Recent advances in the synthesis and application of fl uorescent α-amino acids

Fluorescence spectroscopy has become a powerful technique for probing a range of complex biological processes including enzyme mechanisms and protein–protein interactions. While the application of this technique uses a number of strategies, many of these rely on the use of fluorescent α-amino acids. This review highlights the recent synthetic methods developed for the incorporation of highly conjugated chromophores into the side-chain of α-amino acids and the application of these compounds as probes for imaging in medicine and biology. In particular, the design and synthesis of α-amino acids bearing coumarin, flavone and polyaromatic derived chromophores is described.


Introduction
In the last 20 years, fluorescence spectroscopy has played a key role as an analytical and diagnostic tool in many areas of fundamental and applied biomolecular science. 1 Significant developments in research fields such as molecular and cellular biology, biophysics, biotechnology and medicine have been accelerated due to the application of emerging fluorescent-based experiments. 1,2This is in part due to the high sensitivity, selectivity and fast response time of this technique, as well as the relative ease in tuning the photophysical properties of small molecular probes.
As proteins are crucial for the majority of cellular functions, the investigation of these processes, which involve enzyme mechanisms and protein-protein interactions, can be achieved in combination with fluorescent spectroscopy using a number of strategies.These include expressing the protein of interest with a naturally occurring fluorescent protein (e.g.green fluorescent protein, GFP) 3 or using naturally occurring fluorescent α-amino acids within a protein, such as tyrosine or tryptohan. 4exander H. Harkiss Alexander H. Harkiss was born in 1990 in Ayrshire, Scotland.He graduated with an MSci degree in Chemistry with Medicinal Chemistry from the University of Glasgow in 2013 while carrying out an industrial placement with Systagenix and a final year project on one-pot multibond forming processes for the preparation of drug-like tetralins.Since 2013, he has been part of the Sutherland group at the University of Glasgow working towards his PhD, which is focused on exploring cycloaddition reactions of enone derived amino acids and the development of the heterocyclic products as novel fluorescent imaging agents.
However, there are limitations to such approaches.For example, attachment of fluorescent proteins can alter the stability and functionality of their fusion partner, while the use of intrinsic fluorescent α-amino acids relies on a relative abundance within the protein and multiple residues in different environments can complicate the spectroscopy.][6][7][8][9] These compounds can be readily accessed using standard methods for the asymmetric synthesis of α-amino acids. 10The photophysical properties of unnatural fluorescent α-amino acids can be tuned for a particular application and they can be incorporated into protein structures with a high level of precision using conventional solid phase peptide synthesis (SPPS), expressed protein ligation (EPL) or unnatural amino acid mutagenesis. 82][13] These types of probes have also been used for the investigation of peptidoglycan synthesis and to monitor subsequent bacterial growth. 145][6][7][8][9] For example, in 2009, Katritzky and Narindoshvili described the specific structural features required for the use of fluorescent α-amino acids as effective molecular probes, 6 while more recently Krueger and Imperiali have reviewed the incorporation of unnatural fluorescent α-amino acids into peptides and proteins and their application in various chemical biology studies. 8The aim of this review is to highlight the recent synthetic methods and strategies that have been developed for the preparation of fluorescent α-amino acids incorporating side-chain chromophores.The use of these compounds as biosensors and molecular probes is also described.The review has been organised based on the structure of the chromophore side-chain and this includes compounds derived from natural α-amino acids and wellestablished coumarin, fluorescein and polyaromatic-type chromophores.

