Patent Review
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Fluorescent probes for biomedical applications (2009–2014)
The discovery and subsequent development of fluorescent probes was one of the most exciting innovations in life sciences, which marked the beginning of interpretation of numerous biological phenomena. Today, fluorescent probes are used for a wide range of biomedical applications, such as pharmaceutical research, clinical diagnostics and high-throughput screening, to name a few. Despite the availability of a large number of these probes, efforts to invent newer versions utilizing novel chemistry to address limitations of the current approaches continue. This review article gives a rundown on ‘small-molecule fluorescent probes’ patents/patent applications from January 2009 to March 2014. The patent literature was classified based on ‘preparation’ and ‘biomedical applications’ of these ‘fluorescing wonders’.
Fluorescent probes are an indispensable part of chemical biology research [1] . Over several decades, the design and development of these probes for varied biological applications improved our understanding of biological systems [2,3] . The accidental discovery of mauveine, the first organic chemical dye, dates back to 1856. Adolph Von Baeyer, a German chemist, synthesized fluorescein (1, Figure 1) , one of the most widely used fluorophores today, in 1871. Over the next 30 years, a large number of these probes were synthesized and their fluorescent properties were documented. Paul Erlich, in 1882, used sodium salt of fluorescein to track aqueous humor secretion in the eye. This was the first time that fluorescein was used in vivo. Another derivative of fluorescein that is commonly used as a staining dye is eosin Y, a tetrabromo derivative of fluorescein ( 2, Figure 1) . Eosin Y is used commonly to stain muscle fibres, cytoplasm and collagen. Also, the use of rhodamine, particularly rhodamine B and rhodamine 123 ( 3, 4, Figure 1) , extensively in biotechnology applications, such as fluorescence microscopy, flow cytometry, fluorescence correlation spectroscopy
10.4155/PPA.14.41 © 2014 Future Science Ltd
Sona Warrier1 & Prashant S Kharkar*,1 Department of Pharmaceutical Chemistry, Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management (SPPSPTM), SVKM’s NMIMS, V. L. Mehta Road, Vile Parle (West), Mumbai 400-056, India *Author for correspondence: Tel.: +91 22 4233 2016 Fax: +91 22 2618 5422 [email protected] 1
and ELISA, has gained significance in the field of fluorescence. In 1941, Albert Coons labeled antibodies with fluorescein isothiocyanate, which marks the beginning of the immunofluorescence field. The discovery of green fluorescent protein in Aequorea victoria jellyfish in 1962 by Shimomura, Johnson and Saiga contributed to groundbreaking research in cell and molecular biology [4] . Similarly, the discovery of quantum dots, another type of fluorescent probes, in the 1980s and their subsequent applications in biological research has become another exciting area. Synthetic probes such as SYBR Green I and 3-cyano-7-hydroxy coumarin (5, 6, Figure 1) are some of the many dyes that are used as reference dyes for cell imaging purposes. Overall, fluorescent probes – small molecule organic dyes, biological fluorophores (e.g., green fluorescent protein) and quantum dots – played a critical role in the advancement of biological sciences. The scope of applications of these probes is ever expanding from bioimaging, pharmaceutical research and clinical diagnostics to various areas of material sciences. This review focuses on ‘biomedical applications of small-molecule fluorescent probes’.
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Key terms Fluorophore: Fluorescent organic chemical compound that can reemit light after excitation by light or other electromagnetic radiation. Fluorescent probe: Conjugated molecule, consisting of a fluorophore or fluorochrome group covalently linked to a group having specific interaction profile in an adequate biological environment. Fluorogenic probe: Chemical compound in which fluorescence can be activated by enzymatic or chemical reaction, light or environmental changes. Quenching: Loss of fluorescence signal due to short-range interactions between the fluorophore and the local molecular environment, including other fluorophores (self-quenching). Solvatochromic fluorophores: Fluorophores that possess emission properties that are sensitive to the nature of the local microenvironment.
A thorough analysis of patents related to the subject matter since January 2009 is included. Curious readers are advised to refer to excellent reviews available on the topic for in-depth information [5–9] . Figure 1 shows a few representative examples of widely used small molecule fluorophores. A comprehensive Patents Database search (31 March 2014) that includes Espacenet, European Patent Office, United States Patent and Trademark Office, World Intellectual Property Organisation and others using the keywords ‘fluorescent probes’, ‘fluorogenic probes’, ‘fluorophores’ from 2009 until March 2014 yielded a large number of patents/patent applications. Further refinement of hits using the terms ‘preparation’ and/or ‘biomedical applications’ led to 29 patents/patent applications (Table 1) .
Patents covering preparation of fluorescent probes The patent [10] discusses a fluorescent probe claiming to radiate fluorescence by entrapment of protons, metal ions or active oxygen species. The prior art section of the patent application included the review of fluorescein (1, Figure 1), a molecule known to emit fluorescence with λmax ~500 nm in aqueous solution. This molecule has, therefore, been used as a rudimentary substructure for fluorescent probes. It was also proven in the literature by earlier workers that the carboxyl group of fluorescein is responsible for
O
O
HO
Small-molecule fluorescent probes: patent review (January 2009–March 2014) The novelty of various fluorescent probes discovered over the years makes them patentable for a wide range of industrial applications, namely their use as metal ion sensors in cellular systems, in protein tagging, in livecell imaging to medical diagnostics. There are numerous patents published over several years related to the theme of this review. This confirms, beyond doubt, the fact that fluorescent probes and related technologies have progressed significantly and their utility is increasing continuously in biomedical sciences, almost like a necessity. The article is structured in such a way that it covers all patents published in the past 5 years (2009–2014). The patent applications in languages other than English were reviewed, but a detailed analysis was not possible for obvious reasons. For the ease of reading, this review is divided in two sections – preparation and biomedical applications of fluorescent probes.
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NH+2
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Figure 1. Molecular structures of widely used fluorescent probes. Fluorescein (1), eosin Y (2), rhodamine B (3), rhodamine 123 (4), SYBR Green I (5), 3-cyano-7-hydroxy-coumarin (6).
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Fluorescent probes for biomedical applications (2009–2014)
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Table 1. Patented fluorescent probes. Sr. no.
Patent category
Patent no. (assignee)
Publication date Title
Ref.
1
Preparation
US 7524974 B2
28 Apr 2009
Fluorescent probe
[10]
2
Preparation
WO 2009152102 A2
17 Dec 2009
Pro-fluorescent probes
[11]
3
Preparation
US 20110118441 A1 19 May 2011
Synthesis of highly fluorescent peptidemetallic nanoclusters as bio-probes
[12]
4
Preparation
EP 2389201 A2
30 Nov 2011
Fluorescent probes having a polymeric backbone
[13]
5
Preparation
WO 2012040108 A2
29 Mar 2012
Multivalent fluorescent probes
[14]
6
Preparation
US 8309059 B2
13 Nov 2012
Reactive cyanine compounds
[15]
7
Preparation
US 20120288884 A1
15 Nov 2012
Quinazolinone-based fluorogenic probes
[16]
8
Preparation
US 8540521 B2
24 Sep 2013
Benzoxazole-based fluorescent metal ion indicators
[17]
9
Preparation
US 8557811 B2
15 Oct 2013
Dual-color imaging method of sodium/ calcium activities using two-photon fluorescent probes and preparation method of two-photon fluorescent probes
[18]
10
Preparation
US 8637323 B2
28 Jan 2014
Fluorescent nitric oxide probes and associated methods
[19]
11
Biomedical applications
US 7507395 B2
24 Mar 2009
Methods of using fluorescent pH indicators for intracellular assays
[20]
12
Biomedical applications
US 20090082577 A1
26 Mar 2009
Fluorescent probes for biological studies
[21]
13
Preparation and biomedical applications
US 8232303 B1
31 Jul 2012
Two-photon absorbing water soluble fluorescent probe as a near neutral pH indicator
[22]
14
Biomedical applications
US 8153446 B2
10 Apr 2012
Fluorogenic compounds converted to fluorophores by photochemical or chemical means and their use in biological systems
[23]
15
Biomedical applications
WO 2009121247 A1 8 Oct 2009
Luminescence quenchers and fluorogenic probes for detection of reactive species
[24]
16
Preparation and biomedical applications
US 7601510 B2
13 Oct 2009
Development and use of fluorescent probes of unbound analyte
[25]
17
Biomedical applications
US 7943777 B2
17 May 2011
Fluorescent chemical compounds having high selectivity for double stranded DNA, and methods for their use
[26]
18
Biomedical applications
US 20110293529 A1 1 Dec 2011
Fluorescent nirf activatable probes for disease detection
[27]
19
Biomedical applications
EP 2399920 A1
28 Dec 2011
Fluorescent probe for use in measurement of protease
[28]
20
Biomedical applications
US 8158376 B2
17 Apr 2012
Bisubstrate fluorescent probe binding to protein kinases
[29]
21
Biomedical applications
US 20140057291 A1
27 Feb 2014
Bisubstrate fluorescent probes for protein kinase ck2
[30]
UDP glucuronosyl transferase: Uridine 5’-dephospho glucuronosyl transferase.
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Table 1. Patented fluorescent probes (cont.). Sr. no.
Patent category
Patent no. (assignee)
Publication date Title
Ref.
22
Biomedical applications
US 8206946 B2
26 Jun 2012
Fluorescent virus probes for identification of bacteria
[31]
23
Biomedical applications
US 8309319 B2
13 Nov 2012
Fluorescent probe for measurement of UDP glucuronosyl transferase
[32]
24
Biomedical applications
US 8329413 B2
11 Dec 2012
Glycoproteomic probes for fluorescent imaging of fucosylated glycans in vivo
[33]
25
Biomedical applications
US 20120329085 A1
27 Dec 2012
Fluorescent probes for reactive sulfur species
[34]
26
Biomedical applications
WO 2012159072 A3
11 Jul 2013
Fluorescent molecular probes for use in assays that measure test compound competitive binding with SAM-utilizing proteins
[35]
27
Preparation and biomedical applications
US 8569011 B2
29 Oct 2013
Fluorescent derivatives of polyamines, method for preparing same and applications thereof as diagnosis tools in the treatment of cancerous tumors
[36]
28
Biomedical applications
US 8647119 B1
11 Feb 2014
Methods and kits with fluorescent probes for caries detection
[37]
29
Biomedical applications
US 20140051863 A1
20 Feb 2014
Fluorescent probes for detection of copper
[38]
30
Biomedical applications
US 8431356 B2
30 Apr 2013
Fluorescence resonance energy transfer assays for sarco/endoplasmic reticulum calcium atpase and phospholamban
[39]
31
Biomedical applications
US 8465985 B2
30 Apr 2013
Fluorescent probe
[40]
UDP glucuronosyl transferase: Uridine 5’-dephospho glucuronosyl transferase.
its fluorescent property. However, the inventors of the patent [10] discovered that fluorescence was attributed to the tricyclic xanthene skeleton. In a systematic structure–activity relationship study, the carboxyl
group of the 2-carboxyphenyl moiety was substituted with an electron-donating substituent, such as methyl or methoxy. It was then observed that the fluorescent property was retained with the same intensity as that
O R2
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O
R3 R5
O
R4 N
R6 O
O 7
O
O 8
O
Figure 2. Modifications of fluorescein. (A) General structure of the structure–activity relationship modifications of fluorescein [10] ; (B) representative structure of the pro-fluorescent probes of the invention [11].
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Fluorescent probes for biomedical applications (2009–2014)
NH O
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NH HN O
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OH S
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N+
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OH
O
O NH
HO
O
HO O
HN
OH
HO O
OH 10
HO
OH
Figure 3. Macromolecular fluorescent probes. (A) Representative structure of the quenched fluorescent probes of the invention [13] ; (B) multivalent probe used for studying glucose transport described in patent application [14] .
of fluorescein with almost the same excitation wavelength. However, the absence of any substituents in that position decreases fluorescence due to the free
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rotation between the C–C single bond between the xanthene and the benzene ring. According to this invention, a series of fluorescent probes having the
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O
H N
O
Cl
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OH O
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OH
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Cl
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NH
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N CH3
O
12
CH3
H3C
N
N+
X
X 11
Figure 4. Polycyclic heteroaromatic fluorescent probes. (A) Representative cyanine dye containing reactive 2-cyanobenzothiazole moiety [15] ; (B) quinazoline fluorogenic probe described in the patent application [16].
general structure of 7 (Figure 2A) is created. The structure has R1 and R2 as H, or a substituent for trapping proton, a metal ion or an active oxygen species; R3 as a monovalent substituent; R4 and R5 as H or X and R6 as H, an alkyl carbonyl group or an alkyl carbonyl oxymethyl group. This series of probes is generated in such a way that the overall contribution of R1, R2 and R3 substitution provides the desired fluorescent property of the probe. This was a successful attempt to rationally design excellent fluorescent probes for industrial applications.
