Synthesis of unnatural amino acids by cross-coupled photochemical synthesis: azide, fluorine, and nitroxide containing amino acids

Abstract

The disclosure relates generally to a light driven chemical synthetic method for synthesizing unnatural amino acids.

Description

Synthesis of Unnatural Amino Acids by Cross-Coupled Photochemical Synthesis: Azide, Fluorine, and Nitroxide Containing Amino AcidsCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 657,144 entitled “Photochemical Synthesis of Cross-Coupled Fluorescent Amino Acids,” filed June 7, 2024 the disclosure of which is incorporated herein in its entirety by reference.BACKGROUND

[0002] Unnaturally occurring amino acids, also known as unnatural amino acids (UAAs), are amino acids that are not typically found in nature or involved in protein synthesis within living organisms. These amino acids differ from the 20 standard amino acids that are encoded by the genetic code and used to build proteins in biological systems.

[0003] SUMMARY

[0004] The disclosure relates to a novel light driven synthetic method for synthesizing unnatural amino acids in an easy chemical synthetic procedure with high yields. This method has been used in photochemical synthesis of cross-coupled fluorescent amino acids (CCFAA) through a light initiated key step to link photo-activatable amino acids to fluorophores, a novel method that links a fluorophorc directly to an amino acid derivative in a single step. The disclosure relates to the high yielding photochemical synthesis of RhDYE-Methionine, which displays desired physical and biological properties such as the excitation wavelength being beyond the crucial 500 nm value while emitting strong green fluorescence and having photostability. The new RhDYE-Methionine has excellent biocompatibility and is readily imaged in live cells. The disclosure also relates to a method of imaging a target, the method comprising contacting a live cell with an UAA.BRIEF DESCRIPTION OF THE FIGURES

[0005] The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments of the present invention.

[0006] FIG. 1A shows 'H-NMR spectrum of L-RhDYE-Methionine with zoom-in region between 8 0.9 - 4.0 (ppm).

[0007] FIG. IB shows 'H-NMR spectrum of L-RhDYE-Methionine with zoom-in region between 6 6.6 - 8.2 (ppm).

[0008] FIG. 1C shows proton assignment of 'H-NMR spectrum to the structure of L- RhDYE-Methionine

[0009] FIG. 2A shows ['H, 'H] COSY spectrum of RhDYE-Methionine with zoom-in region between 8 0.9 - 3.9 (ppm).

[0010] FIG. 2B shows COSY spectrum assignment to the structure of RhDYE- Methionine.

[0011] FIG. 3A shows13C-NMR spectrum of L-RhDYE-Methionine.

[0012] FIG. 3B shows H,13C] HSQC spectrum of L-RhDYE-Methionine with zoom-in region between 8 0.9 - 3.9 (ppm) and 8 10 -76 (ppm).

[0013] FIG. 3C shows HSQC spectrum assignment to the structure of L-RhDYE- Methionine.

[0014] FIG. 4 shows ESI mass spectrum of L-RhDYE-Methionine (C26H25N3O5) with molecular ion [M+H]+peak at m / z 460.04.

[0015] FIG. 5 A shows infrared spectrum of L-photo-methionine taken neat.

[0016] FIG. 5B shows infrared spectrum of L-RhDYE-Methionine product taken neat.

[0017] FIG. 6A shows UV-Vis spectra of RhDYE-Met in DMSO-PBS buffer with concentration between 15-75 pM.

[0018] FIG. 6B shows absorbance values at max for the measured concentrations of 6A ranging from 15 M to 75 pM.

[0019] FIG. 6C shows Beer-Lambert graph of Rh-DYE-Met in DMSO-PBS buffer for concentrations from 15 pM up to 75 pM.

[0020] FIG. 7 shows fluorescence spectra of 100 nM RhDYE-Met in DMSO-PBS buffer (SW set to 10 nm, Ex set to 503 nm, Em set to 530 nm).

[0021] FIG. 8 shows the stability of the fluorescence spectra of 100 nM RhDYE-Met in DMSO-PBS buffer from 0.5 to 12 h.

[0022] FIGS. 9A-9F show bioimaging properties of L-RhDYE-Methionine including Confocal microscopy images of human skin fibroblast cells (WS1) incubated with / without 20 pM RhDYE-Met in DMSO for 30 mins. Scale bar, 20 pm. FIG. 9A shows a control: bright filed. FIG 9B shows control: Ex: 515 nm, Em: 549 nm. FIG. 9C shows control: merge. FIG. 9D shows 20 pM RhDYE-Met: Bright field. FIG. 9E shows 20 pM RhDYE-Met: Ex: 515 nm, Em: 549 nm. FIG. 9F shows 20 pM RhDYE-Met: Merge.

[0023] FIG. 10 shows LC-MS spectra of L-Res-Met (CisHisNiOs). The m / z peak at 381.26 is the molecular ion species of Res-Met with K+ion [M+K]+. The peak at m / z 413.2492 is assignable to a methanol adduct.

[0024] FIG. 11 showsJH NMR spectrum of L-Res-Met. The doublet at 3 1.06 is indicative of the formation of the product (L-Resorufin-Met).DETAILED DESCRIPTION

[0025] Reference will now be made in detail to certain embodiments of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0026] Throughout this document, values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of “about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise.

[0027] In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to anonexclusive “or” unless otherwise indicated. The statement “at least one of A and B” or “at least one of A or B” has the same meaning as “A, B, or A and B.” In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. A comma can be used as a delimiter or digit group separator to the left or right of a decimal mark; for example, “0.000,1” is equivalent to “0.0001.” All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0028] In the methods described herein, the acts can be carried out in any order without departing from the principles of the invention, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0029] The term “about” as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range, and includes the exact stated value or range. The term “substantially” as used herein refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%. The term “substantially free of’ as used herein can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that about 0 wt% to about 5 wt% of the composition is the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than or equal to about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less, or about 0 wt%.

[0030] The term “organic group” as used herein refers to any carbon-containing functional group. Examples can include an oxygcn-containing group such as an alkoxy group, aryloxy group, aralkyloxy group, oxo(carbonyl) group; a carboxyl group including a carboxylic acid, carboxylate, and a carboxylate ester; a sulfur-containing group such as an alkyl and aryl sulfide group; and other heteroatom-containing groups. Non-limiting examples of organic groups include OR, OOR, OC(O)N(R)2, CN, CF3, OCF3, R, C(O), methylenedioxy, ethylenedioxy, N(R)2, SR, SOR, SO2R, SO2N(R)2, SO3R, C(O)R, C(O)C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R)2, OC(O)N(R)2, C(S)N(R)2, (CH2)0-2N(R)C(O)R, (CH2)0-2N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, C(=NOR)R, and substituted or unsubstituted (Ci-Cioo)hydrocarbyl, wherein R can be hydrogen (in examples that include other carbon atoms) or a carbon-based moiety, and wherein the carbon-based moiety can be substituted or unsubstituted.

