KR20020037042A - Enantiomerically-enriched compounds having photocleavable bond(s) and methods related thereto - Google Patents

Abstract

The present invention relates to compounds of formula I or II wherein R ¹ is selected from halogen and organic moieties; R ² and R ³ are independently selected from hydrogen and organic residues having a mass of at least 15 daltons, wherein R ² and R ³ may together form a carbonyl group or may be bonded together into a cyclic structure; Z is a (n + 1) -valent atom excluding carbon, where n is an integer of 0 or greater; R ⁴ is independently selected from hydrogen, halogen, and organic moieties having a mass of at least 15 Daltons, provided that at least one R ⁴ (ie, R ^4a ) is an organic moiety having a mass of at least 100 Daltons; R ⁵ is independently at each occurrence an organic moiety having a mass of halogen or less than 500 daltons; m is selected from 0, 1, 2, 3 and 4; When R ² = R ³ = H, when Z (R ⁴ ) _n is -NH (C0) -CH (iBu) -NH (C0)-(CH ₂ Ph or Ot-Bu), R ¹ is CO ₂ ( R ¹ is not CH ₂ CO ₂ t-Bu when H is not H or CH ₃ ) and Z is OH; Wherein if both compounds of formula (I) and (II) are present as a mixture, the molar ratio of formula I: formula II in the mixture is not 50:50. Such compounds are useful as tags, including tags detectable by mass spectrometry.

Description

Enantiomerically-enriched compounds having photocleavable bond (s) and methods related with photocleavable bond (s)

Radiolabeling and fluorescence tagging of DNA is widely used for detection purposes in molecular biology and genetics, while the number of tags is too small for high levels of multiplexing and is not suitable for large scale rapid data acquisition. Cleavable mass spectrometry tags (CMST), a new class of tags containing small molecules, have been developed and used in single nucleotide polymorphism (SNP) genotyping and gene expression measurement applications. See, for example, PCT International Patent Application. One embodiment of WO 99/05319, WO 97/27331, WO 97/27327 and WO 97/27325]. The system is based on covalently binding CMST to oligonucleotides using linkers such as photocleavable linkers. Each tag has a different mass specific to the designated oligonucleotide sequence. Identification of alleles or expressed sequences present is determined by simultaneous detection using standard single quadrupole mass spectroscopy using, for example, photolytic cleavage from oligonucleotides and atmospheric pressure chemical ionization (APCI).

The present invention provides an advantageous tag comprising a tag detectable by mass spectrometry and a method of use thereof, as described more fully herein.

Summary of the Invention

The present invention provides a method for preparing enantiomeric-enriched compounds having photocleavable bonds, and labeled molecules having enantiomeric-enriched photocleavable tags. The present invention includes, for example, the advantage of including the ability to obtain a unique single product when it is necessary to separate members of multiple structurally similar tagged molecules, as well as further related advantages as described more fully herein. To provide.

In one embodiment, the present invention provides a compound of formula (I) or (II).

In the above formula,

R ¹ is selected from halogen and organic residues;

R ² and R ³ are independently selected from hydrogen and organic moieties having a mass of at least 15 daltons, wherein R ² and R ³ together may form a carbonyl group or may be combined together into a cyclic structure;

Z is a (n + 1) -valent atom excluding carbon, where n is an integer of 0 or greater;

R ⁴ is independently selected from hydrogen, halogen, and organic residues having a mass of at least 15 Daltons, provided that at least one R ⁴ (ie R ^4a ) is an organic residue having a mass of at least 100 Daltons;

R ⁵ in each occurrence is independently halogen or an organic moiety having a mass less than 500 Daltons;

m is selected from 0, 1, 2, 3 and 4;

When R ² = R ³ = H, when Z (R ⁴ ) _n is -NH (C0) -CH (iBu) -NH (C0)-(CH ₂ Ph or Ot-Bu), R ¹ is CO ₂ ( R ¹ is not CH ₂ CO ₂ t-Bu when H is not H or CH ₃ ) and Z is OH;

If both compounds of formula (I) and (II) are present as a mixture, then the molar ratio of formula I: formula II in the mixture is not 50:50.

In various specific embodiments of the invention, Z is nitrogen; R ^4a is detectable by mass spectrometry; R ² and R ³ are each hydrogen; R ¹ comprises a synthetic or natural biological material (eg, nucleic acid, protein or saccharide); R ^4a is a mass of less than 10,000 Daltons and the molecular formula C _1-500 N _0-100 O _0-100 S _0-10 P _0-10 H _α F _β I _δ (where the sum of α, β and δ is otherwise Is sufficient to satisfy the unsatisfied valences of C, N, O, P and S).

In another specific embodiment, R ^4a is a chemical formula T ^2- (JT ^3- ) _p- , wherein T ² has a mass of 15 to 500 Daltons, carbon and hydrogen, fluoride, iodide, oxygen, nitrogen, An organic moiety formed from one or more of sulfur and phosphorus; T ³ is an organic moiety formed from one or more of carbon and hydrogen, fluoride, iodide, oxygen, nitrogen, sulfur and phosphorus, having a mass of 50 to 1000 Daltons; J is a direct bond or an amide, ester, amine, sulfide, ether, thioester, disulfide, thioether, urea, thiourea, carbamate, thiocarbamate, Schiff base, reduced Schiff base, imine, oxime, hydra A functional group selected from zones, phosphates, phosphonates, phosphoramides, phosphonamides, sulfonates, sulfonamides or carbon-carbon bonds; p is an integer from 1 to 50; when n is 1 or more, Each T ³ and J is independently selected).

