BACKGROUND OF THE INVENTION While the radiolabeling and fluorescence tagging of DNA is widely used for detection purposes in molecular biology and genetics, the number of tags is too small for high level multiplexing and is not suitable for large scale, rapid data acquisition. A new class of tags, comprised of small molecule, cleavable mass spectrometry tags (CMSTs), has been developed and used in single nucleotide polymorphism (SNP) genotyping and gene expression measurement applications (see, e. g., one aspect of PCT International Publication Nos. WO 99/05319; WO 97/27331; WO 97/27327; and WO 97/27325). The system is based on the covalent attachment of the CMSTs to oligonucleotides with a linker, such as a photocleavable linker. Each tag has a different mass that is specific for a designated oligonucleotide sequence.

Identification of the alleles or expressed sequences present is determined, for example, by photolytic cleavage from the oligonucleotides and simultaneous detection with a standard single quadrupole mass spectrometer using atmospheric pressure chemical ionization (APCI).

The present invention provides advantageous tags, including tags detectable by mass spectrometry, and methods of their use, as disclosed more fully herein.

SUMMARY OF THE INVENTION The present invention affords enantiomerically-enriched compounds having photocleavable bonds, and methods for preparing labeled molecules with enantiomerically-enriched photocleavable tags. The present invention provides

advantages including the ability to obtain a single distinct product in those instances where there is a need to isolate members of a plurality of structurally similar tagged molecules, as well as further related advantages as disclosed more fully herein.

In one aspect, the present invention provides a compound having the formula (1) or (II): wherein R'is selected from halogen and organic moieties; R2 and R3 are independently selected from hydrogen and organic moieties having a mass greater than 15 Daltons where R2 and R3 can together form a carbonyl group or may be joined together within a cyclic structure; Z is an (n+ )-valent atom excluding carbon where n is an integer greater than 0; R4 is independently selected from hydrogen, halogen, and organic moieties having a mass greater than 15 Daltons, with the proviso that at least one R4 (namely R4a) is an organic moiety having a mass greater than 100 Daltons; W at each occurrence is independently either halogen or an organic moiety having a mass of less than 500 Daltons; m is selected from 0,1,2,3 and 4; wherein if R2 = R3= H, then R'is not C02 (H or CH3) when Z (R4) n is either of-NH (CO)-CH (iBu)-NH (CO)- (CH2Ph or 0-t-Bu), and R'is not CH2CO2t-Bu when Z is OH; and wherein if compounds of both formulae (I) and (II) are present in an admixture, the molar formula (I): formula (II) ratio in the admixture is other than 50: 50.

In various embodiments of the invention, Z is nitrogen; R4a is detectable by mass spectrometry; R2 and R3 are each hydrogen; R'comprises synthetic or natural biological material (e. g., nucleic acid, protein or saccharide); and/or R4a has a mass of less than 10,000 Daltons and a molecular formula of C1-500N0-100O0-100S0-10P0-10HαFßI#

wherein the sum of a, p and 8 is sufficient to satisfy the otherwise unsatisfied valencies of the C, N, O, P and S atoms.

In yet another embodiment, R4a has the formula T2- (J-T3-) p-; wherein T2 is an organic moiety formed from carbon and one or more of hydrogen, fluoride, iodide, oxygen, nitrogen, sulfur and phosphorus, having a mass of 15 to 500 Daltons; T3 is an organic moiety formed from carbon and one or more of hydrogen, fluoride, iodide, oxygen, nitrogen, sulfur and phosphorus, having a mass of 50 to 1000 Daltons; J is a direct bond or a functional group selected from amide, ester, amine, sulfide, ether, thioester, disulfide, thioether, urea, thiourea, carbamate, thiocarbamate, Schiff base, reduced Schiff base, imine, oxime, hydrazone, phosphate, phosphonate, phosphoramide, phosphonamide, sulfonate, sulfonamide or carbon-carbon bond; and p is an integer ranging from 1 to 50, and when n is greater than 1, each T3 and J is independently selected.