[0031] The term “substituted” as used herein in conjunction with a molecule or an organic group as defined herein refers to the state in which one or more hydrogen atoms contained therein are replaced by one or more non-hydrogen atoms. The term “functional group” or “substituent” as used herein refers to a group that can be or is substituted onto a molecule or onto an organic group. Examples of substituents or functional groups include, but are not limited to, a halogen (e.g., F, Cl, Br, and I); an oxygen atom in groups such as hydroxy groups, alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups including carboxylic acids, carboxylates, and carboxylate esters; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups; a nitrogen atom in groups such as amines, hydroxyamines, nitriles, nitro groups, N-oxides, hydrazides, azides, and enamines; and other heteroatoms in various other groups. Non-limiting examples of substituents that can be bonded to a substituted carbon (or other) atom include F, Cl, Br, I, OR, OC(O)N(R)2, CN, NO, NO2, ONO2, azido, CF3, OCF3, R, O (oxo), S (thiono), C(O), S(O), methylenedioxy, ethylenedioxy, N(R)2, SR, SOR, SO2R, SO2N(R)2, SO3R, C(O)R, C(O)C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R)2, OC(O)N(R)2, C(S)N(R)2, (CH2)O-2N(R)C(0)R, (CH2)O-2N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2,N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, and C(=NOR)R, wherein R can be hydrogen or a carbon-based moiety; for example, R can be hydrogen, (Ci-Cioo)hydrocarbyl, alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroarylalkyl; or wherein two R groups bonded to a nitrogen atom or to adjacent nitrogen atoms can together with the nitrogen atom or atoms form a heterocyclyl.

[0032] The term “alkyl” as used herein refers to straight chain and branched alkyl groups and cycloalkyl groups having from 1 to 40 carbon atoms, 1 to about 20 carbon atoms, 1 to 12 carbons or, in some embodiments, from 1 to 8 carbon atoms. Examples of straight chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n-butyl, n- pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2- dimethylpropyl groups. As used herein, the term “alkyl” encompasses n-alkyl, isoalkyl, and anteisoalkyl groups as well as other branched chain forms of alkyl. Representative substituted alkyl groups can be substituted one or more times with any of the groups listed herein, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.

[0033] The term “alkenyl” as used herein refers to straight and branched chain and cyclic alkyl groups as defined herein, except that at least one double bond exists between two carbon atoms. Thus, alkenyl groups have from 2 to 40 carbon atoms, or 2 to about 20 carbon atoms, or 2 to 12 carbon atoms or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to vinyl, -CH=CH(CH3), -CH=C(CH3)2, -C(CH3)=CH2, -C(CH3)=CH(CH3), - C(CH2CH3)=CH2, cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl among others.

[0034] The term “alkynyl” as used herein refers to straight and branched chain alkyl groups, except that at least one triple bond exists between two carbon atoms. Thus, alkynyl groups have from 2 to 40 carbon atoms, 2 to about 20 carbon atoms, or from 2 to 12 carbons or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to - C=CH, -C=C(CH3), -C=C(CH2CH3), -CH2C=CH, -CH2C=C(CH3), and -CH2C=C(CH2CH3) among others.

[0035] Synthetic biology, which harnesses innovative approaches for engineering new biological molecules / systems or re-designing existing ones for useful purposes, has beenrevolutionizing scientific research as well as biotechnology and the pharmaceutical industry. As one of its most promising areas, site-specific incorporation of functional unnatural amino acids into peptides or proteins has a wide range of applications in biochemical research and the biopharmaceutical industry. Proteins or peptides can be modified with new physical, chemical or biological properties via the inclusion of unnatural amino acids. These unnatural amino acids resemble the natural building blocks of proteins but contain distinct functional groups. Expanding amino acid building blocks enables peptides or proteins to have new or modified functions, providing great opportunities for innovative research or novel medicines. Of particular interest is the incorporation of fluorescent probes as a mechanism to track protein function, transport, and folding.

[0036] The recent advances in microscopy and the utility of highly sensitive fluorescence-based approaches have significantly accelerated the development in research fields such as molecular and cellular biology, biophysics, biotechnology and medicine. Fluorescent fusion proteins (such as Green Fluorescent Proteins, GFPs), and the more recent enzymatic “tags” (such as the SNAP-tag or CLIP-tag) which are genetically encoded onto the N- or C- terminus of the protein of interest have enabled the visualization of proteins in living systems. However, these fusion-based technologies suffer from restriction to a potentially essential terminus of the protein of interest, and the fluorophores are quite large (>20 kDa) which can cause them to interfere with the expression, localization, and stability or function of the protein to which they are attached. A promising alternative approach is the utilization of unnatural fluorescent a- amino acids (FAA). These a- amino acid analogues are very small in comparison with the GFPs or tags, and can therefore be placed precisely at targeted positions within a peptide or protein, and can therefore act as relatively nonperturbing replacements for the native residues, instead of being restricted to the termini, thereby maintaining the overall native structure and function of a target peptide or protein. Moreover, the photophysical and chemical properties of unnatural fluorescent a-amino acids can be purposely tuned and incorporated into peptides or proteins via traditional solid phase peptide synthesis (SPPS), expressed protein ligation (EPL) or the more recent genetically encoded unnatural amino acid incorporation techniques. Once the unnatural fluorescent a-amino acids are synthesized, new applications in peptide or protein incorporation could be readily adopted.

[0037] Unnatural fluorescent a-amino acids (FAA) have gained particular interest in the field of chemical biology due to their versatile applications such as being used as fluorescent probes to study protein dynamics, folding / unfolding, local conformation changes, biomolecular interactions, and live-cell imaging. However, in applications where an unnatural fluorescent a- amino acid reporter is required or desirable, the choice is rather limited, owing to lack of availability. Since unnatural a-amino acids typically have at least one stereocenter and possess two or more reactive functional groups (the amine and carboxylate), they are challenging synthetic targets and thus their synthetic routes usually require multiple steps. Nevertheless, they have proved to be promising in broad applications, increasing their attraction as synthetic targets, using both chemical and enzymatic approaches.

[0038] A few methods have been developed to chemically synthesize unnatural fluorescent amino acids, which are important tools to monitor and visualize cellular actions. These methods include transition metal catalyzed reactions, cycloaddition reactions, condensation reactions, etc, all involving multistep chemical synthesis with low overall yield. Chemical synthesis of unnatural fluorescent amino acids involves multiple steps of chemical or combined with enzymatic synthesis.