In another specific embodiment, R ^4a is [Wherein, G is (CH _{2) 1-6} (In this case, only one of the hydrogen on the CH ₂ groups of each G and CH ₂ groups - (CH _{2) W} - is replaced with an amide -T ^4), and; T ² and T ⁴ are the C, if the sum is out of the formula _{C 1-25 N 0-9 O 0-9 S 0-3} P 0-3 H α F β I δ ( wherein, α, β and δ, Sufficient to satisfy the unsatisfied valences of N, O, P, and S); Amide is ego; R ¹⁰ is hydrogen or C _1-10 alkyl; w is an integer from 0 to 4; n is an integer from 1 to 50 and when n is 1 or more, G, c, amide, R ¹ and T ⁴ are independently selected.

The invention further provides compositions comprising a compound of Formula I and / or Formula II in which the molar ratio of Formula I: Formula II in the composition is in the range of 95: 5 to 100: 0 or 5:95 to 0: 100. .

The invention is further formulated Wherein Z is selected from oxygen, nitrogen and sulfur, when Z is oxygen or sulfur, R ⁶ is hydrogen, when Z is nitrogen, R ⁶ is selected from hydrogen and C ₁ -C ₂₂ hydrocarbons and two R ⁶ groups Is bonded to Z; R ⁵ is independently at each occurrence an halogen or an organic residue having a mass of less than 500 Daltons; m is selected from 0, 1, 2, 3 and 4; R ⁷ is hydrogen or an organic residue A bond represented by both continuous and interrupted lines represents a single or double bond), which comprises (a) an enzyme, (b) an enzyme, and a compound of formula H ₂ NC (= 0) -CHR ⁸ -NH-R ⁹ , wherein , R ⁸ is an organic moiety and R ⁹ is an amino protecting group), (c) chiral acid, (d) chiral amine, (e) hydrogen and chiral hydrogenation catalyst, and (f) mechanical crystal-separation means By contact with an agent selected from Provided are methods for providing an enantiomerically-enriched compound, including providing an enantiomerically-enriched compound.

In one specific embodiment of the method as defined above, the compound acting in the method is of formula Wherein R ⁵ is independently at each occurrence an organic moiety having a mass of halogen or less than 500 Daltons; m is selected from 0, 1, 2, 3 and 4; R ⁷ is hydrogen or an organic moiety The compound is contacted with an enzyme to To provide enantiomerically-enriched compounds.

In another specific embodiment of the method identified above, the compound acting in the method is of formula Wherein R ⁵ is in each case an organic moiety having a mass of less than halogen or less than 500 daltons; m is selected from 0, 1, 2, 3 and 4; R ⁷ is hydrogen or an organic moiety The compound is contacted with an enzyme and an adjuvant of formula H ₂ NC (= 0) -CHR ⁸ -NH-R ⁹ , wherein R ⁸ is an organic moiety and R ⁹ is an amino protecting group To provide enantiomerically-enriched compounds.

In another specific embodiment of the method identified above, the compound acting in the method is of formula Wherein R ⁵ is independently at each occurrence an organic moiety having a mass of halogen or less than 500 daltons; m is selected from 0, 1, 2, 3 and 4; R ⁷ is hydrogen or an organic moiety , The compound is contacted with chiral acid To provide enantiomerically-concentrated salts.

In another specific embodiment of the method identified above, the compound acting in the method is of formula Wherein R ⁵ is independently at each occurrence an halogen or an organic moiety having a mass of less than 500 Daltons, m is selected from 0, 1, 2, 3 and 4) and the compound is contacted with a chiral amine Chemical formula To provide enantiomerically-concentrated salts.

In another specific embodiment of the method identified above, the compound acting in the method is of formula Wherein R ⁵ is independently at each occurrence a halogen or an organic moiety having a mass of less than 500 Daltons, m is selected from 0, 1, 2, 3 and 4, wherein the compound is in the presence of a chiral hydrogenation catalyst Contact with hydrogen under To provide enantiomerically-enriched compounds.

In another embodiment, the present invention provides a compound of Formula V or VI.

In the above formula,

R ⁵ in each occurrence is independently halogen or an organic moiety having a mass less than 500 Daltons;

m is selected from 0, 1, 2, 3 and 4;

R ⁷ is hydrogen or an organic moiety;

Z is SH or NR ⁸ , wherein R ⁸ is hydrogen or an amine protecting group;

When both compounds of Formulas V and VI are present in the mixture, the molar ratio of Formula V: Formula VI in the mixture is not 50:50.

In another embodiment, the present invention provides a compound of formula VII or VIII.

In the above formula,

R ⁵ in each occurrence is independently halogen or an organic moiety having a mass less than 500 Daltons;

m is selected from 0, 1, 2, 3 and 4;

R ¹¹ is a functional moiety comprising a phosphoramidite or H-phosphonate moiety;

R ¹² is an organic moiety having a mass of 15 to 10,000 Daltons;

In this case, when both compounds of the formulas VII and VIII are present in the mixture, the molar ratio of formula VII: VIII in the mixture is not 50:50.

In one specific embodiment, within the compounds described above, the phosphoramidite residues of R ¹¹ are of the formula —OP (OR ¹³ ) (N (R ¹⁴ ) ₂ ), wherein R ¹³ and R ¹⁴ are each independently Alkyl group, or a substituted alkyl group having one or more substituents selected from halogen and cyano, two R ¹⁴ groups can be joined together to form a heterocycloalkyl group with the nitrogen of phosphoramidite) Has In another specific embodiment, the H-phosphonate moiety of R ¹¹ comprises the formula —OP (═O) (H) (O ^{− +} HN (R ¹⁵ ) ₃ ), wherein R ¹⁵ is independently C _1-6 Alkyl group.