In another embodiment, R4a has a formula comprising: , r4 Amide Amide (CH2) W 'I. N G n Pu''0 wherein G is (CH2)1-6 wherein a hydrogen on one and only one of the CH2 groups of each G is replaced with- (CH2) w-Amide-T4; T2 and T4 are organic moieties of the formula C). 25No-90o-9So-3Po-3HaFpl8 wherein the sum of a, ß and 8 is sufficient to satisfy the otherwise unsatisfied valencies of the C, N, O, S and P atoms; Amide is is hydrogen or C,, 0 alkyl; w is an integer ranging from 0 to 4; and n is an integer ranging from 1 to 50 such that when n is greater than 1, G, c, Amide, Rl and T4 are independently selected.

The invention further provides a composition comprising compounds of formulae (1) and/or (II) wherein the formula (I): formula (II) molar ratio in the composition is within the range of 95: 5 to 100: 0 or within the range of 5: 95 to 0: 100.

The invention further provides a process for providing an enantiomerically-enriched compound comprising contacting a compound of the formula:

wherein Z is selected from oxygen, nitrogen and sulfur, where R is hydrogen when Z is oxygen or sulfur, and when Z is nitrogen then R6 is selected from hydrogen and C-C22 hydrocarbon and two R groups are bonded to Z; R5 at each occurrence is independently either halogen or an organic moiety having a mass of less than 500 Daltons; m is selected from 0,1,2,3 and 4; R is hydrogen or an organic moiety, and the bond represented by both a solid and dashed line represents either a single or double bond; with an agent selected from (a) an enzyme; (b) an enzyme and an adjuvant of the formula H2N-C (=O)-CHR8-NH-R9 wherein R8 is an organic moiety and R9 is an amino protecting group; (c) a chiral acid; (d) a chiral amine; (e) hydrogen and a chiral hydrogenation catalyst; and (f) a mechanical crystal-separating means; to provide an enantiomerically-enriched compound of the formula:

In one embodiment of the above-identified process, the compound to be acted upon in the process has the formula:

wherein R5 at each occurrence is independently either halogen or an organic moiety having a mass of less than 500 Daltons; m is selected from 0,1,2,3 and 4; R7 is hydrogen or an organic moiety; and the compound is contacted with an enzyme to provide an enantiomerically-enriched compound of the formula:

In another embodiment of the above-identified process, the compound to be acted upon in the process has the formula:

wherein R at each occurrence is independently either halogen or an organic moiety having a mass of less than 500 Daltons; m is selected from 3 and 4; R7 is hydrogen or an organic moiety; and the compound is contacted with an enzyme and an adjuvant of the formula H2N-C (=o)-CHR8-NH-R9 wherein R8 is an organic moiety and R9 is an amino protecting group; to provide an enantiomerically-enriched compound of theformula:

In another embodiment of the above-identified process, the compound to be acted upon in the process has the formula:

wherein R5 at each occurrence is independently either halogen or an organic moiety having a mass of less than 500 Daltons; m is selected from 3 and 4; R is hydrogen or an organic moiety; and the compound is contacted with a chiral acid, to provide an enantiomerically-enriched salt of the formula: (R5),, N02 fA < ; > <\ NO2\O NH2 Chiral Acid

In another embodiment of the above-identified process, the compound to be acted upon in the process has the formula:

wherein R at each occurrence is independently either halogen or an organic moiety having a mass of less than 500 Daltons; m is selected from 3 and 4; and the compound is contacted with a chiral amine, to provide an enantiomerically enriched salt of the formula: (R5 111 I N02 Chiral Amine OH" 0" NH2 In another embodiment of the above-identified process, the compound to be acted upon in the process has the formula:

wherein R5 at each occurrence is independently either halogen or an organic moiety having a mass of less than 500 Daltons; m is selected from 3 and 4; and the compound is contacted with hydrogen in the presence of a chiral hydrogenation catalyst, to provide an enantiomerically-enriched compound of the formula: In another aspect, the invention provides compounds of the formula

wherein R at each occurrence is independently either halogen or an organic moiety having a mass of less than 500 Daltons; m is selected from 0,1,2,3 and 4; R7 is hydrogen or an organic moiety; Z is SH, or NW wherein R8 is either hydrogen or an amine protection group; and wherein if compounds of both formulae (V) and (VI) are present in an admixture, the molar formula (V): formula (VI) ratio in the admixture is other than 50: 50.