[0039] There are different ways to obtain unnatural fluorescent amino acids through chemical syntheses. These include but are not limited to tryptophan analogues, flavone-based fluorescent amino acids, pyrazoloquinazoline a-amino acids, rotor-fluorogenic D-amino acids, fluorophores attached to amino acids.

[0040] An unnatural amino acid (UAA) that can be made through chemical synthesis typically relies on utilization of palladium catalysts via multiple steps, or employing enzymes such as transaminases in the synthesis. A few methods have been developed to chemically synthesize UAAs, which are important tools to monitor and visualize cellular actions. These methods include transition metal catalyzed reactions, cycloaddition reactions, condensation reactions, etc, all involving multistep chemical synthesis with low overall yield.

[0041] The disclosure relates to a novel facile photochemical synthetic procedure for synthesis of new UAAs in one step using Photo-Methionine (Photo-Met) or Photo-Leucine (Photo-Leu) (Scheme 1) with a molecule containing a functional group. The disclosure also relates to new fluorescent UAAs.

[0042] This novel facile photochemical procedure may be used to create a broad range of new UAAs with high yield. The new UAAs arc fluorescent amino acids that absorb at long wavelengths in the visible region and emit strong green or yellow fluorescence with high quantum yield. The new fluorescent amino acids are biocompatible and can be used in bioimaging applications. In examples, the fluorescent UAA is L-RhDYE-Methionine (RhDYE- Met). In examples, the fluorescent UAA is L-Res-Methionine (L-Res-Met)

[0043] The disclosure relates to a method of synthesizing an UAA. The method can take any amino acid whether naturally occurring or derived that is modified with a photoreactive group with a compound comprising a reactive group and a fluorophore that can react with the photoreactive group in the presence of light. In general, an amino acid includes an amino group, a carboxyl group, and a unique side chain that determines its properties. The side chain of the starting amino acid in the instant protocols is modified to include group that is photoreactive, such as a group used in photo-reactive cross linking.

[0044] The photoreactive group can include a diazirine groupDiazirines belong to a class of organic compounds characterized by a three-membered ring structure containing one carbon atom and two nitrogen atoms. This unique structure makes them highly reactive, especially under ultraviolet light, which allows them to form carbenes by losing a molecule of nitrogen gas. Due to their reactivity, diazirines are commonly used as photo-reactive crosslinking agents in biochemical research to study interactions between proteins, nucleic acids, and other biomolecules. Other photoreactive groups can include Benzophenones, which initiate crosslinking through hydrogen abstraction, leading to the formation of carbon-centered radicals that can form covalent bonds. Azido-methyl-coumarins can be used. Anthraquinones can also be used to form stable crosslinks under light activation.

[0045] The reactive group that includes the fluorophore (“R”) can include any functional group capable of reacting with the photoreactive group. For example, the reactive group can include R-A-COOH, R-A-OH, R-A-SH, R-A-CH3, R-A-CH0-1CH0-2, or R-A-NH2. In some examples, the reactive group is a double bond. In examples, the reactive group is triple bond. In some examples where “A” is a bond the reactive group has the structure R-A-COOH, R-A-OH, R-A-SH, R-A-CH3, R-A-CH0-1CH0-2, or R-A-NH2.

[0046] In examples, the fluorophore is rhodamine (RhDYE). In examples, the fluorophorc is rcsorufin (Res). In examples the fluorophore is fluorescein. In examples, the fluorophore is a green fluorophore. In examples, the fluorophore is a yellow fluorophore.

[0047] Scheme 1 below shows a representative scheme for synthesizing an unnatural amino acid.Scheme 1: Synthetic routes of Met-derived unnatural amino acids (UAA) from Photo-Met, or Leu-derived unnatural amino acids (UAA) from Photo-Leu using a photochemical method

[0048] In examples, the method comprises dissolving the compound comprising a reactive group (herein after “the compound”) in a solvent. In examples, the solvent can be 1,4- dioxane, DMF or DMSO or a mixture thereof. The solvent can be DMSO.

[0049] In examples, the method comprises dissolving the compound in the solvent under light exposure. In examples, the light exposure is 200-400 mW / cm2. In examples, the light exposure is 250-350 mW / cm2. In examples, the light exposure is 300-350 mW / cm2. In examples, the light exposure is 300 mW / cm2. In examples the light exposure is 320 mW / cm2. In examples, the light has no filter. In examples, the light is not passed through glass.

[0050] In examples, the method comprises adding L-photo-methionine (Photo-Met) or L- photo (Photo-Leu) to the dissolved compound in solvent. In examples, Photo-Met is dissolved ina solvent. Tn examples, Photo-Leu is dissolved in a solvent. In examples, the solvent can be 1 ,4- dioxanc, DMF or DMSO or a mixture thereof. The solvent can be DMSO. In examples, the concentration of the solution is about 0.002M to about 0.02M. In examples, the concentration of the solution is about 0.002M to about 0.0 IM. In examples, the concentration of the solution is about 0.05M to about 0.01M. In examples, the concentration of the solution is about 2mM.

[0051] In examples, the method comprises adding a solution of Photo-Met to a solution of compound. In examples, the addition is done without exposure to light. In examples, the addition is done with exposure to light. In examples, the addition of a solution Photo-Met is repeated, and the resulting solution is stirred under light exposure.

[0052] In examples, the method comprises adding a solution of Photo-Leu to a solution of compound. In examples, the addition is done without exposure to light. In examples, the addition is done with exposure to light. In examples, the addition of a solution Photo-Leu is repeated, and the resulting solution is stirred under light exposure.

[0053] In examples, the ratio of compound to Photo-Met is 1:2. In examples, the ratio of compound to Photo-Met is 1:1.5. In examples, the ratio of compound to Photo-Met is 1:1.

[0054] In examples, the ratio of compound to Photo-Leu is 1:2. In examples, the ratio of compound to Photo-Leu is 1:1.5. In examples, the ratio of compound to Photo-Leu is 1:1.

[0055] In examples, the method further comprises stirring the solution of compound and Photo-Met under light exposure. In examples, the method further comprises stirring the solution of compound and Photo-Leu under light exposure. In examples, the light exposure is 200-400 mW / cm2. In examples, the light exposure is 250-350 mW / cm2. In examples, the light exposure is 300-350 mW / cm2. In examples, the light exposure is 300 mW / cm2. In examples, the light exposure is 320 mW / cm2.