Another specific patent application [11] describes the discovery of fluorescent probes for reactive oxygen species (ROS). The methods of using profluorescent probes to detect analytes are described in the application. In living cells, H2O2 is the major source of ROS responsible for oxidative stress. The distribution of H2O2 in living cells can be mapped by fluorescent probes. The title compounds are selective and sensitive H2O2 chemosensors with properties amenable to biological imaging applications ( 8, Figure 2B) . The basis of this invention is the transformation of profluorescent
A N
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O
13
NH
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N O
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N
NH N R3
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15
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CH3
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N CH3 16
N
Figure 5. Small-molecule fluorescent probes. (A) benzoxazole-based fluorescent probe [17]; (B) two-photon fluorescent probe for dual-color imaging of Na + /Ca2+ activities described in the patent [18]; (C) fluorogenic probe for NO detection [19] and (D) fluorescent species generated from 15.
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Fluorescent probes for biomedical applications (2009–2014)
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O
A
H3C CH3
R1
20
19
Figure 6. Diverse small-molecule fluorogenic probes. (A) General structure of the fluorogenic 2’,7’-dialkylfluorescein derivatives [20] ; (B) general structure of the solvatochromic fluorophores described in [21] ; (C) general structure of a two-photon fluorescent probe described in [22] ; (D) representative photoactivatable fluorogen mentioned in [23] .
species into a fluorescent probe by chemoselective deprotection of the profluorescent species that can be utilized to observe and quantitate the presence and amount of an analyte of interest in an assay. Classical boronate oxidation by H2O2 is the mechanism of action that converts the profluorescent probe into fluorescein- or rhodamine-type dyes. The extensive methods of preparation of fluorescent probes are covered in patents [12] , [13] and [14] . The first patent [12] deals with synthetic bioprobes prepared from peptide template fluorescent nanoclusters. These nanoclusters, owing to their small sizes (less than 10 nm),
are more useful in intracellular probing and imaging than nanoparticles. The use of quenched fluorescent probes that exhibit fluorescence by biochemical processes is covered in patent [13] . The probe is designed in such a way that it has intramolecular quenching and under particular conditions, the quencher cleaves from the probe making it fluorescent (9, Figure 3A) . These probes are suitable for in vivo imaging. In yet another interesting patent application [14] , multivalent fluorescent probes for in vitro and in vivo imaging are described. These probes interact with multiple targets, intra- or extra- cellular, owing to the appropriate
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O
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Cl
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A
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Figure 7. Fluorescent/fluorogenic probes for varied biomedical applications. (A) Fluorogenic probe for detection of peroxynitrite in chemical and biological samples [24] ; (B) a DNA-intercalating fluorescent stain described in patent [26] ; (C) nanoparticle monomer in the probe described in patent [27] ; (D) fluorescent activatable dye discussed in [27] .
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H N
HN
X O
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O
OH H N
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OH
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O
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O
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CH3
O O-
N+
S O
27
Figure 8. Fluorescent probes for important macromolecular targets. (A) General structure of the modified xanthenes fluorescent probe for the detection of proteases [28] ; (B) a bisubstrate probe for kinase inhibition assays [29] ; (C) representative molecule discussed in patent application [30] for protein kinases CK2.
chemical moieties present. The fluorophore molecule is chemically attached to multiple (≥3) targeting structures, such as a small-molecule drugs, sugar, a polypeptide, a cytokine, a neurotransmitter and so on. One of the multivalent probes, 10 (Figure 3B) containing 2-deoxy-d-glucose moieties was used for in vivo imaging of cells/tissues with high rate of glucose uptake (normal and tumor cells) [14] . Novel and unique labeling probes of cyanine groups have been designed and patented for industrial applications of fluorescent probes. Considering the fact that certain carbocyanine dyes have the ability to self quench, modifications have been done in the carbocyanine structure to produce compounds that fluoresce substantially on proteins, nucleic acids or other biopolymers similar to that of cyanine dyes with immense photostability [15] . One such dye, 11 (Figure 4A), with reactive 2-cyanobenzothiazole moiety is shown for reference (where X = CH2, [CH2]2, [CH2]3, N, O, S). The reactive dye can be used for conjugation with a biomolecule from a biological sample, which can then be used for specific applications. Owing to the water solubility of reactive dyes, these probes are suitable for conjugation in aqueous media [15] . Another synthetic fluorogenic probe described in a patent application [16] contains quinazoline as the core structure 12 (Figure 4B). These probes exhibit great potential
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in the molecular imaging of enzyme activity in living cells. The patent describes the use of these probes for enzyme monoamine oxidase. The fluorogenic probe will undergo oxidative deamination by enzymatic reaction leading to fluorescence detection. The probes are druglike, cell-permeable and very stable in aqueous solutions. On completion of the enzymatic reaction, O-dealkylated products are found to be highly fluorescent as opposed to the non-fluorescent original compounds. Benzoxazole-based fluorescent probes for metal ions are described in a patent [17] . The preparation of such probes is particularly for the detection of Ca 2+ in biological systems. According to this patent application, structural modifications on the benzoxazole ring have been carried out to obtain desired fluorometric ratiometric indicators having high affinity towards pertinent metal ions (13, Figure 5A, where A: N [CH2COOCH3]2). Few of these compounds were used for flow cytometry studies involving intracellular calcium response, as described in [17] . Fluorescent probes invention has come a long way also as dual-color imaging probes. One such patent [18] provides the method of preparation of a two-photon fluorescent probe for simultaneous detection of Na+ and Ca2+ in living cells at different channels and wavelengths. The disclosures in this patent include small-molecule probes for staining Ca2+ near cell membrane, selectively
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Fluorescent probes for biomedical applications (2009–2014)
imaging the distribution of Ca2+ near cell membrane and a method for imaging Na+/Ca2+ exchange in living cells. Compound 14 (Figure 5B) represents a two-photon fluorescent probe described in the patent, where A = N (CH2COOH)2 and X = O, S or NH. Patent [19] claims fluorescent probes specifically designed for NO detection. The physiological and pathological significance of NO led to several investigations focused on developing probes for NO. Many of these probes were nonselective and/or difficult to synthesize. The inventors successfully generated compounds (15, Figure 5C) that distinguished NO in cellular systems. The mechanism of action of these fluorogenic probes is their reaction with NO under aerobic conditions that leads to nitrosation of the amine (16, Figure 5D) yielding a nitrosamine. Furthermore, electrophilic aromatic substitution that scavenges the nitrosamine extends conjugation in the product, which displays red-shift that is detectable by fluorescence. The advantages of the patented invention are noninterference with ROS, reactive nitrogen species, ascorbic acid, dehydroascorbic acid, facile synthesis, low pH dependence and fast reaction kinetics.
tions of the key features of fluorescent molecules. There are distinctive characteristics of certain probes that make it exclusive for industrial applications. This section covers, in particular, the biomedical applications of these probes. Patent [20] explores the utility of the fluorescent probes in biological media for intracellular assays. One critical aspect of such assays is the measurement of pH in cells, organelles or other biological fluids that play key role in the enzymatic activity, cell growth, calcium regulation and other processes. Fluorescent probes discussed in this application measures the pH level of cellular fluids with the derivatives of 2′, 7′-dialkylfluorescein that are fluorogenic in nature (17, Figure 6A) . Typically, the nonfluorescent dye was added to the sample, incubated for sufficient time to allow the cleavage of the nonfluorescent spirolactone form of the dye to the fluorescent form and then fluorescence is detected [20] . The detectable change in fluorescence of the compound was then correlated with sample pH. Such assays are highly suitable for automated high-throughput screening, particularly in cases where small sample volumes are desirable. In a similar patent application [21] , the use of compounds for monitoring biological interactions (protein folding, protein–protein interactions, phosphorylation events, ions, small molecules) continuously with
Patents covering biomedical applications of fluorescent probes The utility of fluorescent probes in varied areas of biological and material sciences is attributed to the func-
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Figure 9. Fluorescent/fluorogenic probes for imaging and other biomedical applications (A) One of the several fluorescein derivatives used for measurement of UDP-glucuronosyltransferase activity in patent [32] ; (B) clickactivated fluorogenic probe used for imaging fucosylated glycoconjugates [33] ; (C) representative fluorogenic probe discussed in patent application [34] for detection of H2S in biological systems; (D) representative fluorescent probe used for competitive-binding assays involving S-adenosylmethionine-binding proteins [35].
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N H N
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32
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CH3 N
CH3 S
S
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H3C
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CH3
CH3 33
Figure 10. Metal-binding fluorescent probes. (A) Representative polyamine fluorescent probe containing benzofurazan fluorophore described in patent [36] ; (B) copper sensor 788 (CS788), one of the binding-based fluorescent probes for copper described in patent [38] .
fluorescence readout is described. The spectroscopic behavior of compounds of this invention is dependent on the physicochemical properties of their surrounding environment. One such type is solvatochromic fluorophores, which are sensitive to the polarity of the local environment. These molecules exhibit low quantum yield in aqueous solution. When bound to hydrophobic sites in proteins and membranes or in nonpolar solvents, these dyes become highly fluorescent (18, Figure 6B) . The unique properties of these fluorophores can be used to elucidate the time course, nature and sequence of various cellular and subcellular processes. The effect of inhibitors and/or activators of different signaling pathways and cellular processes can also be studied using the compounds of this invention. Yet another patent [22] claimed to use a two-photon fluorescent probe to measure the pH in biological
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assays. This two-photon approach has the advantages of less scattering, deeper penetration in biological samples, less photodamage and photobleaching, as well as the unique properties of obtaining 3D resolution. This invention takes these advantages into consideration and provides water-soluble fluorene derivatives with pK a value ~ 7 (19, Figure 6C, where R1= Et and R2 = H2CH2COOH). The methods of syntheses of these series of compounds are provided and their utility as two-photon fluorescence pH indicators is evaluated. Their mechanism is due to the change in absorption and emission spectra of the molecule when it comes in contact with different pH. The pH dependence of the absorption spectra is attributed to the protonated form of the probe. Their pKa value makes them ideal for biological and biomedical imaging and diagnostic applications.
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In patent [23] , the use of fluorophores derived from photoactivatable azide-pi-acceptor fluorogens or from a thermal reaction of an azide-pi-acceptor fluorogen with an alkene or alkyne for labeling a biomolecule and subsequent imaging on a single-molecule level in living cells is described. An azide precursor fluorogen has much less fluorescence than the product (amine) fluorophore. Similarly, a thermochemical reaction of an azide with a terminal alkyne leads to formation of a 1, 2, 3-triazole.
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The fluorogen and/or the fluorescent probe can be tagged to any biomolecule using standard techniques. The subsequent photo- or thermal activation leads to visualization of the biomolecule containing these substances. An important example of a fluorogen of the mentioned invention, (E)-2-(4-(4-aminostyryl)-3-cyano-5, 5-dimethylfuran-2(5H)-ylidene) malononitrile, 20 is shown for reference (Figure 6D), where A = N3 and X = CN.
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N OO-
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34 H3C
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35
Figure 11. Fluorescent probes for biophysical/biochemical measurements (A) Representative chromophores used in FRET assays of SERCA described in patent [39] ; (B) a fluorescent probe for the measurement of pH described in patent [40] .
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Table 2. Summary of key features of fluorescent probes for biomedical applications. Compound no.
Biomedical applications
Key features and potential advantages of the probe
Ref.