These and other and specific aspects of the invention will become apparent by reference to the following detailed description. For this purpose, various references are described herein and they are described in more detail specific procedures, compounds and / or compositions, which are hereby incorporated by reference in their entirety.

The present invention generally relates to enantiomerically-concentrated compounds, in particular enantiomerically-concentrated compounds having one or more photocleavable bonds, as well as methods related thereto, including synthetic methods.

The present invention provides a tag suitable for coupling to a nucleic acid or other molecule of interest. When coupled to DNA, the tag provides a means to achieve a high efficiency genotyping system. Preferably, the linker between the tag and the molecule of interest should be cleaved completely under conditions that do not cause tag fragmentation. In addition, the tag preferably yields only one peak per implanted oligonucleotide, providing an optimal signal in terms of ion flow. In addition, the tag is preferably stable against PCR conditions, HPLC, and other manipulations used in the assay form. Reagents that introduce tags into molecules of interest should proceed reproducibly and with good yields. In a preferred method consistent with the above objectives, a modular structure has been developed which allows the CMST components to be optimized independently. The components of the CMST preferably include photolabile linkers, mass spectrometric sensitivity enhancers (MSSEs), variable mass units (VMUs), all of which are bound together through a scaffold.

The present invention provides tagged molecules of interest, precursors of tagged molecules of interest, and methods of making tagged molecules and precursors thereof, wherein the tags are enantiomerically-enriched. By using enantiomerically-enriched precursors, purification of the reaction products of precursors and molecules of interest is facilitated to provide purified tagged molecules of interest.

Accordingly, the present invention provides compounds of formula I or II.

Formula I

Formula II

In the above formula,

R ¹ is selected from halogen and organic residues;

R ² and R ³ are independently selected from hydrogen and organic moieties having a mass of at least 15 daltons, wherein R ² and R ³ together may form a carbonyl group or may be combined together into a cyclic structure;

Z is a (n + 1) -valent atom excluding carbon, where n is an integer of 0 or greater;

R ⁴ is independently selected from hydrogen, halogen, and organic residue having a mass of at least 15 Daltons, provided that at least one R ⁴ is an organic residue having a mass of at least 100 Daltons;

R ⁵ is independently at each occurrence an organic moiety having a mass of halogen or less than 500 daltons;

m is selected from 0, 1, 2, 3 and 4.

The compounds of the present invention are enantiomerically concentrated, with the result that both compounds of formula (I) and (II) are present as mixtures, and the molar ratio of formula (I) to formula (II) in the mixture is not 50:50.

Compounds of the present invention include 2-nitrophenyl groups having a methyl group substituted at position 1 of the phenyl ring, wherein the carbon atoms of the methyl group will be referred to herein as C1 or benzyl carbon atoms. In addition to the 2-nitrophenyl group, the C1 atom is directly bonded to a hydrogen atom (not shown), a carbon atom (herein referred to as C2) and a non-carbon atom.

Due to the 2-nitrophenyl group, the compounds of the present invention are photocleavable. That is, the compound of the present invention reacts to contact with specific electron magnetic radiation by performing a cleavage reaction, whereby the substituted Z atoms are separated from C1 as shown in the following structure (wherein " "Is a photolytically labile bond).

Since C 1 is directly bonded to only one atom and not to carbon or hydrogen atoms, the compounds of the present invention perform a selective photocleavage reaction. That is, C 1 -Z bonds can be selectively cleaved under suitable photodegradable conditions, leaving largely other C 1 bonds if they are not completely intact. Such selective cleavage can occur as long as Z is not a carbon atom. In a preferred specific embodiment, Z is selected from oxygen, nitrogen and sulfur. Oxygen, nitrogen and sulfur are each preferred atoms at position Z.

Photolysis conditions that allow selective cleavage of the C1-Z bond are hardly affected by other substitutions on the 2-nitrophenyl ring. Thus, any one or more hydrogen atoms of the phenyl ring can be replaced with the same number of R ⁵ groups. Thus, the compounds of the present invention may have a plurality of R ⁵ groups, where m is an integer selected from 0, 1, 2, 3 and 4. Preferred photocutting chemistry is hardly influenced by the properties of the R ⁵ group, and consequently R ⁵ can be an inorganic group, for example a halogen, or an organic group. Typically, the organic R ⁵ group is not too large and therefore has a mass of less than about 500 Daltons. Preferred R ⁵ groups are selected from C ₁ -C ₂₂ hydrocarbon groups. In a preferred compound of the invention, m is equal to 0, with the result that the 2-nitrophenyl ring has four hydrogen substituents.

After performing the photocleavage reaction, the resulting Z-containing residues derived from the compounds of the invention will be referred to herein as tags. The tag is or includes a detectable moiety, wherein the term refers to a moiety that can be detected by analytical methods, and preferably can be characterized. Representative analytical methods include spectroscopic or potentiometric methods. Representative spectroscopy methods include mass spectroscopy, infrared spectroscopy, ultraviolet spectroscopy and fluorescence spectroscopy. Representative potentiometer method is the potentiometer ammeter method.

To provide an effective tag, at least one R ⁴ group is an organic moiety having a mass of at least 100 Daltons. This R ⁴ group will be referred to herein as the first R ⁴ group (or R ^4a ). It is desirable for the tag to have a mass of 100 Daltons or more. If the tag has a mass of less than 100 Daltons, it may be difficult to identify the tag. For example, when tags are identified and characterized by mass spectroscopy, tags with molecular weights less than 100 Daltons may be difficult to distinguish from background noise inherent in the operation of the mass spectrometer.