In another aspect, the invention provides compounds of the formula

wherein R5 at each occurrence is independently either halogen or an organic moiety having a mass of less than 500 Daltons; m is selected from 3 and 4; Ri | is a functional moiety that includes a phosphoramidite or H-phosphonate moiety; R'2 is an

organic moiety having a mass of 15-10,000 Daltons; and wherein if compounds of both formulae (VII) and (VIII) are present in an admixture, the molar formula (VII): formula (VIII) ratio in the admixture is other than 50: 50.

In one embodiment, in the compounds described above, the phosphoramidite moiety of R"has the formula-0-P (OR'3) (N (R'4) 2) wherein each of R and Rl4 is independently selected from an alkyl group or a substituted alkyl group having one or more substituents selected from halogen and cyano, and two R'4 groups may be bonded together to form a heterocycloalkyl group with the nitrogen of the phosphoramidite. In another embodiment, the H-phosphonate moiety of Rl l comprises the formula-O-P (=O) (H) (O-+HN (Rl5) 3) and Rl5 is independently a Cl 6alkyl group.

These and other aspects and embodiments of the present invention will be apparent upon reference to the following detailed description. To this end, various references are set forth herein which describe in more detail certain procedures, compounds and/or compositions, and are hereby incorporated by reference in their entirety.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides tags suitable for coupling to nucleic acids or other molecules of interest. When coupled to DNA, the tags provide a means to achieve a high-throughput genotyping system. Preferably, the linker between the tag and the molecule of interest should completely cleave under conditions that do not cause tag fragmentation. Furthermore, the tag preferably yields one peak per oligonucleotide injected, and gives optimal signal in terms of ion current. Also, the tag is preferably stable to PCR conditions, HPLC, and other manipulations used in assay formats. Reagents to introduce tags to a molecule of interest should proceed reproducibly and in good yield. In a preferred approach to meeting these objectives, a modular structure was developed that allows CMST components to be optimized independently. The components of a CMST preferably include a photolabile linker, a mass spectrometry sensitivity enhancer (MSSE), and a variable mass unit (VMU), all connected together through a scaffold.

The present invention provides tagged molecules of interest, precursors to tagged molecules of interest, and methods for generated the tagged molecules and precursors thereto, wherein the tag is enantiomerically-enriched. By using enantiomerically-enriched precursors, the purification of the reaction product of the precursor and the molecule of interest, in order to afford purified tagged molecule of interest, is facilitated.

Thus, the present invention provides a compound having the formula (I) or (II): wherein R'is selected from halogen and organic moieties; R2 and R3 are independently selected from hydrogen and organic moieties having a mass greater than 15 Daltons where R and R3 can together form a carbonyl group or may be joined together within a cyclic structure; Z is an (n+l)-valent atom excluding carbon where n is an integer greater than 0; R4 is independently selected from hydrogen, halogen, and organic moieties having a mass greater than 15 Daltons, with the proviso that at least one R4 is an organic moiety having a mass greater than 100 Daltons; W at each occurrence is independently either halogen or an organic moiety having a mass of less than 500 Daltons; and m is selected from 0,1,2,3 and 4. The compound of the invention is enantiomerically enriched, such that if compounds of both formulae (I) and (II) are present in an admixture, the molar formula (I): formula (II) ratio in the admixture is other than 50: 50.

The compound of the invention includes a 2-nitrophenyl group having, at the 1-position of the phenyl ring, a substituted methyl group, where the carbon atom of

the methyl group will be referred to herein as either Cl or the benzylic carbon atom. In addition to the 2-nitrophenyl group, the C 1 atom is directly bonded to a hydrogen atom (not shown), a carbon atom (referred to herein as C2) and a non-carbon atom.