Fluorescent analogues of natural α-amino acids
Naturally occurring α-amino acids bearing aromatic sidechains, such as L-phenylalanine (1), L-tyrosine (2) and L-tryptophan (3) (Fig. 1) have been used as intrinsic fluorescent probes to study phenomena such as protein dynamics. 15The use of naturally occurring α-amino acids for fluorescent imaging minimises the conformational perturbations of protein structure and loss of stability sometimes observed when using structurally different unnatural α-amino acids.Despite these advantages, their general application in fluorescent studies has been limited by poor optical properties.
For example, phenylalanine has both low quantum yield and molar extinction coefficient, while tyrosine lacks sensitivity to environmental changes such as polarity.For these reasons, a number of novel fluorescent α-amino acids have been developed based on fine-tuning the structures and optical properties of naturally occurring α-amino acids.
A p-cyano-derivative of L-phenylalanine has been synthesised and used to study peptide-membrane interactions. 16he key step of the synthesis involved a phase transfer catalysed alkylation of a benzylidene protected alanine amide (Scheme 1).Following hydrolysis of the benzylidene protecting group, the racemic amino amide was resolved using the genetically modified organism Ochrobactrum anthropi which gave the desired L-phenylalanine derivative 4 in >98% enantiomeric excess.The incorporation of this α-methyl derivative into the decapeptide trichogin GA IV resulted in retention of helical conformation.Using the optical properties of the p-cyanophenyl moiety, the modified peptide was shown to have fluorescence emission around 295-305 nm and this was exploited to investigate the interaction of this modified lipopeptaibol with model membranes.
L-Tyrosine derivatives tend to have significantly higher quantum yields than the corresponding L-phenylalanine derivatives and as such, new fluorescent derivatives have been prepared 17 and used in applications such as the biosensing of hydrogen peroxide. 18g. 1 Structures and quantum yields (QY) 4 of L-phenylalanine (1), L-tyrosine (2) and L-tryptophan (3).
Scheme 1 Synthesis of a fluorogenic L-phenylalanine derivative.
A series of stimuli responsive fluorescent α-amino acids have been designed based on extending the π-conjugation of L-tyrosine. 19Compounds such as the bis-styryl analogue 5 were prepared using a palladium-catalysed double Mizoroki-Heck reaction of a 3,5-diiodo-L-tyrosine derivative with 4-methoxystyrene (Scheme 2).A pyridine derivative from this series displayed distinct red, green and blue emission spectra by control of the solution pH, while the incorporation of bis-styryl analogue 5 into a cell-penetrating peptide and incubation with two different cell lines showed that these compounds could be used as intrinsic labels for fluorescent imaging of cells.
Of the three naturally occurring fluorescent α-amino acids, L-tryptophan has the most favourable photophysical properties, such as more intense brightness.For these reasons, a wide range of indole variants such as azulene and benzothiophenyl have been used to mimic the size and polarity of the L-tryptophan side-chain, resulting in novel fluorescent α-amino acids. 4ore recently, new methodology has been developed that allows C-2 substitution of the indole ring leading to novel fluorescent L-tryptophan analogues. 20,21For example, Chen and co-workers prepared a fluorescent L-tryptophan bearing a 1,2,3triazole at C-2 using a hydrogen-bond mediated coupling reaction, 20 while the group of Fairlamb produced a series of novel, highly fluorescent C-2 arylated L-tryptophan analogues and L-tryptophan-containing peptides using a palladium-mediated direct C-H bond functionalisation (Scheme 3). 21The emission wavelength of these C-2 aryl compounds were found to be redshifted relative to L-tryptophan, with phenyl analogue 6 exhibiting the largest fluorescence intensity.
Hecht and co-workers have reported a series of highly fluorescent L-tryptophan analogues incorporating additional heteroatoms, heterocycles or conjugated substituents within the indole ring system. 22Many of these analogues were found to be significantly brighter and red-shifted in fluorescence emission compared to L-tryptophan.A general asymmetric synthetic route to these compounds was developed by alkylation of the Schöllkopf bis-lactim ether with an indole derivative to give the adducts in high diastereoselectivity.In the case of the 6-cyano analogue 7 (Scheme 4), this was converted to dinucleotide 8 and used to activate a suppressor tRNA transcript for efficient incorporation of the amino acid into two different positions of E. coli DHFR.22c Fluorescent imaging of the 6-cyanotryptophan in DHFR could be performed selectively in the presence of tryptophan analogues and was also shown to form an efficient Förster resonance energy transfer (FRET) pair with a coumarin derived glycine.
The side-chains of other naturally occurring α-amino acids such as aspartic acid, 23 lysine 24 and serine 25 have been labelled with various chromophores, resulting in the preparation of novel fluorescent α-amino acids.For example, in a direct and highly regioselective approach, L-lysine has been condensed with a range of 1-vinylpyrrole-2-carbaldehydes under mild conditions to give the corresponding Schiff bases