The invention also provides compositions containing a plurality of unequal compounds of the invention, wherein unequal compounds contain unequal tags that can be distinguished from one another by analytical methods. In order to identify a number of non-identical tags, the tags must be sufficiently different from each other so that the analysis method can detect the difference. For example, to obtain a suitable number of tags that are distinguished by ammeter methods, the tags must contain a number of atoms that can be bonded together in an analytically identifiable, non-identical manner. If the tag has a mass of less than 100 Daltons, there will be an overly limited number of analytically identifiable tags. Thus, in order to allow sufficient variability in the structure, and to allow for easy effective detection, the tag must have a mass of at least 100 Daltons.

As long as the first R ⁴ group has a mass of at least 100 Daltons, the identification of other R ⁴ groups in the compounds of the present invention may be selected to achieve one or more purposes. For example, the second or more R ⁴ (eg, R ^4b , R ^4c, etc.) group may provide information that complements the information derived from the first R ⁴ group. In this example, the second or more R ⁴ groups also preferably have a mass of at least 100 Daltons. However, in another example, the second or more R ⁴ group is merely satisfying the insufficient valence state of C1 elsewhere, and does not interfere with photocutting reaction or detection of the tag and is easily formed synthetically. It is preferable. In this latter example, the second or more R ⁴ groups can be hydrogen or simple hydrocarbon groups. In a preferred embodiment, the second or more R ⁴ groups, when present, are hydrogen.

In the compounds of the present invention, R ¹ is selected from hydrogen and organic residues, while R ² and R ³ are independently selected from hydrogen and organic residues having a mass of at least 15 daltons. As mentioned above, the compounds of the present invention include detectable moieties in which detection and optionally characterization of detectable moieties provide information for the compounds of the present invention. In one specific embodiment, the R ¹ group contains the properties of the compound of the desired information. For example, R ¹ may comprise a biological material from an individual, and the tag is a characteristic of the individual and / or a specific biological substance. As another example, the R ¹ group may contain a specific synthetic nucleic acid, peptide or saccharide sequence, which is specifically identified by a tag. In another specific embodiment, R ¹ is a reactive chemical group capable of carrying out a chemical reaction that enables the biologically-derived material to be photocleavable. For example, R ¹ can be a halogen atom that can be replaced in a chemical reaction by a biological material or a derived biological material.

The R ² and R ³ groups are independently selected from hydrogen and organic residues having a mass of at least 15 daltons. Typically, the R ² and R ³ groups are selected in terms of their effect on the chemical reaction chosen to bind R ¹ to C ¹ and / or on the photocleavage reaction that causes the tag to separate from C 1. When each of R ² and R ³ is hydrogen, neither R ² nor R ³ has an adverse effect on the photocleavage reaction, and typically they are compatible with all chemical reactions used to couple R ¹ to C1. Therefore, R ² and R ³ are preferably hydrogen. However, R ² and R ³ may be groups other than hydrogen. For example, R ² and R ³ may together form a carbonyl group with C 1. Alternatively, R ¹ and R ² together with C ¹ can form a cyclic structure. Alternatively, either R ² or R ³ can be an organic moiety having a mass of at least 15 Daltons, for example a C ₁ -C ₂₂ hydrocarbon group. Typically, hydrocarbon groups do not interfere with the desired photocleavage reaction and do not interfere with the R ¹ / C1 coupling chemistry.

Another property of the compounds of the present invention is that the C1 atom is chiral. As shown from formulas (I) and (II), the nature of the substituents on C1 essentially requires that C1 is chiral. In addition, when compounds of formula (I) and (II) are present in a mixture, the molar ratio of the compound of formula (I) to the compound of formula (II) is not 50:50. In a preferred specific embodiment of the invention, the compound of formula (I) is not present in mixture with the compound of formula (II) and vice versa. In addition, when the compound of formula I is present in a mixture with the compound of formula II, the molar ratio of the compound of formula I to the compound of formula II is preferably 95: 5, 96: 4, 97: 3, 98: 2 Or greater than 99: 1 or greater than 99.5: 0.5, and vice versa (ie, the molar ratio of the compound of Formula II to the compound of Formula I is preferably 95: 5, 96: 4, 97: 3, 98 : 2, or greater than 99: 1 or greater than 99.5: 0.5).

Since the compounds of formula (I) or (II) do not exist in admixture with the compounds of formula (II) or (I), respectively, or only minimally, the chromatographic separation associated with the compounds of formula (I) or (II) results in significant amounts of It is much easier and more effective than when compounds of II are present as mixtures with one another. The present invention provides compositions containing a plurality of compounds of formula (I) or (II), and because of their isomeric structure, they can be more easily separated from each other than in other cases. This enhanced ability to identify differently tagged compounds allows for the formation of compositions that cannot otherwise be characterized.

For example, two compounds of formula (I) may differ due to slightly different R ¹ groups, while these two compounds (compounds IA and IB) are compounds of formula (II) having the same substitution around C1 (compound IIA). And IIB), when present in admixture with each other. This is because the chromatographic elution time of compound IA may overlap with the elution time of compound IIB and / or the elution time of compound IB may overlap with the elution time of compound IIA. Thus, compounds IA and IB cannot be easily separated from each other due to interference from compounds IIA and IIB. The present invention solves this problem by providing a compound of formula (I) which does not contain or does not contain a compound of formula (II).