8
Mapping of H2O2 in living cells
Mechanism of deprotection; chemoselective boronate deprotection
[11]
Advantages in vivo imaging: • Biologically compatible • Has a near unity quantum yield • A sizeable extinction coefficient • Visible excitation and emission profiles limit photodamage to biological sample • Avoid autofluorescence from native cellular species
9
10
Optical imaging agents suitable for in vivo imaging
Optical imaging agents suitable for in vitro and in vivo imaging
Polypeptide backbone cleaved off only when in contact with enzymes, resistant to fluorescence resonance energy transfer (FRET)
[13]
Advantages in vivo: • Once activated, the relatively high molecular weight fluorochromecontaining portion does not diffuse away readily • The probe remains in proximity to the target of interest, thus giving targetselective imaging
A single fluorochrome is coupled to multiple, spatially and chemically defined entities that are able to interact specifically with their putative targets (e.g., four or more targeting moieties per fluorochrome)
[14]
Advantages in vivo and in vitro: • The multivalency attributes to optimal binding affinity and minimizes steric hindrance • Allows the recording of multiple events or targets • Specificity over single or bivalent probes
11
Detection of proteins, nucleic acids and other biopolymers
Contains modified carbocyanine dyes and their conjugates. Substituted indolium ring system that contains a chemically reactive group or a conjugated substance
[15]
Advantages in vivo: • Greater photostability and/or higher absorbance (extinction coefficients) at the wavelength(s) of peak absorbance than such structurally similar dyes • Better spectral resolution at the far ends of the spectrum
12
13
Determination of presence, amount or activity of an enzyme in living cells
Detection of metal ions analytes
Consist of fluorogenic or fluorescent dyes coupled to a blocking group, cleavable by enzymes generating detectable fluorescence • Insoluble in water therefore not washed away easily at enzyme site and fluorescent only in a solid state at the enzyme activity sites • Rapidly release the fluorescent precipitate with minimum background • Highly photostable with large stokes shift (more than 100 nm), thus easily focused and distinguished from most cell and tissue autofluorescence • Less toxic and more cell permeable without damaging the cellular membrane
Ratiometric indicators froms chelate with metal ions, change in fluorescence intensity
[17]
Advantages in vitro:
Pharm Pat. Anal. (2014) 3(5)
Advantages in vivo:
• Longer excitation and emission wavelengths than many existing metal ion indicators and are fully functional in aqueous solutions, thus compatible with biological systems and assays • Requires no specialized (quartz) optics, such as are required by indicators that are excited or emit at shorter wavelengths • Suitable for use in fluorescence microscopy, flow cytometry, fluoroscopy or any other application that currently utilize fluorescent metal ion indicators
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[16]
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Fluorescent probes for biomedical applications (2009–2014)
Patent Review
Table 2. Summary of key features of fluorescent probes for biomedical applications (cont.). Compound no.
Biomedical applications
Key features and potential advantages of the probe
16
Detection of nitric oxide Advantages in vivo and in vitro: • Highly selective and have not been found to interfere with reactive
Ref. [19]
oxygenated species, reactive nitrogen species, ascorbic acid and dehydroascorbic acid • High specificity, facile synthesis, low pH dependence and fast reaction kinetics
17
19
pH indicators for intracellular assays
A neutral pH indicator
Advantages in vivo:
[20]
• Readily modifiable for applications in various cellular assays • Exhibit strong excitation ratiometric properties and possess ratioable emission spectra Two-photon water-soluble derivatives with pKa value near 7
[22]
Advantages in vivo and in vitro: • Less scattering and deeper penetration in biological samples by using Nearinfrared spectroscopy (NIR) excitation light • Less photodamage and photobleaching • 3D resolution
20
For biological studies
Fluorogens converted to fluorophores either by photoactivation or by a chemical reaction
[23]
Advantages in vivo: • Ability to actively control the fluorescence by photoactivation
25
Selective determination of proteases
Deacylation of acyl group to give fluorescence
[28]
Advantages in vivo and in vitro: • Achieves off/on control of fluorescence • Superior real-time response property • Suitabile for quantification.
27
Selective detection of protein kinases CK2
Combining two fragments targeted to the nucleotide-binding pocket and targeted to the peptide/protein binding site of PK
[30]
Advantages in vivo and in vitro: • Exact determination of binding constants for inhibitors with nanomolar and micromolar affinity in displacement experiments • Selective towards protein kinase CK2 • Affords discrimination of interactions of inhibitors with protein kinase CK2 from their interaction with other kinases
28
Measurement of UDP Substitution of 2-carboxy group on benzene ring of fluorescein increases glucuronosyl transferase uridine 5’- dephospho (UDP) reactivity Advantages in vivo and in vitro:
[32]
• High selectivity for UDP • High molar absorption coefficient
29
Fluorescence imaging of Presence of alkynyl group responsible for Cu (I)- catalyzed [3 + 2] fucosylated glycans azide–alkyne cycloaddition to detect cellular glycans bearing azide groups
[33]
Advantages in vivo: • Higher wavelength fluorescence, no background signals in biological systems
30
For reactive sulfer species
Reaction-based florescent probes for selective imaging of H2S Advantages in vivo and in vitro:
[34]
• Realtime detection • Tunability of colors provide multicolor array strips to rapid quantitation • Varying emission wavelengths, trapped intracellularly to provide signal enhancement
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Table 2. Summary of key features of fluorescent probes for biomedical applications (cont.). Compound no.
Biomedical applications
Key features and potential advantages of the probe
Ref.
33
For detection of copper metal ions
Forms a chelate with Cu metal ions thereby altering optical properties
[38]
Advantages in vivo: • Emits light in the NIR region • Circumvent issues of autofluorescence and signal attenuation by tissue
There are also patent applications that investigate the use of fluorescent probes in detection of specific analytes, such as certain biological species or metal ions in the biological system. In one such patent application, fluorogenic probes are used for measuring ROS, reactive nitrogen species, peroxynitrite or hypochlorite directly or indirectly from chemical and biological samples (cells, tissues in living organisms) [24] . These methods are then applied in a high-throughput manner for detecting or screening peroxynitrite or smallmolecules that can influence their levels in chemical and biological samples. One representative aromatic amine title compound 21 is shown in Figure 7A . Further, few of these compounds were tested in cellular assays for detection of peroxynitrite. As one would expect, these fluorogenic probes, after reacting with peroxynitrite, were found to be localized in the mitochondria, as seen from the green fluorescence of the active fluorophore [24] . In another invention based on fluorescent probes for detection of unbound metabolites in a fluid sample [25] , a method for high-throughput screening of fluorescent probes with diverse signaling properties is described. These probes (proteins tagged with a fluorophore) help in the determination of concentrations of unbound metabolites (e.g., unbound free fatty acids in plasma as a fatty acid profile for an individual). These profiles can be used to diagnose risk factors for diseases such as cardiac diseases, stroke, neurological diseases, such as dementia and Alzheimer’s disease, diabetes, inflammatory diseases and certain cancers. Fluorescent stains and dyes define yet another field in the fluorescent probes technology. The ability of stains to detect the presence or absence of a particular target in a sample makes it valuable in biomedical research for the qualitative or quantitative assays, depending on the compound, target and assay parameters. Dyes, on the other hand, display responses irrespective of the presence or absence of another material and, thus, are useful for labeling a target. A related patent [26] presents specific fluorescent stains that detect double-stranded DNA, thus surpassing the disadvantages of the common stains that are not selective for DNA. These are chemical compounds that fluoresce when they come specifically in contact with double-stranded DNA as
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opposed to RNA or single-stranded DNA. These compounds, which act as stains that illuminate with doublestranded DNA, possess DNA-intercalating moieties, such as quinoline (22, Figure 7B), where X is S, R1, R2, and R4 are H, R5, R6, R7, R8, R9, R10, and R11 independently comprise H, OR, SR, SAr, X, alkyl, alkenyl, alkynyl, aryl or combinations thereof and R12 is alkyl. The patent [27] describes a nanoparticle (as a carrier) with one cleavable spacer consisting of one fluorescence activation site and two types of dyes. The cleavable spacer is attached at one end to the nanoparticle. In the presence of an enzyme, the cleavable spacer gets cleaved emitting fluorescence that can be detected by a detector. A nanoparticle described in the invention comprises of a monomer represented by 23 ( Figure 7C, where n = 10 to 200). Furthermore, the invention also constitutes a fluorescent-activatable dye consisting of a water-dispersible enamine functionalized dye represented by 24 ( Figure 7D, where R1 and R2 are substituted alkyl and may form a ring, A is N–Ra, S, O, Ra–C–Rb, wherein Ra and Rb are substituted or unsubstituted alkyl groups capable of forming a ring, R3 is H, aryl, alkyl, alkoxy, or halogen, R4 is alkyl chain CH2 [n]-CH3 where n = 2 to 30). These probes are used for imaging biological processes and disease detection in vitro and in vivo [27] . Fluorescent probes for detection of a specific species in the cellular systems are obtained by selective modifications in the chemical structures of already known molecules to produce novel fluorescent probes. Therefore, when additions/changes in the structure of xanthenes are made, it produces chemical species that can be used particularly for detection of proteases (25, Figure 8A where R1 represents a hydrogen atom; R2, R3, R4, R5, R6 and R7 independently represent H, OH, halogen, an alkyl group; R8 and R9 represent H, or an alkyl group; X represents a C1–C3 alkylene group; and R10 represents an acyl group, or a salt thereof). The mechanism of fluorescence involves opening of the xanthene ring system by hydrolysis of acyl group causing illumination of the complex, thus quantitating presence of protease [28] . In an interesting patent [29] , utility of fluorescent probes for identification of protein kinase ligand is described. In addition, affinity of kinase inhibitors
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Fluorescent probes for biomedical applications (2009–2014)
and active concentration of kinases binding to the probe are also measured. The distinct feature of the probe is its bisubstrate-analog character, which enables simultaneous evaluation of Type I (ATP-competitive) kinase inhibitors as well as inhibitors targeting substrate protein/peptide-binding domain (26, Figure 8B, where X = [L-Arg]3). The fluorescent probe in this patent is employed for evaluation of inhibitory potency of compounds towards a great number of protein kinases. More precisely, targeting protein kinase CK2 is also covered in another invention that deals with fluorescent probes that selectively bind to CK2 [30] . The interaction of the probe with the binding sites of substrates of a catalytic subunit of CK2 is the characteristic feature of the invention. The representative compound 27 (Figure 8C) depicting the bisubstrate probe is shown for reference. Fluorescently labeled viruses can be used as molecular recognition tools for bacteria in fluid systems [31] . These fluorescently labeled virus probes are highly specific bacteriophages labeled with fluorescent reporters. Problems associated with the use of antibody-based biosensors and DNA probes, such as nonspecific binding and unreliable sensitivity, can be avoided with the method described in this invention. The bacteriophages can be labeled with a variety of fluorescence probes depending on the application. The virus probes are immobilized on optical substrates, such as polymer films. The fluid containing bacteria is then brought in contact with the optical substrate. The response time and selectivity with this method is extremely rapid when compared with conventional methods. Fluorescein, owing to its excellent fluorescent properties in water, has always been the preferred choice. In one instance, it was found that it lacked reactivity with UDP-glucuronosyltransferase (UGT), a Phase II conjugating enzyme. Systematic structure–activity relationship studies led to fluorescein derivatives with improved reactivity at UGT. Using these newly developed fluorescent probes, UGT activity measurement was performed [32] . The major structural modification was to replace the 2-COOH substituent with a monovalent group (H, alkyl, alkenyl, alkynyl, alkoxy, nitro, amino, cyano, alkoxycarbonyl, alkanoylamino, aryl, heteroaryl, aroylamino, heteroaroylamino or similar groups). One such compound 28 is shown in Figure 9A. The patent [33] discloses a novel method for labeling cellular azidoglycans using click chemistry. A nonfluorescent precursor 4-ethynyl-N-ethyl-1,8-naphthalimide (29, Figure 9B) reacts with fucose analogs bearing an azido group (incorporated in the glycoconjugates via fucose salvage pathway) generating a fluorescent probe, thereby allowing fluorescent visualization of the fucosylated cells by flow cytometry. The azido