[0056] In examples, after complete addition of Photo-Met, the resulting solution is stirred under continuous light exposure. In examples, after complete addition of Photo-Leu, the resulting solution is stirred under continuous light exposure. In examples, the light exposure is a direct light exposure (not through glass). In examples, the solution is stirred for 2-12h. In examples, the solution is stirred for l-24h. In examples, the solution is stirred for 4-10h. In examples, the solution is stirred for 5-8h. In examples, the solution is stirred for 6-7h. In examples, the solution is stirred for 6.5h.

[0057] In examples, the method further comprises evaporating the solvent. Evaporation of the solvent can be under reduced pressure.

[0058] In examples, the method further comprises purifying the UAA. The purification can be by flash column chromatography on silica gel. The purification can be by size exclusion column.

[0059] In examples, the UAA is L-Rhodamine-Methionine(L-RhDYE-Methionine).

[0060] In examples, the UAA is L- Resorufin-Methionine (L-Res-Methionine; L-Res- Met).

[0061] In addition to the photochemical synthesis of the green- and yellow-fluorescent amino acids RhDYE-Mct and Rcs-Mct, the synthesis of amino acids containing other desired moieties such as azide-containing amino acids, nitroxide-containing amino acids, and fluorine- containing amino acids via the cross-coupled unnatural amino acid (CCUAA) method are within the scope of this disclosure. These functional UAAs have important applications in chemical biology and medicinal chemistry. Comparing to the traditional chemical synthesis of UAAs (FlAAs, NsAAs, NOAAs, FAAs) which are still quite challenging as the syntheses have a high step count (5-6 steps), low yield, time-consuming, labor-intensive, expensive, inefficient atom economy, and environmentally unfriendly, the photochemical approach to synthesize UAAs via the CCUAA method utilizes a light-driven step to link various desired moieties to photoactivatable amino acids in a single step with high yield. Moreover, this photochemical- driven CCUAA method can be used to synthesis a broad range of UAAs.

[0062] Azides are a class of chemical compounds that contain the azide functional group, which is composed of three nitrogen atoms connected in a linear arrangement (Ns). This group istypically bonded to a carbon atom or a metal. Azides are known for their high reactivity and are often used in organic synthesis, particularly in the formation of other nitrogcn-containing compounds. They can act as precursors to amines, amides, and other nitrogen derivatives through various chemical reactions.

[0063] Azide-containing amino acids are fundamental for click chemistry appications. Azide-bearing amino acids and click chemistry is important for protein chemistry modifications, as it provides a specific, sensitive, rapid, and easy-to-handle method.

[0064] Scheme 2 shows the photochemical Synthesis of 4-azidomethylbenzoicacid- methionine. One of ordinary skill will understand that other unnatural amino acids with azides can be synthesized in a similar manner.Scheme 2

[0065] Scheme 3 shows the photochemical synthesis of azidoaceticacid-methionine. One of ordinary skill will understand that other unnatural amino acids with azides can be synthesized in a similar manner.Scheme 3

[0066] The disclosure also relates to a method of imaging a target. The method comprising contacting a live cell with an unnatural fluorescent amino acid. The method further comprising detecting a fluorescent signal. In examples, the unnatural fluorescent amino acid is RhDYE-Met. In examples, the unnatural fluorescent amino acid is Res-Met.

[0067] Fluorine-containing amino acids are of interest to medicinal chemistry and chemical biology. Since fluorine has a very strong electron withdrawing effect, the number of fluorine atoms and their positions on an amino acid change its properties immensely. The main effects are to decrease the nucleophilicity of the amino group, to increase the acidity of the carboxylic acid functional group, and to modulate steric hindrance and hydrophobicity. Taking advantage of thevery special properties of fluorine, fluorinated analogs of biomolecules are utilized in: (i) tuning an increased hydrophobicity, (ii) controlling conformational constraints, and (iii) high sensitivity F NMR.

[0068] Scheme 4 shows photochemical synthesis of 4-fluorophenylaceticacid -methionine. One of ordinary skill will understand that other unnatural amino acids with fluorine moieties can be synthesized in a similar manner.Scheme 4Scheme 5 shows photochemical synthesis and characterization of trifluoroaceticacid-methionine. One of ordinary skill will understand that other unnatural amino acids with fluorine moieties can be synthesized in a similar manner.Scheme 5Nitroxide functional groups are characterized by the presence of a nitrogen atom bonded to an oxygen atom, typically with an unpaired electron on the nitrogen, giving them a paramagnetic property. These groups are often represented as RiNO*, where R represents organic substituents. Nitroxides are stable radicals and are widely used in various applications, including as spin labels in electron spin resonance (ESR) spectroscopy, which helps in studying molecular structures and dynamics. With unpaired electrons, nitroxide-containing amino acids are important organic radicals that have unique chemical reactivities and display paramagnetic properties with outstanding imaging and therapeutic applications. Through structural fine tuning and molecular design, stable organic radical-containing materials have been implemented with applications in biomedical fields, particularly for bioimaging, biosensing, and photo-triggered therapies.Scheme 6 shows photochemical synthesis and characterization of 4-Carboxy-TEMPO- mcthioninc. One of ordinary skill will understand that other unnatural amino acids with nitroxidc moieties can be synthesized in a similar manner.3Scheme 6EXAMPLES

[0069] Various aspects of the present disclosure can be better understood by reference to the following Examples which are offered by way of illustration. The present invention is not limited to the Examples given herein.1. Experimental section

[0070] Chemicals: Lithium hydroxide was purchased from Alfa Aesar, N- methylmorpholine and anhydrous tert-butanol were purchased from Chem-Impex International, Celite 545 was purchased from GFS chemicals, silica gel SiliaFash® F6040-63 pm (230-400 mesh) was purchased from Silicycle, hydrochloric acid was acquired from Fisher Chemical, glacial acetic acid was purchased from EMD Millipore, hydroxylamine-O-sulfonic acid was obtained from TCI and di-tert-butyl decarbonate was ordered from Oakwood Products. (L)- glutamic acid, (L)-aspartic acid, chlorotrimethylsilane, sodiumbicarbonate, N,N'- dicyclohexylcarbodiimide, 4-(dimethylamino)-pyridine, isobutyl chloroformate, N,O-dimethyl- hydroxylamine hydrochloride, methyllithium, triethylamine, iodine, citric acid, magnesium sulfate, sodium hydroxide, ammonium chloride, methylmagnesium bromide, and calcium hydride were purchased from Sigma-Aldrich. The Rhodamine 110 was purchased from Cayman Chemical and Rcsorufin was purchased from ChcmCruz. L-photo-mcthioninc and L-photo- leucine were synthesized in the lab and characterized by1H,13C NMR and ESI-MS. Allcommercially available chemicals and reagents were used as received and without further purification. The reagents not mentioned as well as the solvents used in the syntheses were purchased commercially and were ACS grade. Solvents were dried according to standard procedures.

