino)-1,2-propanediol, 3 (diisopropylamino) 1,2 propanediol, 2-bis(2 hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediol (also known as bis-tris), N,N,N,N-tetrakis(2-hydroxypropyl)ethylenediamine [1,1,1,1-(ethane-1,2-diylbis(azanetriyl))tetrakis(propan-2-ol)], N,N,N,N-tetrakis(2-Hydroxyethyl)ethylenediamine, and pentrol [1,1,1,1-((((2-hydroxypropyl)azanediyl)bis(ethane-2,1-diyl))bis(azanetriyl))tetrakis(propan-2-ol)].
[0191] Further core molecules may include, but are not limited to, amino-functional tertiary amine cores such as tris(aminoethyl)amine [N,N-bis(2-aminoethyl)ethane-1,2-diamine] and N,N,N,N-tetrakis(3-aminopropyl)-1,4-butanediamine [N.sup.1,N.sup.1-(butane-1,4-diyl)bis(N.sup.1-(3-aminopropyl) propane-1,3-diamine)].
[0192] Additional core molecules may include, but are not limited to, hydroxyl or amino functional iodocores such as 2,4,6-triiodophenol, 2,4,6-triiodophenyl)methanol, 2,4,6-triiodoaniline, (2,4,6-triiodophenyl)methanamine, iohexol [N.sup.1,N.sup.3-bis(2,3-dihydroxypropyl)-5-(N-(2,3-dihydroxypropyl)acetamido)-2,4,6-triiodoisophthalamide], and iodixanol [N.sup.1,N.sup.1-(2-hydroxypropane-1,3-diyl)bis(N.sup.3-(2,3-dihydroxypropyl)-5-(N-(2,3-dihydroxypropyl)acetamido)-2,4,6-triiodoisophthalamide)].
[0193] Further additional core molecules may include, but are not limited to, hydroxyl-functional tertiary amine iodocores such as 2,2-((2,4,6-triiodophenyl)azanediyl)diethanol, 2,2-((2,4,6-triiodobenzyl)azanediyl)diethanol and 3,3-(((5-((2,3-dihydroxypropyl)(ethyl)amino)-2,4,6-triiodo-1,3-phenylene)bis(methylene))bis(azanediyl)) bis(propane-1,2-diol).
Example 22
[0194] General Dendronization Procedure for Preparation of CX-([G-n]Ac.sub.p).sub.z
[0195] The procedure of this EXAMPLE is shown schematically as step i of
Example 23
[0196] General Deprotection Procedure for Preparation of CX-([G-n]OH.sub.).sub.z
[0197] The procedure of this EXAMPLE is shown schematically as step ii of
Example 24
[0198] Synthesis of Iodo-Core Calibrants
[0199] The iodo-core dendrimer species of this EXAMPLE 24 are shown in
[0200] Synthesis of C1-([G-1]Ac).sub.1, dendrimer 1 of
[0201] Synthesis of C1-([G-1]OH.sub.2).sub.1, dendrimer 2 of
[0202] Synthesis of C1-([G-2]Ac.sub.2).sub.1, dendrimer 3 of
[0203] Synthesis of C1-([G-2]OH.sub.4).sub.1, dendrimer 4 of
[0204] Synthesis of C1-([G-3]Ac.sub.4).sub.1, dendrimer 5 of
[0205] Synthesis of C1-([G-3]OH.sub.8).sub.1, dendrimer 6 of
[0206] The dendrimers (dendrimers 2, 4 and 6) made according to the above examples yield a calibrant that is easily distinguishable from naturally occurring peptides and peptidic fragments. First generation dendrimer 2 (as shown in
Example 25
[0207] Internal Calibration TestEndomorphin I
[0208] To further verify the utility of the iodo-core calibrants of the present disclosure in acquiring accurate MALDI-TOF data with high mass resolution, peptide Endomorphin I (H-Try-Pro-Trp-Phe-NH, having the formula C.sub.34H.sub.38N.sub.6O.sub.5 was used in an internal calibration test wherein first generation dendrimer was mixed with Endomorphin I (H-Try-Pro-Trp-Phe-NH.sub.2, C.sub.34H.sub.38N.sub.6O.sub.5). As demonstrated in
[0209] In another embodiment, the dendrimer calibrants may be tuned to include an amine core. During ionization of the analyte, one counterion is attached per molecule. These counterions include, for example, H, Na, and K. It is desirable, as a calibrant, for the core molecule of the dendrimer to be compatible with various counterions. Because amines have an unbounded pair of electrons, they readily attract a hydrogen ion. As such, a core molecule containing an amine will readily attract a hydrogen counterion. Triethanolamine may be used as an amine core, as shown in Formula 10. Alternatives to triethanolamine that are speculated to also readily attract hydrogen counterions are those shown in Formula 11 and Formula 12.
##STR00007##
Example 26
[0210] Synthesis of Amine-Core Calibrants
[0211] The amine-core dendrimer species of this EXAMPLE 26 are shown in
[0212] Synthesis of C3-([G-1]Ph).sub.3, dendrimer 1 of
[0213] Synthesis of C3-([G-1]OH.sub.2).sub.3, dendrimer 2 of
[0214] Synthesis of C3-([G-2]Ph.sub.2).sub.3, dendrimer 3 of
[0215] Synthesis of C3-([G-2]OH.sub.4).sub.3, dendrimer 4 of
[0216] Synthesis of C3-([G-3]Ph.sub.4).sub.3, dendrimer 5 of
[0217] Synthesis of C3-([G-3]OH).sub.3, dendrimer 6 of
[0218] Synthesis of C3-([G-4]Ph.sub.8).sub.3, dendrimer 7 of
[0219]
[0220] An alternate exemplary amine-core dendrimer species is shown in
[0221] Yet another alternate exemplary amine-core dendrimer species is shown in
[0222] All references cited in this specification are herein incorporated by reference as though each reference was specifically and individually indicated to be incorporated by reference. The citation of any reference is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such reference by virtue of prior invention.
[0223] It will be understood that each of the elements described above, or two or more together may also find a useful application in other types of methods differing from the type described above. Without further analysis, the foregoing will so fully reveal the gist of the present disclosure that others can, by applying current knowledge, readily adapt it for various applications without omitting features that, from the standpoint of prior art, fairly constitute essential characteristics of the generic or specific aspects of this disclosure set forth in the appended claims. The foregoing embodiments are presented by way of example only; the scope of the present disclosure is to be limited only by the following claims.