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group and the terminal alkyne can be placed on either fucose or the fluorogen moiety; the other partner will have the corresponding alkyne or the azido group, respectively. Such fluorescent tagging is useful for elucidating the roles of fucosylated glycoconjugates in several cellular processes. Another patent application [34] deals with development of fluorescent probes for H2S, a member of a family of endogenously produced reactive sulfur species that is critical for several signaling pathways and intracellular redox state. Several reaction-based fluorescent probes for sensitive imaging of H2S in cellular systems are disclosed. Various structural modifications are made to generate molecules that cause illumination when H2S is detected (30, Figure 9C) . The azide group in 30, upon reaction with H2S, undergoes reduction, leading to generation of a fluorophore and a signal is detected. The advantages of these probes include spatiotemporal detection, selectivity for H2S over other reactive species, low toxicity and ease of handling. The tunability of fluorescent colors along with readability in the assay makes these probes an important invention to measure H2S levels in the biological systems. A patent application [35] discusses the development of a chemically stable S-adenosylmethionine (SAM) mimetic probe for a fluorescence polarization or TRFRET assay. SAM is a ubiquitous metabolic intermediate that is critical in many biochemical processes. SAMutilizing proteins catalyze important reactions, such as transfer of methyl group from SAM to a substrate for covalent modification. Assays involving displacement of a fluorescence-labeled ligand from SAM-utilizing protein by a test compound are desirable owing to several disadvantages of radioligand-binding assays. The components of fluorescent detection analyte are a fluorophore moiety, a covalent linker and a SAM-utilizing protein ligand moiety (31, Figure 9D) . In an attempt to develop novel fluorescent probes as diagnostic tools in cancer, the inventors of patent [36] exploited the polyamine transport system, which is critical for rapidly proliferating cells, such as cancerous cells. These cells are dependent on the exogenous supply of polyamines by active transport. In the absence of detailed molecular information on such a polyamine transporter, a probe targeting this system is of paramount importance. The patent uses benzoxadiazole or benzofurazan nucleus as the fluorophore attached to an endogenous polyamine, for example, spermine ( 32 , Figure 10A) . These probes are used for detecting tumors expressing the polyamine transport system and selecting patients carrying such tumors. Similarly, patent [37] involves the use of a fluorescent probe for detecting early dental caries in a patient. A variety of fluorescent probes including tetracycline,
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Patent Review Warrier & Kharkar Alexa Fluor 750, indocyanine green, doxorubicin and others are used. The molecule stains the caries in the enamel layer of a tooth, which emits fluorescence at a characteristic excitation wavelength. Overall, such a use of fluorescent probes is for preventive purpose. A recent patent application [38] discloses a method for detecting copper in cells and living animals using fluorescent probes such as derivatives of fluorescein, cyanine, rhodamine, Tokyo Green, BODIPY and rhodol. Given the essential role of copper in the body, such a molecular sensor for copper is highly desirable from many perspectives. In the absence of copper, these probes have little or no fluorescence; in the presence of copper, they fluoresce brightly. Some of these probes emit light in the near-IR region, making them ideal for in vivo imaging. Two types of probes – bindingbased and reaction-based – are described. The former is capable of forming a chelate with metal ions, thereby altering the optical properties (33, Figure 10B) ; the latter forms a chelate with the metal ion, which undergoes a subsequent bond cleavage reaction (responsible for altering the fluorescence behavior). Several of these probes form 1:1 chelates with Cu (I) selectively, are stable over physiological pH range and are able to detect changes in copper levels in living cells. Fluorescent technologies like fluorescence resonance energy transfer (FRET) have taken a significant leap in the biomedical applications field. One of the most recent patent applications [39] has described the use of FRET assays for the identification of molecules that modulate membrane proteins in muscle contraction. Sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA) is a membrane protein that terminates or prevents muscle contraction. It is known that SERCA inhibition increases the concentration of calcium in the cytosolic membrane, which eventually causes apoptosis. This observation was used in the design of drugs as anticancer agents. This patent application discusses the method by which it is identified if the molecule is capable of modulating SERCA. Two chromophores are labeled at both the sites of SERCA, wherein the first and the second chromophores are used for energy transfer. After exciting the second chromophore, FRET is measured between the chromophores. The difference between FRET in the presence of the test compound and FRET in the absence of the test compound indicates that the test compound modulates SERCA, such that the energy transfer between the two chromophores is altered [39] . One of the molecules described in the application for FRET assay is represented by 34 (Figure 11) . Another related patent [40] also describes the mechanism of FRET for wide applications in biological studies. The fluorescent probe designed in the patent
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application is used to induce substantial change in the efficiency of FRET between two fluorophores represented by fluorophore A and fluorophore B before and after a specific reaction with the substance to be measured. The change in the FRET efficiency resulting from contact with the substance to be measured is used to measure the concerned substance. One of the compounds in this patent application represented by 35 (Figure 11) is used for the detection of pH change in the biological microenvironment. For the ease of understanding, Table 2 summarizes the key features of the described fluorescent and/or fluorogenic probes with particular reference to their in vitro and in vivo biomedical applications. In addition, the advantages of these probes have also been outlined in Table 2. Future perspective The versatile nature of small-molecule fluorescent probes makes them useful in several areas of biomedical sciences. As seen from the trends in patent literature, researchers are trying to come out of the conventional approaches and related chemistry of the dyes involved. Newer chemical logic is put to work for detecting so-called ‘tough-nuts-to-crack’. Researchers are trying to substitute radioligand-binding assays with simple ‘fluorescent assays’ with the help of these wondrous molecules. More selective probes are sought after to increase the sensitivity of the measurements. Molecules that emit in the near-IR region are particularly useful for in vivo imaging. Such noninvasive techniques are the need of the hour. The applications of these probes and/or associated fluorophores have transcended into the material sciences area. Fluorescent probes cover a wide spectrum of applications ranging from detection of cytoskeletal proteins, organelles, lipids, proteins, nucleic acids, enzymes, antibodies, avidins and many others in the fields of cell biology, neurobiology, immunology, molecular biology and biophysics. It may very well be anticipated that future enterprises in the field of patents for fluorescent probes will constitute a mix of established classes having unique structural modifications with novel mechanisms. Financial & competing interests’ disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
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Patent Review
Executive summary Background • Design and development of small-molecule fluorescent probes for a variety of biological applications is an ever-expanding field of research. • Several mysteries in biological sciences were solved with the help of these ‘fluorescing wonders’.
Fluorescent probes: patent review • The patents/patent applications covering the field of small-molecule fluorescent probes disclose a wide variety of biomedical uses. • Patents reviewed from January 2009 to March 2014 comment on the industrial applicability of fluorescent probes that ranges from metal ion detection, pH indicator in intracellular assays, disease detection, detection of DNA and detection of reactive species among others.
Future panorama • The scientific community have been progressing significantly in extending and enhancing the quality of human life through the discovery, development and commercialization of medicines, diagnostics and medical devices. • Fluorescent probes are instrumental in the field of medicine because of their unique properties, ability to detect distinguished components of complex molecular structures in the biological systems, such as live cells, with very high sensitivity and selectivity and high temporal and spatial resolution, to detect multiple signals concomitantly, to track single molecules in vivo and to replace radioactive assays whenever possible. 10
Tetsuo Nagano, Daiichi Pure Chemicals Co., Ltd: US7524974 (2009).
11
The Regents of the University of California: WO2009152102 (2009).
12
Gao X: US20110118441 (2011).
13
Ge Healthcare As: EP-2389201-A2 (2011).
14
Caliper Life Sciences Inc.: WO2012040108 (2012).
15
Promega Corporation: US8309059 (2012).
16
Nanyang Technological University: US20120288884 (2012).
17
Life Technologies Corporation: US8540521 (2013).
Mastrogiacomo R, Lovinella I, Napolitano E. New fluorescent probes for ligand-binding assays of odorantbinding proteins. Biochem. Biophys. Res. Comm. 446(1), 137–142 (2014).
18
Ajou University Industry—Academic Cooperation Foundation: US8557811 (2013).
19
Board of Regents, The University Of Texas System: US8637323 (2014).
Shimomura O, Johnson F, Saiga Y. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J. Cell Comp. Physiol. 59(3), 223–239 (1962).
20
Mds Analytical Technologies (USA) Inc.: US7507395 (2009).
21
Massachusetts Institute of Technology: US20090082577 (2009).
5
Gonçalves MS. Fluorescent labelling of biomolecules with organic probes. Chem. Rev. 109(1), 190–212 (2009).
•
Covers the design of probes for biological studies having advantages in vivo.
•
Describes the design of scaffolds of fluorescent probes for labeling purposes.
22
University of Central Florida Research Foundation, Inc.: US8232303 (2012).
6
Han J, Burgess K. Fluorescent indicators for intracellular pH. Chem. Rev. 110(5), 2709–2728 (2010).
23
Kent State University, Leland Stanford Junior University: US8153446 (2012).
7
Cheki M1, Moslehi M, Assadi M. Marvelous applications of quantum dots. Eur. Rev. Med. Pharmacol. Sci. 17(9), 1141–1148 (2013).
24
Versitech Limited, Morningside Ventures Limited: WO2009121247 (2009).
25
Winterbourn CC. The challenges of using fluorescent probes to detect and quantify specific reactive oxygen species in living cells. Biochim. Biophys. Acta. 1840(2), 730–738 (2014).
Ffa Sciences Llc: US7601510 (2009).
8
26
Life Technologies Corporation: US7943777 (2011).
27
Ji T, Warren ML, William EM, Yong Y. US20110293529 (2011).
28
The University of Tokyo. EP-2399920-A1 (2011).
29
University Of Tartu. US8158376 (2012).
30
University Of Tartu. US20140057291 (2014).
References Papers of special note have been highlighted as: • of interest 1
2
• 3
4
9
Drummen GP. Fluorescent probes and fluorescence (microscopy) techniques – illuminating biological and biomedical research. Molecules 17(12), 14067–14090 (2012). Patsenker L, Tatarets A, Kolosova O et al. Fluorescent probes and labels for biomedical applications. Ann. NY Acad. Sci. 1130, 179–187 (2008). Exhaustive review covering wide applications of fluorescent probes in biomedical applications.
Zhang J, Campbell RE, Ting AY, Tsien RY. Creating new fluorescent probes for cell biology. Nat. Rev. Mol. Cell Biol. 3(12), 906–918 (2002).
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Describes the design of probes for selective determination of CK2 kinases as against a variety of kinases.
•
Describes a novel scaffold of polyamines for fluorescent detection of cancer.
31
Tabacco MB, Qian X, Russo J: US8206946 (2012).
37
32
The University Of Tokyo: US8309319 (2012).
President and Fellows of Harvard College: US8647119 (2014).
33
Academia Sinica: US8329413 (2012).
38
34
The Regents of the University Of California: US20120329085 (2012).
The Regents of the University Of California: US20140051863 (2014).
•
35
Cayman Chemical Company, Inc., The Regents Of The University Of Michigan: WO2012159072 (2013).
Covers the binding-based and reaction-based fluorescent probes for metal chelation.
39
Thomas DD, Cornea RL, Zsebo KM: US8431356 (2013).
Pierre Fabre Medicament: US8569011 (2013).
40
The University Of Tokyo: US8465985 (2013).
36
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Fluorescent probes for biomedical applications (2009–2014)
The discovery and subsequent development of fluorescent probes was one of the most exciting innovations in life sciences, which marked the beginning of interpretation of numerous biological phenomena. Today, fluorescent probes are used for a wide range of biomedical applications, such as pharmaceutical research, clinical diagnostics and high-throughput screening, to name a few. Despite the availability of a large number of these probes, efforts to invent newer versions utilizing novel chemistry to address limitations of the current approaches continue. This review article gives a rundown on ‘small-molecule fluorescent probes’ patents/patent applications from January 2009 to March 2014. The patent literature was classified based on ‘preparation’ and ‘biomedical applications’ of these ‘fluorescing wonders’.