In another aspect, the invention relates to a method of providing an enantiomerically-enriched compound using a specific agent. More specifically, the methodWherein Z is selected from oxygen, nitrogen and sulfur; and if Z is oxygen or sulfur⁶Is hydrogen and when Z is nitrogen⁶Silver hydrogen and C_One-C₂₂ Selected from hydrocarbons and two R⁶The group is bonded to Z; R⁵Is independently at each occurrence an organic moiety having a mass of halogen or less than 500 daltons; m is selected from 0, 1, 2, 3 and 4; R⁷Is hydrogen or an organic moiety; Bonds represented by both continuous and interrupted lines represent single or double bonds).

The method of the present invention brings the compound into contact with the particular agent as identified above. The agent comprises (a) an enzyme, (b) an enzyme and an adjuvant of formula H ₂ NC (= 0) -CHR ⁸ -NH-R ⁹ , wherein R ⁸ is an organic moiety and R ⁹ is an amino protecting group, (c) chiral acid, (d) chiral amine, (e) hydrogen and chiral hydrogenation catalyst, and (f) mechanical crystal-separation means. The compound is contacted with the agent to To provide enantiomerically-enriched compounds.

In addition to the specific techniques described herein for preparing compounds having photolabile bonds that bind to enantiomerically concentrated tags, other suitable techniques can be found in the following references: Asymmetric Catalysis in Organic Synthesis, R. Noyori, John Wiley & Sons, New York, NY, 1994; Asymmetric Synthetic Methodology, D. J. Ager and M. B. East, CRC Press, Boca Raton, FL, 1995; Asymmetric Synthesis, R.A. Aitken and S.N. Kilenyi, Eds. Blackie Academic & Professional, Glassglow, U.K., 1992; Asymmetric Synthesis, J. Morrison, Ed., Academic Press, Orlando FL. (series, including volumes issued in 1984 and 1985); Asymmetric Synthesis, R.G. Proctor, Oxford University Press, New York, NH, 1996; Asymmetric Synthesis: Construction of Chiral Molecules using Amino Acids, G.M. Coppola and H.F. Schuster, John Wiley & Sons, New York, NY, 1987; Catalytic Asymmetric Synthesis, I. Ojima, Ed. VCH Publishers, New York, NY, 19933; Chiral Auxiliaries and Ligands in Asymmetric Synthesis, J. Seyden-Penne, John Wiley & Sons, New York, NY, 1995; Chiral Separations; Applications and Technology, S. Ahuja, Ed., American Chemical Society, Washington, D.C., 1996; Chirality in Industry I & II, A.N. Collins, G.N. Sheldrake, and J. Crosby, Eds., John Wiley & Sons, New York, NY, 1995 and 1997; Chirotechnology: Industrial Synthesis of Optically Active Compounds, R.A. Sheldon, Marcel Dekkar, New York, NY, 1993; and Enantiomers, Racemates, and Resolutions, J. Jacques, A. Collet, and S.H. Wilen, John Wiley & Sons, New York, NY, 1981].

Compounds and compositions of the invention, and methods of making the same, can be used to provide tagged molecules in which the tag is optimally concentrated and photocleavably bound to the molecule. Molecules with photocleavable tags, which can be provided in optimally-concentrated form according to the invention, as well as methods which can be carried out with the compounds and compositions of the invention are described, for example, in PCT International. Patent publications WO 95/05319, WO 97/27331, WO 97/27327, WO 97/27325 and WO 95/04160. Thus, the compounds of the present invention may replace other methods and assays using the tagged compounds described in the above four publications, as well as molecules having tags that are photocleavably bound.

The following examples are presented by way of illustration of the invention and are not intended to be limiting.

In the examples below, unless otherwise stated, the chemical reagents and reagents are of standard commercial grade, Aldrich (Milwaukee, WI; www.sigma-aldrich.com), Fluka (a division of Aldrich), and Lancaster Synthesis, Inc. Obtained from commercial sources such as Windham, NH; http://www.lancaster.co.uk.

Example 1

Decomposition with Chiral Acid DTTA

500 mL of (±) -3-amino-3- (2-nitrophenyl) propionic acid methyl ester (24.6 g, 110 mmol) and chiral acid di-p-toluyl-D-tartaric acid (DTTA, 42.4 g, 110 mmol) Heated to 67 ° C. and mixed. The resulting solution was cooled to 56 ° C. and treated with a salt of 3-amino-3- (2-nitrophenyl) propionic acid methyl ester and DTTA. The mixture was cooled to ambient temperature (21 ° C.) and stirred for 16 hours. The resulting crystals were collected by filtration to give a white solid concentrated with one enantiomer (46% enantiomer excess, e.e.). One crystallization in methanol yielded 12 g of material with diastereomeric purity of 88%.

Example 2

Decomposition with Chiral Acid (-) Campanic Acid

(±) -3-amino-3- (2-nitrophenyl) propionic acid (172 mg, 0.82 mmol) and chiral acid (-)-campanoic acid (165 mg, 0.83 mmol by Fluka) were heated in 5 ml of water It was refluxed and mixed. The resulting solution was cooled to ambient temperature (21 ° C.). The crystals formed were collected by filtration to give a white solid (105 mg, 31% yield). Chiral high pressure liquid chromatography (cHPLC, Chirobiotic T column at 5 ° C., 75% buffer of 20 mM ammonium acetate, pH 4.5 and 25% EtOH, detector at 215 nm) shows that the product is enantiomerically pure. It was.