Coumarin derived α-amino acids
2H-Chromen-2-ones, commonly known as coumarins or benzopyranones, are found in a wide range of natural products, pharmaceuticals and new materials. 26This chromophore has also been widely used in optical imaging due to this ring system having high quantum yields, an extended spectropscopic range, photostability and general solubility in a wide range of solvents. 5,6For these reasons, many synthetic methods have been developed to incorporate the coumarin motif and more specifically, 7-methoxy or 6,7-dimethoxy analogues into the side-chain of α-amino acids. 27In 2004, Garbay and co-workers reported a short synthetic approach for the preparation of optically active coumarin derived α-amino acids using a von Pechmann condensation reaction of a β-keto ester with a phenol. 28The side-chains of L-aspartic acid (Scheme 6) and L-glutamic acid were initially activated with carbonyldiimidazole and then reacted with the magnesium salt of monoethyl malonic acid to give the corresponding β-keto esters.
Hydrogenolysis led to the removal of the protecting groups and this was followed by the von Pechmann reaction, performed in the presence of methanesulfonic acid.Using 3-methoxyphenol, allowed the synthesis of L-(7-methoxy- coumarin-4-yl)alanine ( 9), a commonly used imaging agent in 71% yield.
0][31] A phosphate derivative 10 (Fig. 2) was prepared as a mimic of phosphotyrosine and following incorporation into cell penetrating peptides, was used to study the activity of protein tyrosine phosphatases. 29Martin and coworkers developed an optimised protocol based on the von Pechmann reaction for the efficient and scalable synthesis of protected coumarin derived ethylglycine 11. 30 This was found to be an excellent building block for SPPS and following incorporation into truncated peptides of HIV-Tat, was shown to be effective at HeLa cell imaging.Using the Garbay approach, Häuβler and Gütschow prepared a fluorescent bisbenzamidine coumarin derivative 12, which has an emission maximum at 432 nm. 31 The authors propose that this compound could find application in the study of minor grooves of adenine/thyminerich double-strand DNA.
3][34] The groups of Braun 32 and Wang 33 have both shown that (7-hydroxycoumarin-4-yl)ethylglycine can be efficiently prepared by the reaction of a glycine-enolate equivalent with a suitably activated coumarin derived electrophile.In a similar manner, Kazmaier and co-workers prepared a series of novel α-amino acids bearing a coumarin side-chain by the palladium-catalysed allylic alkylation of coumarin activated allylic carbonates with a zinc chelated glycine-enolate (Scheme 7). 34Compounds such as 13 were prepared in good yield with the double bond in conjugation with the coumarin and formed exclusively as the E-isomer.

Fluorescent α-amino acids incorporating other established chromophores
A range of other commonly used fluorophores, such as fluorescein, benzothiadiazole, xanthone and BODIPY have been used to label non-emissive, unnatural α-amino acids and peptides for application in biological imaging.14b,35,36 A good example of this is the work reported by the Xie group who showed that various alkyne-derived chromophores could be attached to azido α-amino acids using click chemistry. 37In an extension of this work, the use of a ruthenium-catalysed cycloaddition reaction of bis-alkyne substituted benzothiadiazole motifs with azidoalanine derivatives gave crosslinked α-amino acids in good yields (Scheme 8).37c An investigation of the photophysical effects of compounds such as 14 showed that conjugating 1,2,3-triazoles with the benzothiadiazole group gave longer excitation and emission wavelengths while maintaining high fluorescence quantum yields.
Other standard coupling techniques such as amide bond formation have been used to attach chromophores to the side chains of unnatural α-amino acids. 38The fluorescein photophore, Tokyo-Green was coupled to an aminopropyl analogue of the kainoid MFPA using a combination of EDCI and HOBt (Scheme 9).Following deprotection, the resulting fluorescent adduct 15, was used to probe the ionotropic glutamate receptor in mice.
As well as simply labelling the side-chain of an α-amino acid with an established chromophore, many fluorescent α-amino acids have been generated by using the chromophore as the entire side-chain.This has the advantage of minimising steric pertubations when incorporated into peptides and proteins.For example, preparation of an xanthone-alanine derivative 16 was achieved by the palladium-catalysed coupling of an xanthone triflate with an alanine derived organozinc reagent (Scheme 10). 39Enzymatic hydrolysis of the ester gave the opti-Scheme 7 Palladium-catalysed allylic alkylation of a glycine-enolate.
Scheme 8 Synthesis of cross-linked α-amino acid 14 using a ruthenium-catalysed cycloaddition reaction.cally pure Boc-protected amino acid, which showed typical xanthone fluorescent characteristics.
The benefits of incorporating a chromophore directly within the side-chain of an α-amino acid is exemplified by L-acridon-2-ylalanine 17, which is small enough to be utilised in ribosomal biosynthesis. 40A highly efficient scalable synthetic route was developed from L-tyrosine using a Buchwald-Hartwig type coupling of a triflate derivative with O-methyl anthranilate (Scheme 11). 40,41Treatment of the coupled product with sulfuric acid resulted in both an intramolecular Friedel-Crafts acylation and deprotection of the amino acid.An aminoacyl tRNA enzyme evolved from Methanococcus janaschii tyrosine synthetase was effective for the milligram synthesis of L-acridon-2-ylalanine-labelled proteins.Characterisation of the photophysical properties of these proteins, demonstrated that 17 was an effective partner in FRET interactions and could be used to monitor peptide binding and protein folding.
Flavone containing α-amino acids have also been prepared as fluorescent probes for a number of applications. 42A general synthesis of 3-hydroxyflavone α-amino acids was developed from an L-tyrosine derivative (Scheme 12).Aldol condensation gave a chalcone intermediate that was then subjected to an Algar-Flynn-Oyamada reaction forming the 3-hydroxyflavone motif. 43These compounds were then typically converted to the Fmoc-derivatives for SPPS.Furan analogue 18, which displays dual emission was incorporated into the zinc finger domain of the HIV-1 nucleocapsid protein in place of a tryptophan residue.42a Despite this change, the folding of the protein was preserved and was used to investigate binding to oligonucleotides.The advantage of placing the chromophore in close proximity to the peptide main-chain was demonstrated with the push-pull 4-dimethylaminophenyl analogue 19.42b This dual fluorescent dye was incorporated into the membrane-active peptide, melittin and used to probe the orientation of the peptide in lipid bilayers.The proximity of the fluorophore in combination with its highly sensitive dual emission properties resulted in detection of the peptide at a much deeper level in the bilayer.