Fluorescent probes are an indispensable part of chemical biology research [1] . Over several decades, the design and development of these probes for varied biological applications improved our understanding of biological systems [2,3] . The accidental discovery of mauveine, the first organic chemical dye, dates back to 1856. Adolph Von Baeyer, a German chemist, synthesized fluorescein (1, Figure 1) , one of the most widely used fluorophores today, in 1871. Over the next 30 years, a large number of these probes were synthesized and their fluorescent properties were documented. Paul Erlich, in 1882, used sodium salt of fluorescein to track aqueous humor secretion in the eye. This was the first time that fluorescein was used in vivo. Another derivative of fluorescein that is commonly used as a staining dye is eosin Y, a tetrabromo derivative of fluorescein ( 2, Figure 1) . Eosin Y is used commonly to stain muscle fibres, cytoplasm and collagen. Also, the use of rhodamine, particularly rhodamine B and rhodamine 123 ( 3, 4, Figure 1) , extensively in biotechnology applications, such as fluorescence microscopy, flow cytometry, fluorescence correlation spectroscopy
10.4155/PPA.14.41 © 2014 Future Science Ltd
Sona Warrier1 & Prashant S Kharkar*,1 Department of Pharmaceutical Chemistry, Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management (SPPSPTM), SVKM’s NMIMS, V. L. Mehta Road, Vile Parle (West), Mumbai 400-056, India *Author for correspondence: Tel.: +91 22 4233 2016 Fax: +91 22 2618 5422 [email protected] 1
and ELISA, has gained significance in the field of fluorescence. In 1941, Albert Coons labeled antibodies with fluorescein isothiocyanate, which marks the beginning of the immunofluorescence field. The discovery of green fluorescent protein in Aequorea victoria jellyfish in 1962 by Shimomura, Johnson and Saiga contributed to groundbreaking research in cell and molecular biology [4] . Similarly, the discovery of quantum dots, another type of fluorescent probes, in the 1980s and their subsequent applications in biological research has become another exciting area. Synthetic probes such as SYBR Green I and 3-cyano-7-hydroxy coumarin (5, 6, Figure 1) are some of the many dyes that are used as reference dyes for cell imaging purposes. Overall, fluorescent probes – small molecule organic dyes, biological fluorophores (e.g., green fluorescent protein) and quantum dots – played a critical role in the advancement of biological sciences. The scope of applications of these probes is ever expanding from bioimaging, pharmaceutical research and clinical diagnostics to various areas of material sciences. This review focuses on ‘biomedical applications of small-molecule fluorescent probes’.
Pharm Pat. Anal. (2014) 3(5), 543–560
part of
ISSN 1758-1869
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Patent Review Warrier & Kharkar
Key terms Fluorophore: Fluorescent organic chemical compound that can reemit light after excitation by light or other electromagnetic radiation. Fluorescent probe: Conjugated molecule, consisting of a fluorophore or fluorochrome group covalently linked to a group having specific interaction profile in an adequate biological environment. Fluorogenic probe: Chemical compound in which fluorescence can be activated by enzymatic or chemical reaction, light or environmental changes. Quenching: Loss of fluorescence signal due to short-range interactions between the fluorophore and the local molecular environment, including other fluorophores (self-quenching). Solvatochromic fluorophores: Fluorophores that possess emission properties that are sensitive to the nature of the local microenvironment.
A thorough analysis of patents related to the subject matter since January 2009 is included. Curious readers are advised to refer to excellent reviews available on the topic for in-depth information [5–9] . Figure 1 shows a few representative examples of widely used small molecule fluorophores. A comprehensive Patents Database search (31 March 2014) that includes Espacenet, European Patent Office, United States Patent and Trademark Office, World Intellectual Property Organisation and others using the keywords ‘fluorescent probes’, ‘fluorogenic probes’, ‘fluorophores’ from 2009 until March 2014 yielded a large number of patents/patent applications. Further refinement of hits using the terms ‘preparation’ and/or ‘biomedical applications’ led to 29 patents/patent applications (Table 1) .
Patents covering preparation of fluorescent probes The patent [10] discusses a fluorescent probe claiming to radiate fluorescence by entrapment of protons, metal ions or active oxygen species. The prior art section of the patent application included the review of fluorescein (1, Figure 1), a molecule known to emit fluorescence with λmax ~500 nm in aqueous solution. This molecule has, therefore, been used as a rudimentary substructure for fluorescent probes. It was also proven in the literature by earlier workers that the carboxyl group of fluorescein is responsible for
O
O
HO
Small-molecule fluorescent probes: patent review (January 2009–March 2014) The novelty of various fluorescent probes discovered over the years makes them patentable for a wide range of industrial applications, namely their use as metal ion sensors in cellular systems, in protein tagging, in livecell imaging to medical diagnostics. There are numerous patents published over several years related to the theme of this review. This confirms, beyond doubt, the fact that fluorescent probes and related technologies have progressed significantly and their utility is increasing continuously in biomedical sciences, almost like a necessity. The article is structured in such a way that it covers all patents published in the past 5 years (2009–2014). The patent applications in languages other than English were reviewed, but a detailed analysis was not possible for obvious reasons. For the ease of reading, this review is divided in two sections – preparation and biomedical applications of fluorescent probes.
O O-
Br
O
O
OH
N+ Br
O
O OH
OBr
Br
1
N
O
2
3
N
O
N
N+
O
S H2N
O
HO
O
NH+2
4
O
N N 5
6
Figure 1. Molecular structures of widely used fluorescent probes. Fluorescein (1), eosin Y (2), rhodamine B (3), rhodamine 123 (4), SYBR Green I (5), 3-cyano-7-hydroxy-coumarin (6).
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Fluorescent probes for biomedical applications (2009–2014)
Patent Review
Table 1. Patented fluorescent probes. Sr. no.
Patent category
Patent no. (assignee)
Publication date Title
Ref.
1
Preparation
US 7524974 B2
28 Apr 2009
Fluorescent probe
[10]
2
Preparation
WO 2009152102 A2
17 Dec 2009
Pro-fluorescent probes
[11]
3
Preparation
US 20110118441 A1 19 May 2011
Synthesis of highly fluorescent peptidemetallic nanoclusters as bio-probes
[12]
4
Preparation
EP 2389201 A2
30 Nov 2011
Fluorescent probes having a polymeric backbone
[13]
5
Preparation
WO 2012040108 A2
29 Mar 2012
Multivalent fluorescent probes
[14]
6
Preparation
US 8309059 B2
13 Nov 2012
Reactive cyanine compounds
[15]
7
Preparation
US 20120288884 A1
15 Nov 2012
Quinazolinone-based fluorogenic probes
[16]
8
Preparation
US 8540521 B2
24 Sep 2013
Benzoxazole-based fluorescent metal ion indicators
[17]
9
Preparation
US 8557811 B2
15 Oct 2013
Dual-color imaging method of sodium/ calcium activities using two-photon fluorescent probes and preparation method of two-photon fluorescent probes
[18]
10
Preparation
US 8637323 B2
28 Jan 2014
Fluorescent nitric oxide probes and associated methods
[19]
11
Biomedical applications
US 7507395 B2
24 Mar 2009
Methods of using fluorescent pH indicators for intracellular assays
[20]
12
Biomedical applications
US 20090082577 A1
26 Mar 2009
Fluorescent probes for biological studies
[21]
13
Preparation and biomedical applications
US 8232303 B1
31 Jul 2012
Two-photon absorbing water soluble fluorescent probe as a near neutral pH indicator
[22]
14
Biomedical applications
US 8153446 B2
10 Apr 2012
Fluorogenic compounds converted to fluorophores by photochemical or chemical means and their use in biological systems
[23]
15
Biomedical applications
WO 2009121247 A1 8 Oct 2009
Luminescence quenchers and fluorogenic probes for detection of reactive species
[24]
16
Preparation and biomedical applications
US 7601510 B2
13 Oct 2009
Development and use of fluorescent probes of unbound analyte
[25]
17
Biomedical applications
US 7943777 B2
17 May 2011
Fluorescent chemical compounds having high selectivity for double stranded DNA, and methods for their use
[26]
18
Biomedical applications
US 20110293529 A1 1 Dec 2011
Fluorescent nirf activatable probes for disease detection
[27]
19
Biomedical applications
EP 2399920 A1
28 Dec 2011
Fluorescent probe for use in measurement of protease
[28]
20
Biomedical applications
US 8158376 B2
17 Apr 2012
Bisubstrate fluorescent probe binding to protein kinases
[29]
21
Biomedical applications
US 20140057291 A1
27 Feb 2014
Bisubstrate fluorescent probes for protein kinase ck2
[30]
UDP glucuronosyl transferase: Uridine 5’-dephospho glucuronosyl transferase.
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Table 1. Patented fluorescent probes (cont.). Sr. no.
Patent category
Patent no. (assignee)
Publication date Title
Ref.
22
Biomedical applications
US 8206946 B2
26 Jun 2012
Fluorescent virus probes for identification of bacteria
[31]
23
Biomedical applications
US 8309319 B2
13 Nov 2012
Fluorescent probe for measurement of UDP glucuronosyl transferase
[32]
24
Biomedical applications
US 8329413 B2
11 Dec 2012
Glycoproteomic probes for fluorescent imaging of fucosylated glycans in vivo
[33]
25
Biomedical applications
US 20120329085 A1
27 Dec 2012
Fluorescent probes for reactive sulfur species
[34]
26
Biomedical applications
WO 2012159072 A3
11 Jul 2013
Fluorescent molecular probes for use in assays that measure test compound competitive binding with SAM-utilizing proteins
[35]
27
Preparation and biomedical applications
US 8569011 B2
29 Oct 2013
Fluorescent derivatives of polyamines, method for preparing same and applications thereof as diagnosis tools in the treatment of cancerous tumors
[36]
28
Biomedical applications
US 8647119 B1
11 Feb 2014
Methods and kits with fluorescent probes for caries detection
[37]
29
Biomedical applications
US 20140051863 A1
20 Feb 2014
Fluorescent probes for detection of copper
[38]
30
Biomedical applications
US 8431356 B2
30 Apr 2013
Fluorescence resonance energy transfer assays for sarco/endoplasmic reticulum calcium atpase and phospholamban
[39]
31
Biomedical applications
US 8465985 B2
30 Apr 2013
Fluorescent probe
[40]
UDP glucuronosyl transferase: Uridine 5’-dephospho glucuronosyl transferase.
its fluorescent property. However, the inventors of the patent [10] discovered that fluorescence was attributed to the tricyclic xanthene skeleton. In a systematic structure–activity relationship study, the carboxyl
group of the 2-carboxyphenyl moiety was substituted with an electron-donating substituent, such as methyl or methoxy. It was then observed that the fluorescent property was retained with the same intensity as that
O R2
R1
B
O
R3 R5
O
R4 N
R6 O
O 7
O
O 8
O
Figure 2. Modifications of fluorescein. (A) General structure of the structure–activity relationship modifications of fluorescein [10] ; (B) representative structure of the pro-fluorescent probes of the invention [11].
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Patent Review
Fluorescent probes for biomedical applications (2009–2014)
NH O
H2N
O
NH
N O
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HN
O NH HN O NH
NH HN O
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n HN
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OH
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NH
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HO
O
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OH S
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N+
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OH
O
O NH
HO
O
HO O
HN
OH
HO O
OH 10
HO
OH
Figure 3. Macromolecular fluorescent probes. (A) Representative structure of the quenched fluorescent probes of the invention [13] ; (B) multivalent probe used for studying glucose transport described in patent application [14] .
of fluorescein with almost the same excitation wavelength. However, the absence of any substituents in that position decreases fluorescence due to the free
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rotation between the C–C single bond between the xanthene and the benzene ring. According to this invention, a series of fluorescent probes having the
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Patent Review Warrier & Kharkar
O
H N
O
Cl
O O
N
OH O
H3C
OO
N
OH
O
S
O
S
Cl
N
S
S
NH
O
N CH3
O
12
CH3
H3C
N
N+
X
X 11
Figure 4. Polycyclic heteroaromatic fluorescent probes. (A) Representative cyanine dye containing reactive 2-cyanobenzothiazole moiety [15] ; (B) quinazoline fluorogenic probe described in the patent application [16].
general structure of 7 (Figure 2A) is created. The structure has R1 and R2 as H, or a substituent for trapping proton, a metal ion or an active oxygen species; R3 as a monovalent substituent; R4 and R5 as H or X and R6 as H, an alkyl carbonyl group or an alkyl carbonyl oxymethyl group. This series of probes is generated in such a way that the overall contribution of R1, R2 and R3 substitution provides the desired fluorescent property of the probe. This was a successful attempt to rationally design excellent fluorescent probes for industrial applications.