By virtue of the 2-nitrophenyl group, compounds of the invention are photocleavable. That is, compounds of the invention will respond to contact with certain electromagnetic radiation by undergoing a cleavage reaction whereby the substituted Z atom separates from Cl as shown in the following structures, where "MM"indicates a bond that is photolytically unstable.

Because C 1 is directly bonded to only one atom that is not a carbon or hydrogen atom, compounds of the invention undergo a selective photocleavage reaction. That is, the Cl-Z bond can be selectively cleaved by appropriate photolytic conditions, leaving the other CI bonds largely if not entirely intact. This selective cleavage can occur so long as Z is not a carbon atom. In preferred embodiments, Z is selected from oxygen, nitrogen and sulfur. Each of oxygen, nitrogen, and sulfur is a preferred atom for position Z.

The photolytic conditions that allow selective cleavage of the Cl-Z bond are largely unaffected by other substitution on the 2-nitrophenyl ring. Accordingly, any one or more of the hydrogen atoms of the phenyl ring may be replaced with an equal number of R 5 groups. Accordingly, compounds of the invention may have a number ("m") of Rs groups, where m is an integer selected from 3 and 4. The desired photocleavage chemistry is largely independent of the nature of the Rs group, so that R 5 may be an inorganic group, e. g., halogen, or an organic group. Typically, an organic R group is not too large, and accordingly has a mass of less than about 500 Daltons.

Preferred R5 groups are selected from C,-C22 hydrocarbon groups. In preferred compounds of the invention, m is equal to zero, so that the 2-nitrophenyl ring has four hydrogen substituents.

After undergoing a photocleavage reaction, the resulting Z-containing residue which derived from a compound of the invention will be referred to herein as a tag. The tag is, or includes a detectable moiety, where this term refers to a moiety that can be detected, and preferably characterized, by an analytical method. Representative analytical methods include spectrometric or potentiometric methods. Representative spectrometric methods include mass spectrometry, infrared spectrometry, ultraviolet spectrometry and fluorescent spectrometry. A representative potentiometric method is potentiostatic amperometry.

In order to provide an effective tag, at least one R4 group is an organic moiety having a mass greater than 100 Daltons. This R4 group will be referred to herein as the first R4 group (or R4a). That tags should have masses greater than 100 Daltons is desirable for a number of reasons. When the tags have masses of less than 100 Daltons, it may be difficult to identify the tags. For instance, where the tags are identified and characterized by mass spectrometry, tags of molecular weight less than 100 Daltons may be difficult to distinguish from the background noise inherent in the operation of a mass spectrometer.

In addition, the present invention provides compositions containing a number of non-identical compounds of the invention, where the non-identical compounds contain non-identical tags that may be distinguished from one another by an analytical method. In order to distinguish a number of non-identical tags, the tags must be sufficiently different from one another that the analytical method can detect the difference. In order to obtain a suitable number of tags that are distinct by, e. g., potentiometry, the tags must contain a number of atoms that can be bonded together in non-identical ways which are analytically distinct. When tags have a mass of less than 100 Daltons, there are an unduly limiting number of analytically distinct tags.

Accordingly, to allow sufficient variability in structure, and to allow easy and effective detection, the tags should have masses in excess of 100 Daltons.

So long as the first R4 group has a mass in excess of 100 Daltons, the identity of the other R4 groups in a compound of the invention may be selected to achieve one or more goals. For instance, a second or more R4 (e. g., R4b, R4C, etc.) group may impart information supplementing the information derived from the first R4 group.

In this instance, a second or more R4 group also preferably has a mass of at least 100 Daltons. However, in other instances, it may be desirable that the second or more R4 groups do no more than satisfy the otherwise unsatisfied valence state of C1, not interfere with the photocleavage reaction or detection of the tag, and be synthetically easy to form. In this later instance, the second or more R4 groups might be hydrogen or simple hydrocarbon groups. In a preferred embodiment, the second or more R4 groups, if present, is/are hydrogen.