Aminonaphthyl and aminophthalimide-type fluorescent α-amino acids
Over the last two decades α-amino acids bearing various pushpull chromophores such as aminonaphthyl and aminophthalimide have been developed as highly environmentally sensitive probes for biological processes (Fig. 3).The charge transfer dye, dimethylaminonaphthyl 20, simultaneously developed by the Imperiali and Cohen groups, was used to study peptideprotein interactions associated with the S-peptide of RNase S, 44 as well as the electrostatic character of the B1 domain of streptococcal protein G. 45 The dimethylaminophthalimide α-amino acid 21, as a component of a short peptide sequence, was shown to be an excellent reporter for binding with a 14-3-3 protein. 46A 6-fold increase in fluorescence intensity and a 40 nm shift of the emission maximum was observed on binding to the 14-3-3 protein.More recently, the Imperiali group reported a new member of the dimethylaminophthalimide family of fluorophores, the 4-N,N-dimethylamino-1,8-naphthalimide analogue 22. 47 This solvatochromic α-amino acid was found to have a number of advantages over other members of this family, including greater chemical stability and longer wavelength of excitation.A demonstration Scheme 11 Synthesis of acridon-2-ylalanine 17.
of its use to monitor protein-protein interactions by incorporation into a peptide recognised by calcium-activated calmodulin showed a 900-fold increase in fluorescence emission on binding.Modified versions of this family of fluorescent α-amino acids have recently been reported.A chain-extended D-configured version of 22 has been prepared and incorporated into peptides. 48The synthesis of this novel analogue, which is typical for most phthalimide and naphthalimide compounds involved the condensation of an anhydride with a suitably protected α-amino acid with an amine side chain (Scheme 13).The amino protecting group was replaced with Fmoc to give 23 and this was incorporated into short peptides using SPPS.
The fluorescent unnatural amino acid L-3-(6-acetylnaphtha- len-2-ylamino)-2-aminopropanoic acid (24) has been genetically incorporated into various proteins to study proteinligand and protein-protein interactions in Saccharomyces cerevisiae and mammalian cells. 12,49,50The relatively small size of 24 and the close proximity of the chromophore to the protein backbone means it is a good substitute for natural α-amino acids, with minimal perturbation to protein conformation.A new synthesis of 24 has been reported by Xiang and Wang, who used a Fukuyama-Mitsunobu reaction to implement the key step (Scheme 14). 51This allowed the efficient coupling of a 2-aminonaphthyl unit with a serine derivative.Stepwise deprotection of the various protecting groups under mild conditions gave 24 in high optical purity.
Novel fluorescent α-amino acids bearing aminonaphthyl side-chains have been prepared using strategies such as clickchemistry to couple the chromophore with the amino acid core. 52Other approaches include the use of a mild Horner-Wadsworth-Emmons (HWE) reaction between an L-aspartic acid derived β-keto phosphonate ester and 4-dimethylamino-1naphthaldehyde (Scheme 15). 53Following deprotection, E-enone 25 displayed solvatochromic properties and an emission maximum at 552 nm in water.