Another specific patent application [11] describes the discovery of fluorescent probes for reactive oxygen species (ROS). The methods of using profluorescent probes to detect analytes are described in the application. In living cells, H2O2 is the major source of ROS responsible for oxidative stress. The distribution of H2O2 in living cells can be mapped by fluorescent probes. The title compounds are selective and sensitive H2O2 chemosensors with properties amenable to biological imaging applications ( 8, Figure 2B) . The basis of this invention is the transformation of profluorescent
A N
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O
A
Ar
N X
O N O
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13
NH
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N O
A
14
R1
N
NH N R3
R2
15
R4
CH3
N
N CH3 16
N
Figure 5. Small-molecule fluorescent probes. (A) benzoxazole-based fluorescent probe [17]; (B) two-photon fluorescent probe for dual-color imaging of Na + /Ca2+ activities described in the patent [18]; (C) fluorogenic probe for NO detection [19] and (D) fluorescent species generated from 15.
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Fluorescent probes for biomedical applications (2009–2014)
O
R4
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O
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R1
R8 R2 R7
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O OH
N HN
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Patent Review
18 O 17
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X
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S
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O
A
H3C CH3
R1
20
19
Figure 6. Diverse small-molecule fluorogenic probes. (A) General structure of the fluorogenic 2’,7’-dialkylfluorescein derivatives [20] ; (B) general structure of the solvatochromic fluorophores described in [21] ; (C) general structure of a two-photon fluorescent probe described in [22] ; (D) representative photoactivatable fluorogen mentioned in [23] .
species into a fluorescent probe by chemoselective deprotection of the profluorescent species that can be utilized to observe and quantitate the presence and amount of an analyte of interest in an assay. Classical boronate oxidation by H2O2 is the mechanism of action that converts the profluorescent probe into fluorescein- or rhodamine-type dyes. The extensive methods of preparation of fluorescent probes are covered in patents [12] , [13] and [14] . The first patent [12] deals with synthetic bioprobes prepared from peptide template fluorescent nanoclusters. These nanoclusters, owing to their small sizes (less than 10 nm),
are more useful in intracellular probing and imaging than nanoparticles. The use of quenched fluorescent probes that exhibit fluorescence by biochemical processes is covered in patent [13] . The probe is designed in such a way that it has intramolecular quenching and under particular conditions, the quencher cleaves from the probe making it fluorescent (9, Figure 3A) . These probes are suitable for in vivo imaging. In yet another interesting patent application [14] , multivalent fluorescent probes for in vitro and in vivo imaging are described. These probes interact with multiple targets, intra- or extra- cellular, owing to the appropriate
O
R1
O
O
Cl HO
Cl
O
F
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N
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R11
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R9 R8
n
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CH3
21
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22
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CH3 O
H 2C
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R10 R12
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O n 23
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NH2
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A
R4
N R4
24
Figure 7. Fluorescent/fluorogenic probes for varied biomedical applications. (A) Fluorogenic probe for detection of peroxynitrite in chemical and biological samples [24] ; (B) a DNA-intercalating fluorescent stain described in patent [26] ; (C) nanoparticle monomer in the probe described in patent [27] ; (D) fluorescent activatable dye discussed in [27] .
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R1
H N
HN
X O
R7
R2
O
N R9
R4
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N H
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R6 R8
X NH2
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N OH
N
N
26
25
O HO S
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HN O
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N N
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OH O H N
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N H
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OH
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O OH
N H
N
H N
N H
O
O
OH H N
O
OH
N H
O
OH O
O
H3C
O
H3C
CH3 CH2
CH3
O O-
N+
S O
27
Figure 8. Fluorescent probes for important macromolecular targets. (A) General structure of the modified xanthenes fluorescent probe for the detection of proteases [28] ; (B) a bisubstrate probe for kinase inhibition assays [29] ; (C) representative molecule discussed in patent application [30] for protein kinases CK2.
chemical moieties present. The fluorophore molecule is chemically attached to multiple (≥3) targeting structures, such as a small-molecule drugs, sugar, a polypeptide, a cytokine, a neurotransmitter and so on. One of the multivalent probes, 10 (Figure 3B) containing 2-deoxy-d-glucose moieties was used for in vivo imaging of cells/tissues with high rate of glucose uptake (normal and tumor cells) [14] . Novel and unique labeling probes of cyanine groups have been designed and patented for industrial applications of fluorescent probes. Considering the fact that certain carbocyanine dyes have the ability to self quench, modifications have been done in the carbocyanine structure to produce compounds that fluoresce substantially on proteins, nucleic acids or other biopolymers similar to that of cyanine dyes with immense photostability [15] . One such dye, 11 (Figure 4A), with reactive 2-cyanobenzothiazole moiety is shown for reference (where X = CH2, [CH2]2, [CH2]3, N, O, S). The reactive dye can be used for conjugation with a biomolecule from a biological sample, which can then be used for specific applications. Owing to the water solubility of reactive dyes, these probes are suitable for conjugation in aqueous media [15] . Another synthetic fluorogenic probe described in a patent application [16] contains quinazoline as the core structure 12 (Figure 4B). These probes exhibit great potential
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Pharm Pat. Anal. (2014) 3(5)
in the molecular imaging of enzyme activity in living cells. The patent describes the use of these probes for enzyme monoamine oxidase. The fluorogenic probe will undergo oxidative deamination by enzymatic reaction leading to fluorescence detection. The probes are druglike, cell-permeable and very stable in aqueous solutions. On completion of the enzymatic reaction, O-dealkylated products are found to be highly fluorescent as opposed to the non-fluorescent original compounds. Benzoxazole-based fluorescent probes for metal ions are described in a patent [17] . The preparation of such probes is particularly for the detection of Ca 2+ in biological systems. According to this patent application, structural modifications on the benzoxazole ring have been carried out to obtain desired fluorometric ratiometric indicators having high affinity towards pertinent metal ions (13, Figure 5A, where A: N [CH2COOCH3]2). Few of these compounds were used for flow cytometry studies involving intracellular calcium response, as described in [17] . Fluorescent probes invention has come a long way also as dual-color imaging probes. One such patent [18] provides the method of preparation of a two-photon fluorescent probe for simultaneous detection of Na+ and Ca2+ in living cells at different channels and wavelengths. The disclosures in this patent include small-molecule probes for staining Ca2+ near cell membrane, selectively
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imaging the distribution of Ca2+ near cell membrane and a method for imaging Na+/Ca2+ exchange in living cells. Compound 14 (Figure 5B) represents a two-photon fluorescent probe described in the patent, where A = N (CH2COOH)2 and X = O, S or NH. Patent [19] claims fluorescent probes specifically designed for NO detection. The physiological and pathological significance of NO led to several investigations focused on developing probes for NO. Many of these probes were nonselective and/or difficult to synthesize. The inventors successfully generated compounds (15, Figure 5C) that distinguished NO in cellular systems. The mechanism of action of these fluorogenic probes is their reaction with NO under aerobic conditions that leads to nitrosation of the amine (16, Figure 5D) yielding a nitrosamine. Furthermore, electrophilic aromatic substitution that scavenges the nitrosamine extends conjugation in the product, which displays red-shift that is detectable by fluorescence. The advantages of the patented invention are noninterference with ROS, reactive nitrogen species, ascorbic acid, dehydroascorbic acid, facile synthesis, low pH dependence and fast reaction kinetics.
tions of the key features of fluorescent molecules. There are distinctive characteristics of certain probes that make it exclusive for industrial applications. This section covers, in particular, the biomedical applications of these probes. Patent [20] explores the utility of the fluorescent probes in biological media for intracellular assays. One critical aspect of such assays is the measurement of pH in cells, organelles or other biological fluids that play key role in the enzymatic activity, cell growth, calcium regulation and other processes. Fluorescent probes discussed in this application measures the pH level of cellular fluids with the derivatives of 2′, 7′-dialkylfluorescein that are fluorogenic in nature (17, Figure 6A) . Typically, the nonfluorescent dye was added to the sample, incubated for sufficient time to allow the cleavage of the nonfluorescent spirolactone form of the dye to the fluorescent form and then fluorescence is detected [20] . The detectable change in fluorescence of the compound was then correlated with sample pH. Such assays are highly suitable for automated high-throughput screening, particularly in cases where small sample volumes are desirable. In a similar patent application [21] , the use of compounds for monitoring biological interactions (protein folding, protein–protein interactions, phosphorylation events, ions, small molecules) continuously with
Patents covering biomedical applications of fluorescent probes The utility of fluorescent probes in varied areas of biological and material sciences is attributed to the func-
CH3
CH3
O
N
O
CH3
Patent Review
H N
O
H3C
O
N N+
O
N-
O
O
O
HO
O
O O
R1
28
R1 = N3
30
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29
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NH2
N
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NH2
N OH
O N N
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N+
N S H O O
O O N
N
31
Figure 9. Fluorescent/fluorogenic probes for imaging and other biomedical applications (A) One of the several fluorescein derivatives used for measurement of UDP-glucuronosyltransferase activity in patent [32] ; (B) clickactivated fluorogenic probe used for imaging fucosylated glycoconjugates [33] ; (C) representative fluorogenic probe discussed in patent application [34] for detection of H2S in biological systems; (D) representative fluorescent probe used for competitive-binding assays involving S-adenosylmethionine-binding proteins [35].
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N H N
H2N
H N
O N
N H
O S
CH3
O
32
H3C
CH3 N
CH3 S
S
S
S N
H3C
CH3
H3C
CH3
O N
N+ I-
CH3
CH3 33
Figure 10. Metal-binding fluorescent probes. (A) Representative polyamine fluorescent probe containing benzofurazan fluorophore described in patent [36] ; (B) copper sensor 788 (CS788), one of the binding-based fluorescent probes for copper described in patent [38] .
fluorescence readout is described. The spectroscopic behavior of compounds of this invention is dependent on the physicochemical properties of their surrounding environment. One such type is solvatochromic fluorophores, which are sensitive to the polarity of the local environment. These molecules exhibit low quantum yield in aqueous solution. When bound to hydrophobic sites in proteins and membranes or in nonpolar solvents, these dyes become highly fluorescent (18, Figure 6B) . The unique properties of these fluorophores can be used to elucidate the time course, nature and sequence of various cellular and subcellular processes. The effect of inhibitors and/or activators of different signaling pathways and cellular processes can also be studied using the compounds of this invention. Yet another patent [22] claimed to use a two-photon fluorescent probe to measure the pH in biological
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assays. This two-photon approach has the advantages of less scattering, deeper penetration in biological samples, less photodamage and photobleaching, as well as the unique properties of obtaining 3D resolution. This invention takes these advantages into consideration and provides water-soluble fluorene derivatives with pK a value ~ 7 (19, Figure 6C, where R1= Et and R2 = H2CH2COOH). The methods of syntheses of these series of compounds are provided and their utility as two-photon fluorescence pH indicators is evaluated. Their mechanism is due to the change in absorption and emission spectra of the molecule when it comes in contact with different pH. The pH dependence of the absorption spectra is attributed to the protonated form of the probe. Their pKa value makes them ideal for biological and biomedical imaging and diagnostic applications.
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In patent [23] , the use of fluorophores derived from photoactivatable azide-pi-acceptor fluorogens or from a thermal reaction of an azide-pi-acceptor fluorogen with an alkene or alkyne for labeling a biomolecule and subsequent imaging on a single-molecule level in living cells is described. An azide precursor fluorogen has much less fluorescence than the product (amine) fluorophore. Similarly, a thermochemical reaction of an azide with a terminal alkyne leads to formation of a 1, 2, 3-triazole.