In compounds of the invention, R'is selected from halogen and organic moieties, while R2 and R3 are independently selected from hydrogen and organic moieties having a mass greater than 15 Daltons. As stated above, compounds of the invention include a detectable moiety where detection, and optionally characterization of the detectable moiety provides information about the compound of the invention. In one embodiment, the R'group contains the feature of the compound about which information is desired. For instance, R'may include biological material from an individual, and the tag is characteristic of that individual and/or the specific biological material. As another example, the R'group may contain a particular synthetic nucleic acid, peptide or saccharide sequence, which is uniquely identified by the tag. In another embodiment, R'is a reactive chemical group which can undergo a chemical reaction whereby biologically-derived material may be photocleavably linked to a tag. For instance, R'may be a halogen atom which may be displaced in a chemical reaction by biological material or derivatized biological material.

The R2 and R3 groups are independently selected from hydrogen and organic moieties having a mass greater than 15 Daltons. Typically, the W and R3

groups are selected with a view to their impact on the chemistry selected for joining R' to Cl, and/or for their impact on the photocleavage reaction whereby the tag is separated from Cl. When each of R2 and R3 is hydrogen, then neither R2 nor R3 adversely impacts the photocleavage reaction, and typically they are compatible with any chemistry used to couple Rl to C l. Accordingly, R2 and R3 are preferably hydrogen. However, R2 and R3 could be groups other than hydrogen. For instance, R2 and R together might fonn a carbonyl group with C 1. Alternatively, R'and R2 might, together with Cl, form a cyclic structure. As another alternative, either of R2 or R3 might be an organic moiety of mass greater than 15 Daltons, e. g., a Cl-C22 hydrocarbon group. Typically, hydrocarbon groups do not interfere with the desired photocleavage reaction, and do not interfere with R'/C ! coupling chemistry.

Another feature of a compound of the invention is that the Cl atom is chiral. As seen from the formulae (I) and (11), the nature of the substituents on Cl necessarily require that Cl is chiral. In addition, if compounds of formulae (I) and (II) are in admixture, the molar ratio of the formula (I) compounds to the formula (II) compounds is other than 50: 50. In a preferred embodiment of the invention, compounds of formula (I) are not in admixture with compounds of formula (II), and vice versa. Furthermore, in the event compounds of formula (I) are in admixture with compounds of formula (II), the molar ratio of the formula (I) compounds to the formula (II) compounds is preferably in excess of 95: 5, or 96: 4, or 97: 3, or 98: 2, or 99: 1, or in excess of 99.5: 0.5, or vice versa (i. e., the molar ratio of the formula (II) compounds to the formula (I) compounds is preferably in excess of 95: 5, or 96: 4, or 97: 3, or 98: 2, or 99: 1, or in excess of 99.5: 0.5).

Because compounds of formulae (I) or (II) are either not, or are only minimally, in admixture with compounds of formulae (II) or (I), respectively, chromatographic separations involving compounds of formulae (I) or (II) are much easier and effective than if significant amounts of compounds of formulae (I) and (II) were in admixture with each other. The present invention provides compositions containing a plurality of compounds of formulae (I) or (II) which, because of their

isomeric constitution, may be more readily separated from one another than would otherwise be the case. This enhanced ability to distinguish differently tagged compounds allows the formation of compositions that could otherwise not be characterized.

For instance, while two compounds of formula (I) might be different by virtue of slightly different R'groups, these two compounds (compounds IA and IB) might not be effectively separable if they were in admixture with compounds of formula (II) having the identical substitution around Cl (compounds IIA and IIB). This is because the chromatography elution time of compound IA may overlap that of compound IIB and/or the elution time of compound IB may overlap that of compound IIA. Accordingly, compounds IA and IB may not be readily resolved from one another, due to interference from compounds IIA and IIB. The present invention addresses this problem by providing compounds of formula (I) free of, or effectively free of, compounds of formula (II).