Fluorescent polyaromatic α-amino acids
Fluorescent α-amino acids with conjugated polyaromatic sidechains tend to have similar spectroscopic properties (e.g.quantum yields) in polar and apolar environments.While this limits the application of these compounds as reporting probes, they can be used as fluorescent tags.
A range of general methods have been developed for the synthesis of this class of fluorescent α-amino acid that involve a coupling reaction between an activated amino acid core and a suitably functionalised polyaromatic group. 54A number of approaches utilise dehydroalanine derivatives in combination with either conjugate addition 55,56 or Diels-Alder reactions. 57 Heck reaction between a vinylglycinol derivative and polyaromatic bromides has also been shown to be an effective key step for the synthesis of fluorescent polyaromatic α-amino acids (Scheme 16). 58For example, the palladium acetate catalysed reaction of vinylglycinol 26 with 1-bromopyrene gave the Heck adduct in 87% yield.Hydrogenation, protecting group manipulation and oxidation gave the brightly fluorescent pyrene derived α-amino acid 27 in good overall yield.Tripeptide 28 formed from α-amino acid 27 was shown to bind to the viral RNA trans-activation response element with a K d value of 50 nM and exhibited antibacterial activity against Bacillus subtilis.
0][61][62][63] This has resulted in the preparation of a range of novel pyrene or terphenyl derived α-amino acids that have been incorporated into proteins and used as fluorescent tags or in FRET experiments. 61,63For example, Hecht and co-workers used a Suzuki-Miyaura reaction of iodinated phenylalanine derivatives with biphenylboronic acids to give regioisomeric terphenyl analogues such as 29 (Scheme 17). 60These compounds were used to activate suppressor tRNA transcripts and incorporated into sterically accessible positions of DHFR from E. coli, with minimal disruption to structure or function.The potential of these compounds to act as a FRET pair with a coumarin derived α-amino acid within the mutant reductase was also demonstrated.
Fluorescent polyaromatic α-amino acids have also been prepared using HWE reactions of phosphonate ester derived α-amino acids with various polyaromatic aldehydes. 64,65arbay and co-workers used such a process to prepare naphthyl-derived dehydroalanine derivatives, which were then subjected to an asymmetric hydrogenation reaction to give the L-amino acids. 64Many of these compounds displayed higher quantum yields and emission maximum wavelengths than tryptophan or tyrosine.A phosphonate ester derived L-aspartic acid derivative has been used in a HWE reaction for the synthesis of α-amino acids bearing aromatic and polyaromatic enone side-chains (Scheme 18). 65The enones were found to be excellent substrates for a highly regioselective condensation/ aza-Michael reaction with phenylhydrazine.The resulting pyrazolines were oxidised with DDQ and following deprotection, gave a range of aromatic and polyaromatic pyrazole containing α-amino acids.Compounds such as naphthyl analogue 30 showed intense fluorescence at an emission maximum of 356 nm.

Conclusions
With a better understanding of how molecular structure affects the intensity and wavelength of fluorescent characteristics, 66 significant progress has been made in designing unnatural α-amino acids for specific applications in medicine and biology.The continued development of new synthetic methodology as well as application and optimisation of wellestablished transition metal catalysed coupling technology has allowed the fine-tuning of the fluorescent properties of a diverse range of next generation unnatural α-amino acids.The combination of these developments with the advances in chemical biology methods for site-specific incorporation of these dyes into proteins has allowed the unparalleled investigation of a range of fundamental biological processes at the molecular level.In particular, the use of small unnatural α-amino acids that mimic tyrosine or tryptophan has resulted in labelled active proteins with minimal disruption to structure and local conformation.These constructs in combination with processes such as FRET experiments 67 have led to significant insight into a wide range of enzyme mechanisms and cellular processes.Despite these advances, challenges still remain.Research is underway to further refine the structure and photophysical properties of fluorescent unnatural α-amino acids for application in a wider range of environmentsensitive processes and this includes producing compounds with higher quantum yields with fluorescence at longer wavelengths for new applications in medical research. 68

Scheme 4
Scheme 4 Asymmetric synthesis of a fluorescent cyanotryptophan.

Scheme 9
Scheme 9 Synthesis of a fluorescent ionotropic glutamate receptor probe.