[0071] Instrumentation and characterization: The 'l l NMR and13C NMR spectra were obtained on a Bruker AVANCE III HD 400 MHz High-Performance Digital NMR spectrometer. Chemical shifts were analyzed on Topspin 3.5 software. Chemical shifts for1H and13C NMR were reported in delta (5) unit parts per million (ppm) downfield to internal standard 0.03% tetramethyl silane (TMS).1H-NMR spectra are referenced to signals for TMS (0 ppm) and residual non-deuterated solvents (chloroform 7.26 ppm; deuterium oxide 4.80 ppm; methanol-cU 3.31 ppm; dimethylsulfoxide-de 2.50 ppm). Chemical shifts (5) for13C-NMR spectra are referenced to signals for residual deuterated solvents (chloroform-d 77.16 ppm, methanol-ck 49.15 ppm, dimethylsulfoxide-de 39.51 ppm). Multiplicities are reported by the following abbreviations: s (singlet), d (doublet), t (triplet), q (quartet), quint, (quintet), m (multiplet), dd (double doublet), dt (double triplet), br s (broad singlet). Coupling constants (J) are represented in hertz (Hz). The mass spectra were obtained on a Waters ACQUITY UPLC (Ultra-Pure Liquid chromatograph) Xevo QTOF (Quadrupole Time of Flight) high resolution mass spectrometer in a mobile phase of methanol : water with a solvent gradient of 100% to 50% over 10 minutes of time and the spectra were analyzed on the MassLynx software. The infrared spectra were obtained on a Bruker Alpha Platinum-ATR spectrometer in neat form and the spectra were analyzed on OPUS 7.5 software. UV-VIS spectra were acquired on a Perkin Elmer Precisely Lambda 25 UV / VIS Spectrometer and the spectra were analyzed via PerkinElmer UV Winlab software. The fluorescence spectra were obtained with a Perkin Elmer Precisely LS 55 Luminescence Spectrometer and were evaluated with PerkinElmer FL Winlab software. The confocal images were acquired on a Zeiss LSM T-PMT, LSM 710 microscope and were analyzed on ZEN 2012 SPS software. The optical rotation values were obtained with Rudolph Research Analytical Autopol III Automatic Polarimeter.

[0072] Experimental general techniques: All solution-phase reactions were monitored by thin-layer chromatography (TLC) carried out on E. Merck silica gel plates (60 F254) with UV light, visualized by potassium permanganate or vanillin stain. Column chromatography was carried out with silica gel pore size 60 A (Sigma Aldrich, 40-63 pm, 230-400 mesh particle size).2. Synthesis of L-RhDYE-Methionine (RhDYE-Met)

[0073] Rhodamine 110 (56.5 mg, 0.1526 mmol, 1.05 eqv.) was dissolved in 36.5 ml dry DMSO and stirred under 300 mW / cm2light exposure until completely dissolved. While stirring, the solution of L-photo-methionine (23 mg, 0.1465 mmol, 1.00 eqv.) dissolved in 1 ml dry DMSO was added to the rhodamine solution over 2 h in 6 min intervals by adding each time 50 L without exposing the reactants to light. After every addition, the mixture was continued to stir under 300 mW / cm2light exposure. Once all the photo-met was added, the reaction mixture was stirred under continuous light exposure for an additional 6.5 h. The solvent was evaporated and the product was isolated by flash column chromatography on silica gel with dichloromethane:methanol (10:1) and 1 % acetic acid which provided 22 mg L-RhDYE- Methionine product as a red-solid with 31% yield. The reaction was repeated, and L-RhDYE- Methionine was isolated through a size exclusion Sephadex column. Due to solubility issues of the L-RhDYE-Methionine in D2O, the proton NMR was taken under acidic conditions (HC1).

[0074] 1H NMR (400 MHz, D2O) 5 8.09 (d, J = 6.7 Hz, 1H), 8.03 (s, 2H), 7.67 (d, J =7.4, 1H), 7.64-7.59 (m, 1H), 7.49 (dd, J = 5.8, 3.3 Hz, 1H), 7.21 (d, J = 7.7 Hz, 1H), 7.03 (d, J = 9.1 Hz, 1H), 6.81 (d, J = 1.9 Hz, 1H), 6.75 (d, J = 9.0 Hz, 1H), 3.97 - 3.90 (m, 1H), 3.66 (m, 1H), 1.86 - 1.74 (m, 2H), 1.44 - 1.32 (m, 2H), 0.99 (d, J = 6.3 Hz, 3H).

[0075] 13C NMR (109 MHz, DMSO-d6) 5 169.4, 152.9, 152.7, 151.6, 135.7, 130.2,128.8, 127.4, 124.8, 124.5, 111.3, 106.4, 99.6, 85.9, 49.1, 47.5, 23.7, 22.9, 14.4.IR (neat, cm1): 3405, 3383, 3360, 3344, 3332, 3282, 3216, 3206, 3186, 3170, 3155, 3133, 3122, 3095, 3077, 3060, 3030, 3012, 2920, 2858, 2361, 2342, 2322, 1733, 1654, 1597, 1567, 1558, 1540, 1507, 1465, 1406, 1357, 1329, 1287, 1277, 1188, 1111, 1066, 1011. HRMS (ESI): for C26H25N3O5 459.3726 m / z. Specific rotation: [OI]25D = + 22° (c 0.05, H2O).3. Optimization of reaction conditions:

[0076] Because the RhDYE fluorophore was extremely poorly soluble and no quantitative solubility data was available for polar aprotic solvents, we screened some polar aprotic solvents to determine a suitable solvent. Because of the fluorophore’ s poor solubility, we only could dissolve the dye in very dilute concentrations (~2 mM). Different solvents were screened such as dichloromethane, acetone, tetrahydrofuran, acetonitrile, 1,4-dioxane, dimethylformamide, and dimethyl sulfoxide with the relative results as indicated in Table la, whereby 1 ,4-dioxane, DMF, and DMSO yielded the best results with increasing relative solubilities. The lower boiling solvents like 1,4-dioxane and dimethylformamide were eliminated because of evaporation concerns. DMSO was determined to be the best solvent to dissolve the RhDYE fluorophore and was taken forward. In the next set of experiments, the effect of using a wavelength filter on the light source was studied and presented in Table lb, with the result that the desired product formed under both conditions with 320 nm filter and without, however the reaction condition without employing a filter was much more effective. Since the reaction reached temperatures of 70°C due to the light source we investigated whether the activation is based on a photochemical or thermal reaction, Table 1c, it was determined that the reaction progressed exclusively photochemically, and heat is not the driving force.Table 1: Screening of reaction conditions (where, X: ineffective, • / : effective).