In another aspect, the present invention is directed to a process for providing an enantiomerically-enriched compound using a specified agent. More specifically, the process acts on a compound of the formula: wherein Z is selected from oxygen, nitrogen and sulfur; where R6 is hydrogen when Z is oxygen or sulfur, and when Z is nitrogen then R6 is selected from hydrogen and C,-C22 hydrocarbon and two R6 groups are bonded to Z; R 5 at each occurrence is independently either halogen or an organic moiety having a mass of less than 500 Daltons; m is selected from 3 and 4; R is hydrogen or an organic moiety, and the bond represented by both a solid and dashed line represents either a single or double bond.

The inventive process contacts a compound as identified above with an agent. The agent is selected from (a) an enzyme; (b) an enzyme and an adjuvant of the formula H2N-C (=O)-CHR8-NH-R9 wherein R8 is an organic moiety and R9 is an amino protecting group; (c) a chiral acid; (d) a chiral amine; (e) hydrogen and a chiral hydrogenation catalyst ; and (f) a mechanical crystal-separating means. The compound is contacted with the agent so as to provide an enantiomerically-enriched compound of theformula: In addition to specific techniques disclosed herein to prepare compounds having photolabile bonds to enantiomerically enriched tags, other suitable techniques may 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, Glasglow, 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.

The present compounds and compositions, and methods for their preparation, may be used to provide tagged molecules where the tag is both optically enriched and is photocleavably linked to the molecule. Molecules with photocleavably attached tags, which may be provided in optically-enriched form according to the present invention, as well as methods that may be practiced with compounds and compositions of the present invention, are set forth in, for example, PCT International Publication Nos. WO 99/05319; WO 97/27331; WO 97/27327; WO 97/27325; and WO 95/04160. Thus, the compounds of the present invention may be substituted for the tagged compounds disclosed in these four publications, as well as other methods and assays where molecules having photocleavably linked tags are utilized.

The following examples are set forth as a means of illustrating the present invention and are not to be construed as a limitation thereon.

EXAMPLES In the following examples, and unless otherwise noted, the chemical reactants and reagents were of standard commercial grade, obtained from commercial supply houses such as Aldrich (Milwaukee, WI; www. sigma-aldrich. com), Fluka (a division of Aldrich) and Lancaster Synthesis, Inc. (Windham, NH; http://www. lancaster. co. uk).

EXAMPLE I RESOLUTION WITH CHIRAL ACID DTTA The ()-3-amino-3- (2-nitrophenyl) propionic acid methyl ester (24.6 g, 110 mmol) and the chiral acid di-p-toluoyl-D-tartaric acid (DTTA, 42.4 g, 110 mmol) were taken up in 500 mL of methanol by heating to 67°C. The resulting solution was

cooled to 56°C and seeded with the salt of 3-amino-3- (2-nitrophenyl) propionic acid methyl ester and DTTA. The mixture was allowed to cool to ambient temperature (21°C) and stirred for 16 hours. The crystals that were formed were collected by filtration giving a white solid that was enriched in one enantiomer (46% enantiomeric excess, e. e.). One crystallization in methanol resulted in 12 g of material with an diastereomeric purity of 88%.

EXAMPLE 2 RESOLUTION WITH CHIRAL ACID (-) CAMPHANIC ACID The ()-3-amino-3- (2-nitrophenyl) propionic acid (172 mg, 0.82 mmol) and the chiral acid (-)-camphanic acid (165 mg, 0.83 mmol, Fluka) were taken up in 5 mL of water by heating to reflux. The resulting solution was cooled to ambient temperature (21 °C). The crystals that formed were collected by filtration giving a white solid (105 mg, 31% yield). Analysis by 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) indicated that the product was enantiomerically pure.

EXAMPLE 3 SELECTIVE HYDROLYSIS WITH ENZYME The ()-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. Then Amano PS Enzyme (6 g, 10 wt%, Amano Enzyme (Milton Keynes, U. K.; Lombard IL, USA; www. amano-enzyme. cojp)) as a slurry in phosphate buffer (100 mL) was added to the solution, and the reaction mixture stirred at ambient temperature (21 °C) for 24 hours.