Example 3

Selective Hydrolysis Using Enzymes

(±) -3-amino-3- (2-nitrophenyl) propionic acid methyl ester (60 g, 270 mmol) was dissolved in phosphate buffer (50 mM, 500 mL) and adjusted to pH 7. Amano PS enzyme (Amano PS Enzyme, 6 g, 10 wt%, manufactured by Amano Enzyme, Milton Keynes, UK; Lombard IL, USA: www.amano-enzyme.co.jp) as a slurry in phosphate buffer (100 mL). To the solution was added and the reaction mixture was stirred at ambient temperature (21 ° C.) for 24 hours. After completion, the amino acid precipitate was filtered off, the aqueous filtrate was removed and the solid was washed with dichloromethane (DCM, 2 x 10 mL). The solid was dissolved in aqueous HCl (6M, 150 mL) but a fine unknown precipitate still remained and was filtered off. The resulting aqueous mixture was extracted with DCM and adjusted to pH 7 to reprecipitate the solid. This gave the desired amino acid (23 g, 90% yield).

The first filtrate was basified to pH 8 and first extracted with the collected DCM washes and secondly with fresh DCM. The organic layers were mixed, dried and evaporated to afford amino ethyl ester (23 g, 80% yield). Optical rotation of the ethyl ester HCl salt in methanol gave + 127.55 ° (c = 1, 20 ° C., 589 nm) of α _D. Enantiomeric excess (ee) of both amino acids and amino ethyl esters was determined by cHPLC and both were more than 98% enantiomeric pure.

Example 4

Synthesis of Reactive Molecules to Deliver Optically-Enriched Tags to the Molecule of Interest

The tag described herein is preferably bound to the molecule of interest via photolabile binding. Preferred photolabile bonds are present in the o-nitrobenzyl group. See, for example, (a) Greenberg, M.M .; Gilmore, J.L. J. Org. Chem. 1994, 59, 746-753; (b) Yoo, D. J.,; Greenberg, M.M. J. Org. Chem. 1995, 60, 3358-3364; and (c) Venkatesan, H .; Greenberg, M.M. J. Org. Chem. 1996, 61, 525-529. Intramolecular photoreduction reactions in the o-nitrobenzyl group (e.g. Pillai, VNRSynthesis 1980, 1-26) rapidly raise without any fragmentation under neutral conditions (6 sec exposure with 254 nm Hg lamp). The tag can be cleaved from the nucleotide.

Thus, the synthetic route (Scheme 1) is based on the photosensitive linker 3-amino-3- (2-nitrophenyl) propionic acid [ANP, 1 , see, eg, Brown, BB; Wagner, DS; Geysen, HM Molecular Diversity 1995, 1, 4-12; and (b) Rodebaugh, R .; Fraser-Reid, B .; Geysen, HM Tetrahedron Letters 1997, 38, 7653-7656, to give ethyl ester hydrochloride 2 in 84% yield. Enzymatic conversion of 2 then provides ethyl ester as the sole isomer, which facilitates HPLC purification in the oligonucleotide binding step. Ethyl ester hydrochloride was dissolved in water and adjusted to neutral pH with 2N HCl. Amano PS esterase enzyme was added as a phosphate buffer slurry. After completion of the reaction, base post-treatment removed the hydrolyzed ANP by-product ( 4 ) and recovered the homo-isomeric ethyl ester ( 3 ,> 99% ee) (92% of useful material).

Lysine provided a very suitable scaffold for tag synthesis in the modular method. Two amines and one carboxylic acid group present in lysine provided an opportunity for potent peptide chemistry while causing amide bonds between tag components. Peptides have foreseeable action in mass spectrometry and are also relatively stable against 254 nm light. As shown in Scheme 2, EDACI (1- [3- (dimethylamino) propyl] -3-ethyl-carbodiimide hydrochloride (Aldrich Chemical Co., Milwaukee, WI) and 1-hydroxybenzotriazole (HOBT) was used to couple 3 and α-BOC-ε-Alloc-lysine ( 5 ) to obtain protected ANP lysine ( 6 ), which was white in 81% yield from 3 by removal of BOC using HCl. Ε-Alloc-lysine ANP ester 7 was obtained as a solid.

Perfluorinated aromatics can be used as electrical labels for analytical purposes in anion mode mass spectrometry. See, for example, (a) Abdel-Baky, S .; Klempier, N .; Giese, R. W. Tetrahedron 1990, 46, 5859; (b) Abdel-Baky, S .; Allam, K .; Giese, R. W. Anal. Chem. 1993, 65, 498-499; (c) Trainor, T; Giese, R. W .; Vouros, P. Journal of Chromatography 1988, 452, 369-376; and (d) Saha, M .; Saha, J .; Giese, R. W. Journal of Chromatography 1993, 641, 400-404]. Our initial strategy for MSSE candidates, the ionization component of the tag, consisted of attaching various electron deficient highly fluorinated carboxylic acids to representative scaffolds and measuring relative ion flow with anion mode APCI. When fragmentation occurred, multiple peaks appeared with weak signals. Thus, enhancers for reverse ionization methods have been developed. The tag was prepared by introducing pyridyl, prolinyl and piperidyl structures as MSSE and tested in positive mode APCI. For all criteria, tags with N-methyl isonifecotic acid (INA) showed good results.

INA hydrochloride was coupled to 7 using EDAC and triethylamine to give an Alloc-protected crude structure 8 which was deprotected with diethylamine, triphenylphosphine, and palladium acetate. The resulting core structure ( 9 ) was crystallized from the reaction mixture and recovered by filtration as a yellow solid in 95% yield.