[0077] Because of the poor solubility we started to investigate the influence of dilution on the reaction progress by keeping the reactant ratio constant at 1:1, shown in Table 2a. The more diluted the reaction was conducted in the direction from 0.2 M towards 0.002 M, to the contrary the more product formed with a gradual increase in product yield from 0% to 36%. Further improvement of the product formation to 64% was achieved under the same reaction conditions except when no wavelength filter was used.

[0078] The first entry of Table 2b shows the effect of different reaction concentrations on the product formation without using a filter, while the reactant ratio was kept 1:1. As the reaction concentration increased steadily from 0.002 M to 0.02 M, the product formation decreased inversely proportional from 64% to 8%. The second entry of Table 2b presents the impact of various RhDYE fluorophore to photo-met ratios on the product yield without using a filter. As the ratio increased from 1:1 to 10:1, the product yield decreased accordingly from 64% to 7%.

[0079] Finally, the influence of different reactant ratios on the product formation was analyzed with a wavelength filter and without and the data were shown in Table 2c. Reducing the L-RhDYE fluorophore to photo-met ratio from 1:1 to 1:2 resulted in a reduction of the product yield from 36% to 23% when filter was used. Under the same conditions when no filter was used the L-RhDYE fluorophore to photo-met ratio of 1:1 formed 64% product whereas when the ratio was 1:2 the product yield was 29%. Therefore, the first entry of Table 2c shows increasing the photo-met concentration from 2 mM to 4 mM while keeping the L-RhDYE fluorophore concentration constant decreases the product yield when a light filter is used, and the second entry conveys the same trend but by excluding the wavelength filter the product formation improves significantly.Table 2: Screening and optimization of the reaction conditions.

[0080] Since the exposure of the reaction mixture to the light source over several hours increased the temperature to 70°C, it was investigated whether the carbene species forms through a thermal or photochemical activation. Therefore, a control experiment was conducted under the same reaction condition except for using heat (70°C) instead of light, shown in Table 3a. Since no desired fluorescent amino acid formed, it was determined that the diazirine activation of the photo-amino acid and the reaction progress is exclusively photochemically driven, and heat is not required to initiate the reaction. This explained the mechanism of diazirine initiation as photochemically activated and that the temperature of the solution has no direct effect on the activation and progression of this reaction. This showed that the fluorescent amino acid was exclusively photo-chcmically synthesized. Furthermore, Table 3a shows that light passing through glass is not effective to activate the photo-amino acid because no desired product was detected. Therefore, photoactivation only occurs under direct light exposure, without light passing through a glass. The combined effects of activation at the liquid-gas interphase and the direct light exposure contributed to the long reaction times. This kinetic explanation of the reaction progress was further supported by the observation that the reaction completes faster when light is provided directly and when the gas-liquid surface area is increased. Additional determining factors affecting the reaction kinetics of photo-activation are based on three steps. First, Phot-Met needs to come to the solvent surface and needs to be activated by the light at the surface, after that it needs to collide and react with the fluorophore which can be at the surface or deeper in the solution. Each Photo-met and fluorophore must react in this way. Furthermore, photo-activation does not happen in the solution due to fluorophores shielding effect on the photons with increasing depth, additionally the light losses its ability to activate the diazirine in the solution with increasing depth.

[0081] Next, the effect on the product yield of the different photo-methionine addition times to the RhDYE fluorophorc solution was examined, the results arc presented in Table 3b. Combining the reactants together at the beginning and letting them react for 2h formed 23% product, adding the amino acid slowly over 60 min increased the product yield to 32% after 2 h reaction time, and the reduction of the amino acid addition rate to 120 min increased the product formation yield further to 64% after a reaction time of 2 h. The outcome of these experiments was as the product formation was directly proportional with the addition time and the highest product formation was obtained over a 2 h addition.

[0082] The manipulation of the reaction conditions rendered the highest product yield as 64%. To further increase the product yield, the effect of the light exposure on the product formation rate was studied. After the photo-met addition was complete and the subsequent stirring of the reaction mixture with light for an additional 2 h, the solution was stirred without light in the darkness for up to 2 days, the evaluation of this study in 12 h intervals is shown in Table 3c. As soon as the light source was removed the reaction was kept on halt with 64% product conversion and no progress towards the completion of the reaction was determined when light is absent. This confirmed that an essential component for the reaction progress is the uninterrupted light exposure. Since the reaction required light for completion, the reaction mixture was left to stir under light for longer while closely monitoring the reaction progress as outlined in Table 3d. After adding the amino acid to the fluorophore solution over 2 hours followed by additional light irradiation, the product development monitoring conclusion is with increasing light exposure time, the product formation also increased gradually and reached a 100% completion after additional 6.5 hours.Table 3: Determination and optimization of the reaction parameters.4. Characterization of L-RhDYE-Methionine:

[0083] Figure 1 A) shows the aliphatic region of the1H-NMR spectrum for RhDYE- Methionine, and B) shows the aromatic region of the proton NMR. Figure 1 C) displays the structure of RhDYE-Methionine with the assigned protons on the structure. The assignment is assisted by the 2D NMR COSY and HSQC spectra. The 2D COSY spectrum of the RhDYE- Met is shown in Figure 2 A). The spectral evaluation confirmed the assigned structure of RhDYE-Met as drawn in Figure 2 B). The spectrum contains solvent residues of methanol, acetic acid and methyl acetate from the isolation step. The singlet methanol peak in D2O appeal's at 3.30 ppm (ref. 3.34 ppm). The singlet peak at 2.71 ppm stems from the dimethyl sulfoxide in D2O (ref. 2.71 ppm) and displays satellite peaks.

[0084] The singlet peak of acetic acid in D2O shows up at 1 .80 ppm due to the different pH of the NMR solvent (ref. 2.08 ppm). The methanol reacted with acetic acid and formed methyl acetate which resulted in two singlet peaks at 3.70 and 2.00 ppm in D2O as the pH of NMR solvent was slightly acidic (ref. 3.69 and 2.09 ppm, respectively). The13C-NMR spectrum of L-RhDYE-Methionine is shown in Figure 3 A). Figure 3 B) shows the ['H,13C] HSQC spectrum of L-RhDYE-Methionine. The HSQC experiment enabled the assignment of the five aliphatic amino acid carbon signals at 21.9, 26.8, 33.3, 54.5, 67.3 ppm. Based on HSQC spectrum the structural assignment of L-RhDYE-Methionine is displayed in Figure 3 C).