At completion, the amino acid precipitate was filtered off and the aqueous filtrate removed before the solid was washed with dichloromethane (DCM, 2 x 10 mL). The solid was dissolved in aqueous HCl (6 M, 150 mL) but a fine unknown precipitate still remained which was filtered off. The resulting aqueous mixture was extracted with DCM before the

solid was reprecipitated by adjusting the pH to 7. This gave the desired amino acid (23 g, 90% yield).

The initial filtrate was basified to pH 8 and extracted first with the DCM washings collected above; and secondly with fresh DCM. The organic layers were combined, dried and evaporated to give the amino ethyl ester (23 g, 80% yield). Optical rotation of the ethyl ester HCI salt in methanol gave an aD of +127. 55° (c=l, 20°C, 589 nm). The enantiomeric excess (e. e.) of both the amino acid and amino ethyl ester were determined by cHPLC and were both in excess of 98% enantiomerically pure.

EXAMPLE 4 SYNTHESIS OF REACTIVE MOLECULE FOR DELIVERING OPTICALLY-ENRICHED TAG TO MOLECULE OF INTEREST The tags described herein are preferably conjugated to a molecule of interest through a photolabile bond. A preferred photolabile bond is present within an o-nitrobenzyl group (see, e. g., (a) Greenberg, M. M.; Gilmore, J. L. J. Org. Chem. 1994, 59,746-753; (b) Yoo, D. J.; Greenberg, M. M. J. Org. Client. 1995,60,3358-3364; and (c) Venkatesan, H.; Greenberg, M. M. J. Org. Chem. 1996,61,525-529). The intramolecular photoredox reaction in an o-nitrobenzyl group (see, e. g., Pillai, V. N. R.

Synthesis 1980,1-26) enables the tag to cleave from the oligonucleotide quickly under neutral conditions (six second exposure with a 254nm Hg lamp) and with no fragmentation.

Accordingly, the synthetic route begins (Scheme 1) with esterification of the photosensitive linker 3-amino-3- (2-nitrophenyl) propionic acid, (ANP, 1, see, e. g., Brown, B. B.; Wagner, D. S.; Geysen, H. M. Moleclular Diversity and (b) Rodebaugh, R.; Fraser-Reid, B.; Geysen, H. M. Tetrahedron Letters 1997,38,7653- 7656) to give the ethyl ester hydrochloride (2) in 84% yield. This is followed by the enzymatic transformation of 2 to provide the ethyl ester as a single isomer, which facilitates HPLC purification in the oligonucleotide conjugation step. The ethyl ester

hydrochloride was dissolved in water and adjusted to neutral pH with 2N HCI. The Amano PS esterase enzyme was added as a phosphate buffer slurry. After completion of the reaction, a basic workup removed the hydrolyzed ANP by-product (4), and the single-isomer ethyl ester (3, >99% e. e.) was recovered (92% of available material).

Scheme 1 H2NHC!. H2NHN N- H2N CO2H HCI. HZN COZEt HZN COZEt H2N C02H N02 HCI N02 1. NAOH N02 N02 W EtOH W 2. Amano PS W W i i 1 2 3 4 Lysine provided a very suitable scaffold for the modular approach to the synthesis of the tags. The two amine and one carboxylic acid groups present in lysine provided handles for robust peptide chemistries with resulting amide linkages between the tag components. Peptides have a predictable behavior in mass spectrometry and are also relatively stable to 254 nm light. As seen in Scheme 2, coupling of 3 with a-BOC- s-Alloc-lysine (5), using EDACI (l- [3- (Dimethylamino) propyl]-3-ethyl-carbodiimide hydrochloride, obtained from Aldrich Chemical Co., Milwaukee, Wl) and 1- hydroxybenzotriazole (HOBT), gave the protected ANP lysine (6). Removal of BOC with HCI provided the £-Alloc-lysine ANP ester (7) as a white solid in 81 % yield from 3.