In order to provide a large set of detectable tags with unique molecular weights, core structure 9 was derivatized with a set of 45 carboxylic acids, referred to as variable mass units (VMUs). The mass of the VMU is placed at least 4 amu apart to minimize isotope redundancy between the tags. The molecular weight of the VMU is about 90 to 298 amu. Preferred VMUs do not have the following properties or characteristics: (1) functional groups that are not compatible with synthetic sequences such as esters; (2) factors with multiple isotopes (Cl, Br, S); (3) functional groups (iodide, acyl- and aryl-phenones) that can result in competitive light action; (4) racemic acid; And (5) lack of commercial availability. VMU was coupled to 9 using HATU and N-methyl morpholine. After purification by column chromatography, CMST ethyl ester ( 10 ) was recovered in various yields. Saponification of 10 with NaOH gave CMST acid ( 11 ) in quantitative yield. For the final step, the formation of active esters is carried out using tetrafluorophenyl trifluoroacetate and base-catalyzed transesterification of 11 [TFP TFA, see, eg, Green, M .; Berman, J. Tetrahedron Letters 1990, 31, 5851-5852; TFP TFA was prepared and used in a similar manner to pentafluorophenyl trifluoroacetate, because the protons on the tetrafluorophenyl ring act as diagnostic NMR inhibitors. ( 12 ) was obtained. Tetrafluorophenyl active esters were chosen because of their easily removed by-products and their relative stability and compatibility with oligonucleotide binding conditions. CMST TFP esters are described in, for example, Lukhtanov EA; Kutyavin, IV; Gamper, HB; Meyer, RB Jr. Bioconjug. Chem. 1995, 4, 418-426] to 5'-aminohexyl-terminated oligonucleotides (TriLink Biotechnology, San Diego, Calif.).

The number of tags further comprises tyrosine derivatives [GMA, and more specifically N-Boc-O-ethyl-L-tyrosine, which act as total mass regulators, which are purchased from Bachem California, Torrance, CA, or N-Boc-L -Tyrosine-OMe can be prepared by alkylation with iodoethane and cesium carbonate in 86% yield followed by quantitative hydrolysis with NaOH] (which increases the molecular weight of the cleaved tag by about 191 amu and results in a higher molecular weight of the VMU set. Enable reuse with tags). GMA was coupled to core structure 9 as shown in Scheme 4. After base workup, the BOC-protected GMA structure was isolated by filtration. Deprotection with TFA gave the GMA monomer core structure ( 13 , n = 1) in quantitative yield. The VMU was coupled in the same manner as before to obtain an additional set of tags 14 . Using four units of mass control agent, a total of about 200 tags can be synthesized.

From the foregoing, while certain specific aspects of the invention have been described herein for illustrative purposes, it will be apparent that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is limited only by the appended claims.

Claims (20)