Patent Review
The fluorogen and/or the fluorescent probe can be tagged to any biomolecule using standard techniques. The subsequent photo- or thermal activation leads to visualization of the biomolecule containing these substances. An important example of a fluorogen of the mentioned invention, (E)-2-(4-(4-aminostyryl)-3-cyano-5, 5-dimethylfuran-2(5H)-ylidene) malononitrile, 20 is shown for reference (Figure 6D), where A = N3 and X = CN.
H2N
N
N OO-
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P
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P
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34 H3C
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35
Figure 11. Fluorescent probes for biophysical/biochemical measurements (A) Representative chromophores used in FRET assays of SERCA described in patent [39] ; (B) a fluorescent probe for the measurement of pH described in patent [40] .
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Table 2. Summary of key features of fluorescent probes for biomedical applications. Compound no.
Biomedical applications
Key features and potential advantages of the probe
Ref.
8
Mapping of H2O2 in living cells
Mechanism of deprotection; chemoselective boronate deprotection
[11]
Advantages in vivo imaging: • Biologically compatible • Has a near unity quantum yield • A sizeable extinction coefficient • Visible excitation and emission profiles limit photodamage to biological sample • Avoid autofluorescence from native cellular species
9
10
Optical imaging agents suitable for in vivo imaging
Optical imaging agents suitable for in vitro and in vivo imaging
Polypeptide backbone cleaved off only when in contact with enzymes, resistant to fluorescence resonance energy transfer (FRET)
[13]
Advantages in vivo: • Once activated, the relatively high molecular weight fluorochromecontaining portion does not diffuse away readily • The probe remains in proximity to the target of interest, thus giving targetselective imaging
A single fluorochrome is coupled to multiple, spatially and chemically defined entities that are able to interact specifically with their putative targets (e.g., four or more targeting moieties per fluorochrome)
[14]
Advantages in vivo and in vitro: • The multivalency attributes to optimal binding affinity and minimizes steric hindrance • Allows the recording of multiple events or targets • Specificity over single or bivalent probes
11
Detection of proteins, nucleic acids and other biopolymers
Contains modified carbocyanine dyes and their conjugates. Substituted indolium ring system that contains a chemically reactive group or a conjugated substance
[15]
Advantages in vivo: • Greater photostability and/or higher absorbance (extinction coefficients) at the wavelength(s) of peak absorbance than such structurally similar dyes • Better spectral resolution at the far ends of the spectrum
12
13
Determination of presence, amount or activity of an enzyme in living cells
Detection of metal ions analytes
Consist of fluorogenic or fluorescent dyes coupled to a blocking group, cleavable by enzymes generating detectable fluorescence • Insoluble in water therefore not washed away easily at enzyme site and fluorescent only in a solid state at the enzyme activity sites • Rapidly release the fluorescent precipitate with minimum background • Highly photostable with large stokes shift (more than 100 nm), thus easily focused and distinguished from most cell and tissue autofluorescence • Less toxic and more cell permeable without damaging the cellular membrane
Ratiometric indicators froms chelate with metal ions, change in fluorescence intensity
[17]
Advantages in vitro:
Pharm Pat. Anal. (2014) 3(5)
Advantages in vivo:
• Longer excitation and emission wavelengths than many existing metal ion indicators and are fully functional in aqueous solutions, thus compatible with biological systems and assays • Requires no specialized (quartz) optics, such as are required by indicators that are excited or emit at shorter wavelengths • Suitable for use in fluorescence microscopy, flow cytometry, fluoroscopy or any other application that currently utilize fluorescent metal ion indicators
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[16]
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Fluorescent probes for biomedical applications (2009–2014)
Patent Review
Table 2. Summary of key features of fluorescent probes for biomedical applications (cont.). Compound no.
Biomedical applications
Key features and potential advantages of the probe
16
Detection of nitric oxide Advantages in vivo and in vitro: • Highly selective and have not been found to interfere with reactive
Ref. [19]
oxygenated species, reactive nitrogen species, ascorbic acid and dehydroascorbic acid • High specificity, facile synthesis, low pH dependence and fast reaction kinetics
17
19
pH indicators for intracellular assays
A neutral pH indicator
Advantages in vivo:
[20]
• Readily modifiable for applications in various cellular assays • Exhibit strong excitation ratiometric properties and possess ratioable emission spectra Two-photon water-soluble derivatives with pKa value near 7
[22]
Advantages in vivo and in vitro: • Less scattering and deeper penetration in biological samples by using Nearinfrared spectroscopy (NIR) excitation light • Less photodamage and photobleaching • 3D resolution
20
For biological studies
Fluorogens converted to fluorophores either by photoactivation or by a chemical reaction
[23]
Advantages in vivo: • Ability to actively control the fluorescence by photoactivation
25
Selective determination of proteases
Deacylation of acyl group to give fluorescence
[28]
Advantages in vivo and in vitro: • Achieves off/on control of fluorescence • Superior real-time response property • Suitabile for quantification.
27
Selective detection of protein kinases CK2
Combining two fragments targeted to the nucleotide-binding pocket and targeted to the peptide/protein binding site of PK
[30]
Advantages in vivo and in vitro: • Exact determination of binding constants for inhibitors with nanomolar and micromolar affinity in displacement experiments • Selective towards protein kinase CK2 • Affords discrimination of interactions of inhibitors with protein kinase CK2 from their interaction with other kinases
28
Measurement of UDP Substitution of 2-carboxy group on benzene ring of fluorescein increases glucuronosyl transferase uridine 5’- dephospho (UDP) reactivity Advantages in vivo and in vitro:
[32]
• High selectivity for UDP • High molar absorption coefficient
29
Fluorescence imaging of Presence of alkynyl group responsible for Cu (I)- catalyzed [3 + 2] fucosylated glycans azide–alkyne cycloaddition to detect cellular glycans bearing azide groups
[33]
Advantages in vivo: • Higher wavelength fluorescence, no background signals in biological systems
30
For reactive sulfer species
Reaction-based florescent probes for selective imaging of H2S Advantages in vivo and in vitro:
[34]
• Realtime detection • Tunability of colors provide multicolor array strips to rapid quantitation • Varying emission wavelengths, trapped intracellularly to provide signal enhancement
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Patent Review Warrier & Kharkar
Table 2. Summary of key features of fluorescent probes for biomedical applications (cont.). Compound no.
Biomedical applications
Key features and potential advantages of the probe
Ref.
33
For detection of copper metal ions
Forms a chelate with Cu metal ions thereby altering optical properties
[38]
Advantages in vivo: • Emits light in the NIR region • Circumvent issues of autofluorescence and signal attenuation by tissue
There are also patent applications that investigate the use of fluorescent probes in detection of specific analytes, such as certain biological species or metal ions in the biological system. In one such patent application, fluorogenic probes are used for measuring ROS, reactive nitrogen species, peroxynitrite or hypochlorite directly or indirectly from chemical and biological samples (cells, tissues in living organisms) [24] . These methods are then applied in a high-throughput manner for detecting or screening peroxynitrite or smallmolecules that can influence their levels in chemical and biological samples. One representative aromatic amine title compound 21 is shown in Figure 7A . Further, few of these compounds were tested in cellular assays for detection of peroxynitrite. As one would expect, these fluorogenic probes, after reacting with peroxynitrite, were found to be localized in the mitochondria, as seen from the green fluorescence of the active fluorophore [24] . In another invention based on fluorescent probes for detection of unbound metabolites in a fluid sample [25] , a method for high-throughput screening of fluorescent probes with diverse signaling properties is described. These probes (proteins tagged with a fluorophore) help in the determination of concentrations of unbound metabolites (e.g., unbound free fatty acids in plasma as a fatty acid profile for an individual). These profiles can be used to diagnose risk factors for diseases such as cardiac diseases, stroke, neurological diseases, such as dementia and Alzheimer’s disease, diabetes, inflammatory diseases and certain cancers. Fluorescent stains and dyes define yet another field in the fluorescent probes technology. The ability of stains to detect the presence or absence of a particular target in a sample makes it valuable in biomedical research for the qualitative or quantitative assays, depending on the compound, target and assay parameters. Dyes, on the other hand, display responses irrespective of the presence or absence of another material and, thus, are useful for labeling a target. A related patent [26] presents specific fluorescent stains that detect double-stranded DNA, thus surpassing the disadvantages of the common stains that are not selective for DNA. These are chemical compounds that fluoresce when they come specifically in contact with double-stranded DNA as
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opposed to RNA or single-stranded DNA. These compounds, which act as stains that illuminate with doublestranded DNA, possess DNA-intercalating moieties, such as quinoline (22, Figure 7B), where X is S, R1, R2, and R4 are H, R5, R6, R7, R8, R9, R10, and R11 independently comprise H, OR, SR, SAr, X, alkyl, alkenyl, alkynyl, aryl or combinations thereof and R12 is alkyl. The patent [27] describes a nanoparticle (as a carrier) with one cleavable spacer consisting of one fluorescence activation site and two types of dyes. The cleavable spacer is attached at one end to the nanoparticle. In the presence of an enzyme, the cleavable spacer gets cleaved emitting fluorescence that can be detected by a detector. A nanoparticle described in the invention comprises of a monomer represented by 23 ( Figure 7C, where n = 10 to 200). Furthermore, the invention also constitutes a fluorescent-activatable dye consisting of a water-dispersible enamine functionalized dye represented by 24 ( Figure 7D, where R1 and R2 are substituted alkyl and may form a ring, A is N–Ra, S, O, Ra–C–Rb, wherein Ra and Rb are substituted or unsubstituted alkyl groups capable of forming a ring, R3 is H, aryl, alkyl, alkoxy, or halogen, R4 is alkyl chain CH2 [n]-CH3 where n = 2 to 30). These probes are used for imaging biological processes and disease detection in vitro and in vivo [27] . Fluorescent probes for detection of a specific species in the cellular systems are obtained by selective modifications in the chemical structures of already known molecules to produce novel fluorescent probes. Therefore, when additions/changes in the structure of xanthenes are made, it produces chemical species that can be used particularly for detection of proteases (25, Figure 8A where R1 represents a hydrogen atom; R2, R3, R4, R5, R6 and R7 independently represent H, OH, halogen, an alkyl group; R8 and R9 represent H, or an alkyl group; X represents a C1–C3 alkylene group; and R10 represents an acyl group, or a salt thereof). The mechanism of fluorescence involves opening of the xanthene ring system by hydrolysis of acyl group causing illumination of the complex, thus quantitating presence of protease [28] . In an interesting patent [29] , utility of fluorescent probes for identification of protein kinase ligand is described. In addition, affinity of kinase inhibitors
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Fluorescent probes for biomedical applications (2009–2014)
and active concentration of kinases binding to the probe are also measured. The distinct feature of the probe is its bisubstrate-analog character, which enables simultaneous evaluation of Type I (ATP-competitive) kinase inhibitors as well as inhibitors targeting substrate protein/peptide-binding domain (26, Figure 8B, where X = [L-Arg]3). The fluorescent probe in this patent is employed for evaluation of inhibitory potency of compounds towards a great number of protein kinases. More precisely, targeting protein kinase CK2 is also covered in another invention that deals with fluorescent probes that selectively bind to CK2 [30] . The interaction of the probe with the binding sites of substrates of a catalytic subunit of CK2 is the characteristic feature of the invention. The representative compound 27 (Figure 8C) depicting the bisubstrate probe is shown for reference. Fluorescently labeled viruses can be used as molecular recognition tools for bacteria in fluid systems [31] . These fluorescently labeled virus probes are highly specific bacteriophages labeled with fluorescent reporters. Problems associated with the use of antibody-based biosensors and DNA probes, such as nonspecific binding and unreliable sensitivity, can be avoided with the method described in this invention. The bacteriophages can be labeled with a variety of fluorescence probes depending on the application. The virus probes are immobilized on optical substrates, such as polymer films. The fluid containing bacteria is then brought in contact with the optical substrate. The response time and selectivity with this method is extremely rapid when compared with conventional methods. Fluorescein, owing to its excellent fluorescent properties in water, has always been the preferred choice. In one instance, it was found that it lacked reactivity with UDP-glucuronosyltransferase (UGT), a Phase II conjugating enzyme. Systematic structure–activity relationship studies led to fluorescein derivatives with improved reactivity at UGT. Using these newly developed fluorescent probes, UGT activity measurement was performed [32] . The major structural modification was to replace the 2-COOH substituent with a monovalent group (H, alkyl, alkenyl, alkynyl, alkoxy, nitro, amino, cyano, alkoxycarbonyl, alkanoylamino, aryl, heteroaryl, aroylamino, heteroaroylamino or similar groups). One such compound 28 is shown in Figure 9A. The patent [33] discloses a novel method for labeling cellular azidoglycans using click chemistry. A nonfluorescent precursor 4-ethynyl-N-ethyl-1,8-naphthalimide (29, Figure 9B) reacts with fucose analogs bearing an azido group (incorporated in the glycoconjugates via fucose salvage pathway) generating a fluorescent probe, thereby allowing fluorescent visualization of the fucosylated cells by flow cytometry. The azido