Perfluorinated aromatics may be used as electrophoric labels for analytical purposes in negative ion mode mass spectrometry (see, e. g., (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 Chromatograply 1988,452,369-376; and (d) Saha, M.; Saha, J.; Giese, R. W. Journal of Chromatography 1993,641,400-404). Our initial strategy for a MSSE candidate, the ionization component of the tag, consisted of attaching various electron poor, highly fluorinated carboxylic acids to a representative scaffold and measuring relative ion current with negative ion mode APCI. Fragmentation occurred, giving

multiple peaks along with poor signal. Enhancers for the opposite ionization regime were then explored. Tags were made incorporating pyridyl, prolinyl and piperidyl structures as the MSSE and tested in positive mode APCI. By all criteria, a tag using N- methyl isonipecotic acid (INA) gave excellent results.

INA hydrochloride was coupled to 7, using EDAC and triethylamine, to give crude Alloc-protected 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 in 95% yield as a yellow solid.

Scheme 2 To provide a large set of detectable tags with distinct molecular weights, core structure 9 was derivatized with a set of 45 carboxylic acids referred to as variable mass units (VMUs). The masses of the VMUs are spaced a minimum of 4 a. m. u. apart, to minimize isotopic overlap between tags. The molecular weights of the VMUs ranged from 90-298 a. m. u. Preferred VMUs did not have the following properties or features: (1) functionalities incompatible with the synthetic sequence, such as esters; (2) elements

with multiple isotopes (Cl, Br, S); (3) functionalities that might lead to competing photoprocesses (iodides, acyl-and aryl-phenones); (4) racemic acids, and (5) lack of commercial availability. VMUs were coupled to 9, using HATU and N-methyl morpholine. After purification by column chromatography, the CMST ethyl ester (10) was recovered in variable yields. Saponification of 10 with NaOH gave the CMST acid (11) in quantitative yield. For the final step, formation of the active ester was achieved using a base-catalyzed transesterification of tetrafluorophenyl trifluoroacetate (TFP TFA, see, e. g., Green, M.; Berman, J. Teti-ahedi-oti Lettei-s 1990, 31, 5851-5852; TFP TFA was prepared in a similar manner to pentafluorophenyl trifluoroacetate, and was used because the proton on the tetrafluorophenyl ring acts as a diagnostic NMR check) and 11, resulting in the CMST TFP ester (12) in variable yields. The tetrafluorophenyl active ester was chosen because of easily removed side products and relative stability and compatibility towards oligonucleotide conjugation conditions. The CMST TFP esters were conjugated to 5'-aminohexyl-tailed oligonucleotides (obtained from TriLink Biotechnology, San Diego, CA) according to the method of Lukhtanov et al. (see, e. g., Lukhtanov, E. A.; Kutyavin, I. V.; Gamper, H. B.; Meyer, R. B. Jr. Bioconjllg. Cliem.

1995,4,418-426).

Scheme 3

The number of tags was further expanded by the incremental use of a tyrosine derivative which functioned as a gross mass adjuster (GMA, and more specifically N-Boc-O-ethyl-L-tyrosine, which can be ordered commercially from

Bachem California (Torrance, CA) or prepared by alkylation of N-Boc-L-Tyrosine-OMe with iodoethane and cesium carbonate in 86% yield, followed by hydrolysis with NaOH in quantitative) which increases the molecular weight of the cleaved tag by 191 a. m. u. and allows the set of VMUs to be re-used for higher molecular weight tags. The GMA was coupled to the core structure 9 as shown in Scheme 4. After basic workup, the BOC-protected GMA structure was isolated by filtration. Deprotection with TFA resulted in the GMA monomer core structure (13, n=l) in quantitative yield. The VMUs were coupled in the same manner as before, resulting in additional sets of tags (14). Using four units of the mass adjuster, a total of nearly 200 tags may be synthesized.

Scheme 4 a

' (a) EDAC, HOBT, THF; (b) TFA, DCM; (c) VMU, HATU, NMM; (d) IN NaOH, THF; (e) TFP TFA, DIEA, DMF From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.