Compounds of formula (I) or (II). Formula I Formula II In the above formula, R ¹ is selected from halogen and organic residues; R ² and R ³ are independently selected from hydrogen and organic moieties having a mass of at least 15 daltons, wherein R ² and R ³ together may form a carbonyl group or may be combined together into a cyclic structure; Z is a (n + 1) -valent atom excluding carbon, where n is an integer of 0 or greater; R ⁴ is independently selected from hydrogen, halogen, and organic residues having a mass of at least 15 Daltons, provided that at least one R ⁴ (ie R ^4a ) is an organic residue having a mass of at least 100 Daltons; R ⁵ in each occurrence is independently halogen or an organic moiety having a mass less than 500 Daltons; m is selected from 0, 1, 2, 3 and 4; When R ² = R ³ = H, when Z (R ⁴ ) _n is -NH (C0) -CH (iBu) -NH (C0)-(CH ₂ Ph or Ot-Bu), R ¹ is CO ₂ ( R ¹ is not CH ₂ CO ₂ t-Bu when H is not H or CH ₃ ) and Z is OH; If both compounds of formula (I) and (II) are present as a mixture, then the molar ratio of formula I: formula II in the mixture is not 50:50. The compound of claim 1, wherein Z is nitrogen. The compound of claim 1, wherein R ^4a is detectable by mass spectrometry. The _compound of claim 1, wherein R ^4a is less than 10,000 Daltons of mass and molecular formula C _1-500 N _0-100 O _0-100 S _0-10 P _0-10 H _α F _β I _δ , wherein α, β, and δ The sum of is otherwise sufficient to meet the unsatisfied valences of C, N, O, P and S). The compound of claim 1, wherein R ^4a is of the formula T ^2- (JT ^3- ) _p- , wherein T ² has a mass of 15 to 500 Daltons, carbon and hydrogen, fluoride, iodide, oxygen, nitrogen, An organic moiety formed from one or more of sulfur and phosphorus; T ³ is an organic moiety formed from one or more of carbon and hydrogen, fluoride, iodide, oxygen, nitrogen, sulfur and phosphorus, having a mass of 50 to 1000 Daltons; J is a direct bond or an amide, ester, amine, sulfide, ether, thioester, disulfide, thioether, urea, thiourea, carbamate, thiocarbamate, Schiff base, reduced Schiff base, imine, oxime, hydra A functional group selected from zones, phosphates, phosphonates, phosphoramides, phosphonamides, sulfonates, sulfonamides or carbon-carbon bonds; p is an integer from 1 to 50; when n is 1 or more, T ³ And J is independently selected. The compound of claim 1, wherein R ^4a is Wherein G is (CH ₂ ) _1-6 , wherein only one hydrogen and CH ₂ group on each CH ₂ group of each G is replaced with-(CH ₂ ) _W -amide-T ⁴ ; T ² and T ⁴ are the C, if the sum is out of the formula _{C 1-25 N 0-9 O 0-9 S 0-3} P 0-3 H α F β I δ ( wherein, α, β and δ, Sufficient to satisfy the unsatisfied valences of N, O, P, and S); Amide is ego; R ¹⁰ is hydrogen or C _1-10 alkyl; w is an integer from 0 to 4; n is an integer from 1 to 50 and when n is 1 or more, G, c, amide, R ¹ and T ⁴ are independently selected. The compound of claim 1, wherein R ² and R ³ are each hydrogen. The compound of claim 1, wherein R ¹ comprises a synthetic or natural biological material. The compound of claim 8, wherein R ¹ comprises a nucleic acid, protein or saccharide. A composition comprising the compound of claim 1, wherein the molar ratio of formula I: formula II falls within the range of 95: 5 to 100: 0 or 5:95 to 0: 100. Chemical formula Wherein Z is selected from oxygen, nitrogen and sulfur, when Z is oxygen or sulfur, R ⁶ is hydrogen, when Z is nitrogen, R ⁶ is selected from hydrogen and C ₁ -C ₂₂ hydrocarbons and two R ⁶ groups Is bonded to Z; R ⁵ is independently at each occurrence an halogen or an organic residue having a mass of less than 500 Daltons; m is selected from 0, 1, 2, 3 and 4; R ⁷ is hydrogen or an organic residue A bond represented by both continuous and interrupted lines represents a single or double bond), which comprises (a) an enzyme, (b) an enzyme, and a compound of formula H ₂ NC (= 0) -CHR ⁸ -NH-R ⁹ , wherein , R ⁸ is an organic moiety and R ⁹ is an amino protecting group), (c) chiral acid, (d) chiral amine, (e) hydrogen and chiral hydrogenation catalyst, and (f) mechanical crystal-separation means In contact with an agent selected from A method for obtaining an enantiomerically-enriched compound, comprising obtaining an enantiomerically-enriched compound. The compound of claim 11, wherein Wherein R ⁵ is independently at each occurrence an organic moiety having a mass of less than halogen or less than 500 daltons; m is selected from 0, 1, 2, 3 and 4; R ⁷ is hydrogen or an organic moiety Is contacted with an enzyme to To obtain an enantiomerically-enriched compound. The compound of claim 11, wherein Enzyme a compound of (wherein, R ⁵ is in each case an organic moiety having a mass of halogen or lower than 500 daltons, and; R ⁷ is hydrogen or an organic moiety; m is 0, is selected from 1, 2, 3 and 4) And an adjuvant of formula H ₂ NC (═O) —CHR ⁸ -NH-R ⁹ , wherein R ⁸ is an organic moiety and R ⁹ is an amino protecting group. To obtain an enantiomerically-enriched compound. The compound of claim 11, wherein Wherein R ⁵ is independently at each occurrence an organic moiety having a mass of less than halogen or less than 500 daltons; m is selected from 0, 1, 2, 3 and 4; R ⁷ is hydrogen or an organic moiety By contacting with chiral acid To obtain enantiomerically-concentrated salts of a. The compound of claim 11, wherein Wherein R ⁵ is independently at each occurrence a halogen or an organic moiety having a mass of less than 500 Daltons and m is selected from 0, 1, 2, 3 and 4) by contacting a compound of the formula with a chiral amine To obtain enantiomerically concentrated salts. The compound of claim 11, wherein Wherein R ⁵ is independently at each occurrence an halogen or an organic moiety having a mass of less than 500 Daltons and m is selected from 0, 1, 2, 3 and 4) with a hydrogen in the presence of a chiral hydrogenation catalyst By contacting To obtain an enantiomerically concentrated compound. Compounds of Formula V or VI. Formula V Formula VI In the above formula, R ⁵ in each occurrence is independently halogen or an organic moiety having a mass less than 500 Daltons; m is selected from 0, 1, 2, 3 and 4; R ⁷ is hydrogen or an organic moiety; Z is SH or NR ⁸ , wherein R ⁸ is hydrogen or an amine protecting group; When both compounds of Formulas V and VI are present in the mixture, the molar ratio of Formula V: Formula VI in the mixture is not 50:50. Compounds of Formula VII or VIII. Formula VII Formula VIII In the above formula, R ⁵ in each occurrence is independently halogen or an organic moiety having a mass less than 500 Daltons; m is selected from 0, 1, 2, 3 and 4; R ¹¹ is a functional moiety comprising a phosphoramidite or H-phosphonate moiety; R ¹² is an organic moiety having a mass of 15 to 10,000 Daltons; In this case, when both compounds of the formulas VII and VIII are present in the mixture, the molar ratio of formula VII: VIII in the mixture is not 50:50. The compound of claim 18, wherein the phosphoramidite moiety of R ¹¹ is of the formula —OP (OR ¹³ ) (N (R ¹⁴ ) ₂ ), wherein R ¹³ and R ¹⁴ are each independently an alkyl group, or halogen and cyan. A substituted alkyl group having one or more substituents selected from the furnace, and two R ¹⁴ groups can be joined together to form a heterocycloalkyl group with the nitrogen of the phosphoramidite. The compound of claim 18, wherein the H-phosphonate moiety of R ¹¹ comprises the formula —OP (═O) (H) (O ^{− +} HN (R ¹⁵ ) ₃ ), wherein R ¹⁵ is independently C _1-6 Compounds that are alkyl groups.