[0085] The infrared absorption spectrum for the N=N stretching frequency have typical values of 1560-1585 cm1for a variety of dialkyl diazirine structures. This characteristic mildstretch is visible in the infrared spectrum of the photo-methionine starting material at 1 ,583 cm' as shown in Figure 5 A), whereas this distinctive peak at 1,583 cm'1has disappeared in the spectrum of the RhDYE-Met product, Figure 5 B), supporting the formation of the RhDYE-Met product. Furthermore, the RhDYE-Methionine product displays in the infrared spectrum both features of the RhDYE fluorophore and methionine amino acid analog, which are as shown in Figure 5 B): the characteristic strong aliphatic amino acid C-H stretches at 2,920-2,858 cm1. The broad and strong amine N-H stretches are present between 3,405-3,206 cm1, the broad carboxylic acid O-H stretch is between 3,186-3012 cm1, the aromatic mild C-H stretches are visible between 3,095-3,012 cm1, the aliphatic C-H stretches of strong intensities are present between at 2,920 and 2,858 cm1, the C=N stretch is visible at 2,361 cm1, the ester C=O stretch is at 1,733 cm1(or 1,654 cm'1), the carboxylic acid C=O stretch is at 1,654 cm1(or l,597cm-l), the aromatic C=C stretches are present at 1,567-1465 cm1, and the strong phenolic, ester and carboxylic acid C-0 stretches are visible between 1,287-1,011 cm1.Specific rotation

[0086] The optical rotation values were obtained with a Rudolph Research Analytical Autopol III Automatic Polarimeter. Polarimeter measurement parameters: 25°C temperature, 50 mm cell length with wavelength of 589 nm. The sample concentration of the probe was 0.05 g L- RhDYE-Met / 100 ml H2O and the specific rotation value was obtained as +22°. The PubChem literature value of L-Methionine’s specific rotation is +22°.[8,9]5. Physical properties of the L- RhDYE-Methionine

[0087] After synthesizing the L-RhDYE-Methionine, its physical and biological features were investigated. Figure 6 shows the UV-Vis spectra and its Beer-Lambert graph of the L- RhDYE-Methionine in DMSO-PBS buffer. The UV-vis properties of L-RhDYE-Methionine in DMSO-PBS buffer were studied by analyzing the UV-vis absorption spectrum with concentrations ranging from 15 pM, 30 pM, 45 pM, 60 pM up to 75 pM. The L-RhDYE-Met’ s absorbance plot between 400-600 nm for different concentrations from 15 pM to 75 pM is displayed in figure 6 A) and has a maximum absorption wavelength of 502 nm. Figure 6 B) lists the absorbance values at Z.maxfor the measured concentrations between 15 pM and 75 pM.Figure 6 C) shows the Beer-Lambert graph of the RhDYE-Met probe in DMSO-PBS buffer and resulted in a high molar extinction coefficient value of 13,229 M‘l cm" ' .

[0088] The L-RhDYE-Met’s emit strong green fluorescence with a peak at 530 nm and excitation peak at 503 nm (Figure 7) with high quantum yield (0.94) in DMSO-PBS buffer. It is photostable over a 12 h exposure to the light (Figure 8), being well suitable for bioimaging in live cells or tissues.6. Biological properties of the RhDYE-Methionine probe

[0089] After obtaining the physical properties of the L-RhDYE-Met probe, biological studies were conducted. First the probe’s operating conditions were determined and optimized. The screening experiments were performed with bioimaging studies on live human skin fibroblasts by applying different concentrations (5 pM, 10 pM and 20 pM), different incubation times (10 min, 30 min) and different solvents (DMSO and DMSO-PBS buffer). The localization studies resulted in the conclusion that the fluorescent probe works best with 20 pM concentration , an incubation time of 30 min, and DMSO-PBS buffer or DMSO alone. After determining the L- RhDYE-Met’s operating conditions, localization studies were conducted through live cell imaging. Figure 9 shows the confocal microscopy images of human skin fibroblast cells (WS1) incubated with and without the RhDYE-Methionine probe, scale bar is 20 pm. The probe’s excitation wavelength in the cell is 514 nm and the emission wavelength is 549 nm, the confocal microscopy images show that the fluorescent RhDYE-methionine probe is located within the cell and is homogeneously and well distributed in the cell cytoplasm.7. Synthesis and characterization of L-Resorufin-Methionine (L-Res-Met)

[0090] Resorufin (3.3 mg, 0.01548 mmol, 1.0 eqv.) was dissolved in 7.6 ml dry DMSO and stirred under 300 mW / cm2light exposure until completely dissolved. While stirring, thesolution of L-photo-methionine (3 mg, 0.01549 mmol, 1.0 eqv.) dissolved in 100 pl dry DMSO was added to the rcsorufin solution over 2 h in 6 min intervals by adding each time 5 pL without exposing the reactants to light. After every addition, the mixture was continued to stir under 300 mW / cm2light exposure. Once all the L-photo-met was added, the reaction mixture was stirred under continuous light exposure for an additional 2 h. The solvent was evaporated and crude product was obtained with a -14% yield. The reaction was repeated and let stir after the photomet addition was complete for an additional 6.5 h. NMR showed -70% crude product formation.

[0091] ' H NMR (400 MHz, DMSO) 8 1.06 (d, J = 6.2 Hz, 3H) product (resorufin-met) and 1.01 (s, 3H) starting material (photo-met). Product / Starting Material = 1.00 / 0.43 HRMS (ESI): M=Ci8Hi8N2O5=342.1216 m / z, [M+K]+= 381.2548 m / z.

[0092] L-Res-Met is a much-desired fluorescent amino acid that absorbs above 500 nm and emits strong yellow fluorescence at a longer wavelength.Exemplary Embodiments.

[0093] The following exemplary embodiments are provided, the numbering of which is not to be construed as designating levels of importance:

[0094] Aspect 1 provides a method of synthesizing an unnatural amino acid comprising: contacting an amino acid comprising a photoreactive group with a reactive group comprising a fluorophore; and exposing the amino acid and reactive group to light.

[0095] Aspect 2 provides the method of Aspect 1, wherein the amino acid comprising the photoreactive group comprises the structure according to formula I or II:R1OH R1— 4, J— CO9HH2N O (I), H2N (jj), wherein R1is substituted or unsubstituted (Ci-C2o)hydrocarbyl, substituted or unsubstituted (Ci-C2o)alkyl, substituted or unsubstituted (Ci-C2o)alkenyl, of substituted or unsubstituted (C1-C20) alkynyl.