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group and the terminal alkyne can be placed on either fucose or the fluorogen moiety; the other partner will have the corresponding alkyne or the azido group, respectively. Such fluorescent tagging is useful for elucidating the roles of fucosylated glycoconjugates in several cellular processes. Another patent application [34] deals with development of fluorescent probes for H2S, a member of a family of endogenously produced reactive sulfur species that is critical for several signaling pathways and intracellular redox state. Several reaction-based fluorescent probes for sensitive imaging of H2S in cellular systems are disclosed. Various structural modifications are made to generate molecules that cause illumination when H2S is detected (30, Figure 9C) . The azide group in 30, upon reaction with H2S, undergoes reduction, leading to generation of a fluorophore and a signal is detected. The advantages of these probes include spatiotemporal detection, selectivity for H2S over other reactive species, low toxicity and ease of handling. The tunability of fluorescent colors along with readability in the assay makes these probes an important invention to measure H2S levels in the biological systems. A patent application [35] discusses the development of a chemically stable S-adenosylmethionine (SAM) mimetic probe for a fluorescence polarization or TRFRET assay. SAM is a ubiquitous metabolic intermediate that is critical in many biochemical processes. SAMutilizing proteins catalyze important reactions, such as transfer of methyl group from SAM to a substrate for covalent modification. Assays involving displacement of a fluorescence-labeled ligand from SAM-utilizing protein by a test compound are desirable owing to several disadvantages of radioligand-binding assays. The components of fluorescent detection analyte are a fluorophore moiety, a covalent linker and a SAM-utilizing protein ligand moiety (31, Figure 9D) . In an attempt to develop novel fluorescent probes as diagnostic tools in cancer, the inventors of patent [36] exploited the polyamine transport system, which is critical for rapidly proliferating cells, such as cancerous cells. These cells are dependent on the exogenous supply of polyamines by active transport. In the absence of detailed molecular information on such a polyamine transporter, a probe targeting this system is of paramount importance. The patent uses benzoxadiazole or benzofurazan nucleus as the fluorophore attached to an endogenous polyamine, for example, spermine ( 32 , Figure 10A) . These probes are used for detecting tumors expressing the polyamine transport system and selecting patients carrying such tumors. Similarly, patent [37] involves the use of a fluorescent probe for detecting early dental caries in a patient. A variety of fluorescent probes including tetracycline,
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Patent Review Warrier & Kharkar Alexa Fluor 750, indocyanine green, doxorubicin and others are used. The molecule stains the caries in the enamel layer of a tooth, which emits fluorescence at a characteristic excitation wavelength. Overall, such a use of fluorescent probes is for preventive purpose. A recent patent application [38] discloses a method for detecting copper in cells and living animals using fluorescent probes such as derivatives of fluorescein, cyanine, rhodamine, Tokyo Green, BODIPY and rhodol. Given the essential role of copper in the body, such a molecular sensor for copper is highly desirable from many perspectives. In the absence of copper, these probes have little or no fluorescence; in the presence of copper, they fluoresce brightly. Some of these probes emit light in the near-IR region, making them ideal for in vivo imaging. Two types of probes – bindingbased and reaction-based – are described. The former is capable of forming a chelate with metal ions, thereby altering the optical properties (33, Figure 10B) ; the latter forms a chelate with the metal ion, which undergoes a subsequent bond cleavage reaction (responsible for altering the fluorescence behavior). Several of these probes form 1:1 chelates with Cu (I) selectively, are stable over physiological pH range and are able to detect changes in copper levels in living cells. Fluorescent technologies like fluorescence resonance energy transfer (FRET) have taken a significant leap in the biomedical applications field. One of the most recent patent applications [39] has described the use of FRET assays for the identification of molecules that modulate membrane proteins in muscle contraction. Sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA) is a membrane protein that terminates or prevents muscle contraction. It is known that SERCA inhibition increases the concentration of calcium in the cytosolic membrane, which eventually causes apoptosis. This observation was used in the design of drugs as anticancer agents. This patent application discusses the method by which it is identified if the molecule is capable of modulating SERCA. Two chromophores are labeled at both the sites of SERCA, wherein the first and the second chromophores are used for energy transfer. After exciting the second chromophore, FRET is measured between the chromophores. The difference between FRET in the presence of the test compound and FRET in the absence of the test compound indicates that the test compound modulates SERCA, such that the energy transfer between the two chromophores is altered [39] . One of the molecules described in the application for FRET assay is represented by 34 (Figure 11) . Another related patent [40] also describes the mechanism of FRET for wide applications in biological studies. The fluorescent probe designed in the patent
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application is used to induce substantial change in the efficiency of FRET between two fluorophores represented by fluorophore A and fluorophore B before and after a specific reaction with the substance to be measured. The change in the FRET efficiency resulting from contact with the substance to be measured is used to measure the concerned substance. One of the compounds in this patent application represented by 35 (Figure 11) is used for the detection of pH change in the biological microenvironment. For the ease of understanding, Table 2 summarizes the key features of the described fluorescent and/or fluorogenic probes with particular reference to their in vitro and in vivo biomedical applications. In addition, the advantages of these probes have also been outlined in Table 2. Future perspective The versatile nature of small-molecule fluorescent probes makes them useful in several areas of biomedical sciences. As seen from the trends in patent literature, researchers are trying to come out of the conventional approaches and related chemistry of the dyes involved. Newer chemical logic is put to work for detecting so-called ‘tough-nuts-to-crack’. Researchers are trying to substitute radioligand-binding assays with simple ‘fluorescent assays’ with the help of these wondrous molecules. More selective probes are sought after to increase the sensitivity of the measurements. Molecules that emit in the near-IR region are particularly useful for in vivo imaging. Such noninvasive techniques are the need of the hour. The applications of these probes and/or associated fluorophores have transcended into the material sciences area. Fluorescent probes cover a wide spectrum of applications ranging from detection of cytoskeletal proteins, organelles, lipids, proteins, nucleic acids, enzymes, antibodies, avidins and many others in the fields of cell biology, neurobiology, immunology, molecular biology and biophysics. It may very well be anticipated that future enterprises in the field of patents for fluorescent probes will constitute a mix of established classes having unique structural modifications with novel mechanisms. Financial & competing interests’ disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
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Fluorescent probes for biomedical applications (2009–2014)
Patent Review
Executive summary Background • Design and development of small-molecule fluorescent probes for a variety of biological applications is an ever-expanding field of research. • Several mysteries in biological sciences were solved with the help of these ‘fluorescing wonders’.
Fluorescent probes: patent review • The patents/patent applications covering the field of small-molecule fluorescent probes disclose a wide variety of biomedical uses. • Patents reviewed from January 2009 to March 2014 comment on the industrial applicability of fluorescent probes that ranges from metal ion detection, pH indicator in intracellular assays, disease detection, detection of DNA and detection of reactive species among others.
Future panorama • The scientific community have been progressing significantly in extending and enhancing the quality of human life through the discovery, development and commercialization of medicines, diagnostics and medical devices. • Fluorescent probes are instrumental in the field of medicine because of their unique properties, ability to detect distinguished components of complex molecular structures in the biological systems, such as live cells, with very high sensitivity and selectivity and high temporal and spatial resolution, to detect multiple signals concomitantly, to track single molecules in vivo and to replace radioactive assays whenever possible. 10
Tetsuo Nagano, Daiichi Pure Chemicals Co., Ltd: US7524974 (2009).
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The Regents of the University of California: WO2009152102 (2009).
12
Gao X: US20110118441 (2011).
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Ge Healthcare As: EP-2389201-A2 (2011).
14
Caliper Life Sciences Inc.: WO2012040108 (2012).
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Promega Corporation: US8309059 (2012).
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Nanyang Technological University: US20120288884 (2012).
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Life Technologies Corporation: US8540521 (2013).
Mastrogiacomo R, Lovinella I, Napolitano E. New fluorescent probes for ligand-binding assays of odorantbinding proteins. Biochem. Biophys. Res. Comm. 446(1), 137–142 (2014).
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Ajou University Industry—Academic Cooperation Foundation: US8557811 (2013).
19
Board of Regents, The University Of Texas System: US8637323 (2014).
Shimomura O, Johnson F, Saiga Y. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J. Cell Comp. Physiol. 59(3), 223–239 (1962).
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Mds Analytical Technologies (USA) Inc.: US7507395 (2009).
21
Massachusetts Institute of Technology: US20090082577 (2009).
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Gonçalves MS. Fluorescent labelling of biomolecules with organic probes. Chem. Rev. 109(1), 190–212 (2009).
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Covers the design of probes for biological studies having advantages in vivo.
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Describes the design of scaffolds of fluorescent probes for labeling purposes.
22
University of Central Florida Research Foundation, Inc.: US8232303 (2012).
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Han J, Burgess K. Fluorescent indicators for intracellular pH. Chem. Rev. 110(5), 2709–2728 (2010).
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Kent State University, Leland Stanford Junior University: US8153446 (2012).
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Cheki M1, Moslehi M, Assadi M. Marvelous applications of quantum dots. Eur. Rev. Med. Pharmacol. Sci. 17(9), 1141–1148 (2013).
24
Versitech Limited, Morningside Ventures Limited: WO2009121247 (2009).
25
Winterbourn CC. The challenges of using fluorescent probes to detect and quantify specific reactive oxygen species in living cells. Biochim. Biophys. Acta. 1840(2), 730–738 (2014).
Ffa Sciences Llc: US7601510 (2009).
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26
Life Technologies Corporation: US7943777 (2011).
27
Ji T, Warren ML, William EM, Yong Y. US20110293529 (2011).
28
The University of Tokyo. EP-2399920-A1 (2011).
29
University Of Tartu. US8158376 (2012).
30
University Of Tartu. US20140057291 (2014).
References Papers of special note have been highlighted as: • of interest 1
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Drummen GP. Fluorescent probes and fluorescence (microscopy) techniques – illuminating biological and biomedical research. Molecules 17(12), 14067–14090 (2012). Patsenker L, Tatarets A, Kolosova O et al. Fluorescent probes and labels for biomedical applications. Ann. NY Acad. Sci. 1130, 179–187 (2008). Exhaustive review covering wide applications of fluorescent probes in biomedical applications.
Zhang J, Campbell RE, Ting AY, Tsien RY. Creating new fluorescent probes for cell biology. Nat. Rev. Mol. Cell Biol. 3(12), 906–918 (2002).
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Patent Review Warrier & Kharkar •
Describes the design of probes for selective determination of CK2 kinases as against a variety of kinases.
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Describes a novel scaffold of polyamines for fluorescent detection of cancer.
31
Tabacco MB, Qian X, Russo J: US8206946 (2012).
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32
The University Of Tokyo: US8309319 (2012).
President and Fellows of Harvard College: US8647119 (2014).
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Academia Sinica: US8329413 (2012).
38
34
The Regents of the University Of California: US20120329085 (2012).
The Regents of the University Of California: US20140051863 (2014).
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35
Cayman Chemical Company, Inc., The Regents Of The University Of Michigan: WO2012159072 (2013).
Covers the binding-based and reaction-based fluorescent probes for metal chelation.
39
Thomas DD, Cornea RL, Zsebo KM: US8431356 (2013).
Pierre Fabre Medicament: US8569011 (2013).
40
The University Of Tokyo: US8465985 (2013).
36
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