[0096] Aspect 3 provides the method of any of Aspects 1 or 2, wherein the amino acid comprising the photoreactive group comprises the structure according to formula II or III:

[0097] Aspect 4 provides the method of any of Aspects 1-3, wherein the reactive group comprising a fluorophore comprises R-A-COOH, R-A-OH, R-A-SH, R-A-CHs, R-A-CHo-iCHo- 2, or R-A-NH2, wherein R is the fluorophore, and A is a bond, substituted or unsubstituted (Ci- C2o)hydrocarbyl, substituted or unsubstituted (Ci-C2o)alkyl, substituted or unsubstituted (Ci- C2o)alkenyl, or substituted or unsubstituted (C1-C20) alkynyl.

[0098] Aspect 5 provides the method of any of Aspects 1-4, wherein the fluorophore is rhodamine (RhDYE) or resorufin (Res).

[0099] Aspect 6 provides the method of any of Aspects 1-5, wherein the compound is dissolved in a solvent.

[0100] Aspect 7 provides the method of any of Aspects 1-6, wherein the unnatural amino acid is

[0101] Aspect 8 provides the method of any of Aspects 1-7, wherein the unnatural amino acid is

[0102] Aspect 9 provides a compound of formulaPage 1

[0103] Aspect 10 provides a method of imaging a target comprising imaging a biotarget comprising an unnatural fluorescent amino acid.

[0104] Aspect 11 provides the method of Aspect 10, wherein the biotarget comprises a live cell comprising the unnatural fluorescent amino acid.

[0105] Aspect 12 provides the method of any of Aspects 10 or 11, wherein the biotarget comprises a polypeptide, having the unnatural fluorescent amino acid incorporated into the polypeptide.

[0106] Aspect 13 provides the method of Aspect 12, wherein the polypeptide is a protein.

[0107] Aspect 14 provides the method of any of Aspects 10-13, wherein the unnatural fluorescent amino acid is a first unnatural fluorescent amino acid and the biotarget further comprises a second unnatural fluorescent amino acid.

[0108] Aspect 15 provides the method of Aspect 14, wherein the first unnatural fluorescent amino acid and the cell are exposed to electromagnetic radiation.

[0109] Aspect 16 provides the method of any of Aspects 10-15, further comprising detecting a fluorescent signal from the unnatural fluorescent amino acid.

[0110] Aspect 17 provides the method of any of Aspects 14-16, further comprising detecting a fluorescent signal from the first unnatural fluorescent amino acid and the first unnatural fluorescent amino acid.Aspect 18 provides the method of any of Aspects 10-17, wherein the unnatural amino acid isAspect 19 provides the method of any of Aspects 10-18, wherein the unnatural amino acid is

[0111] Aspect 20 provides the method of any of Aspects 10-19, wherein the unnatural amino acid is synthesized according to the method of Aspect 1.

[0112] Aspect 21 provides a method of synthesizing an unnatural amino acid comprising reacting Photo-Met or Photo-Leu with a compound comprising a reactive group in the presence of light.

[0113] Aspect 22 provides the method of Aspect 21, wherein the compound is dissolved in a solvent.

[0114] Aspect 23 provides the method of Aspect 21 or 22, wherein the compound is a fluorophore.

[0115] Aspect 24 provides the method of any of Aspects 21-23 wherein the unnatural amino acid

[0116] Aspect 25 provides the method of any of Aspects 21-23, wherein the unnatural

[0117] Aspect 26 provides a compound of formula

[0118] Aspect 27 provides a compound of formula

[0119] Aspect 28 provides a method of imaging a target comprising a live cell with a unnatural fluorescent amino acid.

[0120] Aspect 29 provides the method of Aspect 28 further comprising detecting a fluorescent signal.

[0121] Aspect 30 provides the method of Aspect 28 or 29 wherein the amino acid is L- RhDYE-Met or L-Res-Met.

[0122] The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the embodiments of the present invention. Thus, it should be understood that although the present invention has been specifically disclosed by specific embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those of ordinary skill in the art, and that such modifications and variations are considered to be within the scope of embodiments of the present invention.

Claims

We claim:

1. A method of synthesizing an unnatural amino acid comprising: contacting an amino acid comprising a photoreactive group with a reactive group comprising a fluorophore; and exposing the amino acid and reactive group to light.

2. The method of claim 1, wherein the amino acid comprising the photoreactive group comprises the structure according to formula I or II:wherein R1is substituted or unsubstituted (Ci-C2o)hydrocarbyl, substituted or unsubstituted (Ci-C2o)alkyl, substituted or unsubstituted (Ci-C2o)alkenyl, of substituted or unsubstituted (C1-C20) alkynyl, a substituted or unsubstituted azide, a substituted or unsubstituted fluorine, or a substituted or unsubstituted nitroxide.

3. The method of claim 1, wherein the amino acid comprising the photoreactive group comprises the structure according to formula II or III:

4. The method of claim 1, wherein the reactive group comprising a fluorophore comprises R-A-COOH, R-A-OH, R-A-SH, R-A-CH3, R-A-CH0 1CH0-2, or R-A-NH2, wherein R is the fluorophore, and A is a bond, substituted or unsubstituted (Ci-C2o)hydrocarbyl, substituted or unsubstituted (Ci-C2o)alkyl, substituted or unsubstituted (Ci-C2o)alkenyl, or substituted or unsubstituted (C1-C20) alkynyl.

5. The method of claim 1 , wherein the fluorophore is rhodamine (RhDYE) or resorufin (Res).

6. The method of claim 1, wherein the compound is dissolved in a solvent.

7. The method of claim 1 wherein the unnatural amino acid is8. The method of claim 1, wherein the unnatural amino acid is9. A compound of formula10. A method of imaging a target comprising imaging a biotarget comprising an unnatural fluorescent amino acid.

11. The method of claim 10, wherein the biotarget comprises a live cell comprising the unnatural fluorescent amino acid.

12. The method of claim 10, wherein the biotarget comprises a polypeptide, having the unnatural fluorescent amino acid incorporated into the polypeptide.

13. The method of claim 12, wherein the polypeptide is a protein.

14. The method of claim 10, wherein the unnatural fluorescent amino acid is a first unnatural fluorescent amino acid and the biotarget further comprises a second unnatural fluorescent amino acid.

15. The method of claim 14, wherein the first unnatural fluorescent amino acid and the cell are exposed to electromagnetic emission.

16. The method of claim 10, further comprising detecting a fluorescent signal from the unnatural fluorescent amino acid.

17. The method of claim 14, further comprising detecting a fluorescent signal from the first unnatural fluorescent amino acid and the first unnatural fluorescent amino acid.

18. The method of claim 10, wherein the unnatural amino acid is19. The method of claim 10, wherein the unnatural amino acid is20. The method of claim 10, wherein the unnatural amino acid is synthesized according to the method of claim 1.

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