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Quinoline based sulfonyl derivatives, and especially sulfonamides, are relevant and promising structures for drug design. We have developed a new convenient protocol for the synthesis of 3-sulfonyl substituted quinolines (sulfonamides and sulfones). The approach is based on Knoevenagel condensation/aza-Wittig reaction cascade involving o-azidobenzaldehydes and ketosulfonamides or ketosulfones as key building blocks. The protocol is appropriate both for ketosulfonyl reagents and α-sulfonyl alkyl acetates providing the target quinoline derivatives in good to excellent yields.

The last decade has been characterized by the development and approval of pretomanid and delamanid, which are nitroimidazole based drugs for multidrug -resistant tuberculosis. This attracted renewed attention to the nitroheterocyclic scaffolds as a source of safe and efficient antimicrobial agents. While the primary focus is still on nitrofurans and nitroimidazoles, well known as bioreducible prodrugs, a number of studies have been published on other 5-membered nitroheteroaromatic compounds. The latter not only show promising antimicrobial activity but also demonstrate modes of action different from the conventional reductive activation of the nitro group. Considering the potential of these efforts to impact the continuing race against drug-resistant pathogens, herein we review non-furan/imidazole-based 5-membered nitroheteroaromatics investigated as antimicrobial agents in 2010-2020.

Aims: To synthesize novel sulfonamide inhibitors of carbonic anhydrase and develop in vitro prioritization workflow to select compounds for in vivo evaluation Background: Carbonic anhydrase (CA) inhibitors gain significant attention in the context of drug discovery research for glaucoma, hypoxic malignancies, and bacterial infections. In previous works, we have successfully used direct sulfochlorination approach to develop diverse heterocyclic primary sulfonamides with remarkable activity and selectivity against therapeutically relevant CA isoforms. Objective: Synthesis and investigation of the CA inhibitory properties of novel trifluoromethylisoxazolyl- and trifluoromethylpyrazolyl-substituted (hetero)aromatic sulfonamides. Methods: Thirteen trifluoromethylisoxazolyl- and thirteen trifluoromethylpyrazolyl-substituted (hetero)aromatic sulfonamides were synthesized by direct sulfochlorination of hydroxyisoxazolines and pyrazoles followed by reaction with ammonia. The compound structures were confirmed by 1 H and 13C NMR as well as element analysis. The obtained compounds were evaluated, using the CA esterase activity assay, for their potential to block the catalytic activity of bovine CA (bCA). Results: Eight most potent compounds selected based on the esterase activity assay data were tested for direct affinity to the enzyme using the thermal shift assay (TSA). These compounds displayed Kd values (measured by TSA) in the double-digit nanomolar range, thus showing comparable activity to the reference drug acetazolamide. Conclusion: Coupling the bCA esterase activity assay with thermal shift assay represents a streamlined and economical strategy for the prioritization of sulfonamide CA inhibitors for subsequent evaluation in vivo.

Combination therapy is becoming imperative for the treatment of many cancers, as it provides a higher chance of avoiding drug resistance and tumor recurrence. Among the resistance-conferring factors, the tumor microenvironment plays a major role, and therefore, represents a viable target for adjuvant therapeutic agents. Thus, hypoxia and extracellular acidosis are known to select for the most aggressive and resilient phenotypes and build poorly responsive regions of the tumor mass. Carbonic anhydrase (CA, EC 4.2.1.1) IX isoform is a surficial zinc metalloenzyme that is proven to play a central role in regulating intra and extracellular pH, as well as modulating invasion and metastasis processes. With its strong association and distribution in various tumor tissues and well-known druggability, this protein holds great promise as a target to pharmacologically interfere with the tumor microenvironment by using drug combination regimens. In the present review, we summarized recent publications revealing the potential of CA IX inhibitors to intensify cancer chemotherapy and overcome drug resistance in preclinical settings.

Conformational dynamics of biomolecules are of fundamental importance for their function. Single-molecule F\"orster Resonance Energy Transfer (smFRET) is a powerful approach to inform on the structure and the dynamics of labeled molecules. If the dynamics occur on the sub-millisecond timescale, capturing and quantifying conformational dynamics can be challenging by intensity-based smFRET. Multiparameter fluorescence detection (MFD) addresses this challenge by simultaneously registering intensities and fluorescence lifetimes. Together, the mean donor fluorescence lifetime and the fluorescence intensities inform on the variance, and the mean FRET efficiency tells the conformational dynamics. Here, we present a general framework that relates average fluorescence lifetimes and intensities in smFRET counting histograms. Using this framework, we show how to compute parametric relations (FRET-lines) of these observables that facilitate a graphical interpretation of experimental data, can be used to test models, identify conformational states, resolve the connectivity of states, and can be applied to unstructured systems to infer properties of polymer chains or study fast protein folding. To simplify the graphical analysis of complex kinetic networks, we derive a moment-based representation of the experimental data and show how to decouple the motion of the fluorescence labels from the conformational dynamics of the biomolecule., Comment: Main Text (47 pages, 14 figures, 3 tables) with Supporting Information (22 pages, 2 figures)

Herein we report the synthesis of a set of seventeen 3-sulfonamide substituted coumarin derivatives. Prepared compounds were tested in vitro for inhibition of four physiologically relevant isoforms of the metalloenzyme human carbonic anhydrase (hCA, EC 4.2.1.1). Several coumarin sulfonamides displayed low nanomolar KI values against therapeutically relevant hCA II, IX, and XII, whereas they did not potently inhibit hCA I. Some of these compounds exerted a concentration-dependent antiproliferative action toward RT4 human bladder cancer and especially A431 human epidermoid carcinoma cell lines. In the meantime, the viability of non-tumorigenic hTERT immortalized human foreskin fibroblast cell line Bj-5ta was not significantly affected by the obtained derivatives. Interestingly, compound 10q (2-oxo-2H-benzo [h]chromene-3-sulfonamide) showed a profound and selective dose-dependent inhibition of A431 cell growth with low nanomolar IC50 values. We demonstrated that 10q possessed a concentration-dependent apoptosis induction activity associated with caspase 3/7 activation in cancer cells. As carbonic anhydrase isoforms in question were not potently inhibited by this compound, its antiproliferative effects likely involve other mechanisms, such as DNA intercalation. Compound 10q clearly represents a viable lead for further development of new-generation anticancer agents.

Mucoadhesive formulations capable of situ gelation are promising for improving ocular drug delivery. Here we investigated two types of nanogels based on anionic glycosaminoglycans with grafted thermo-responsive poly(N-isopropylacrylamide) chains. One type of nanogels were formed by thermo-induced gelling of heparin-graft-poly(N-isopropylacrylamide) and chondroitin sulfate-graft-poly(N-isopropylacrylamide) copolymers. Another type of nanogels was based on the same copolymers, but terminal groups of thermosensitive macromolecular chains were modified to form covalent disulfide cross-links. All types of nanogels were studied towards their ability to encapsulate and release model drug – dexamethasone. Mucoadhesivity of both thermo-gelled and covalently cross-linked polymeric systems, as well as their ability to interact with dexamethasone, was assessed by microscale thermophoresis (MST). Mucoadhesion properties were also evaluated by isothermal titration calorimetry (ITC), which were in good correlation with MST data. The presence of disulfide linkages and thiol groups were shown to favor improved binding of cross-linked nanogels to mucin. Moreover, in vivo intraocular pressure studies showed that presence of polymers in solution can alter the ocular absorption of carbonic anhydrase inhibitor from eyedrops. The pharmacological effect was in line with mucoadhesive properties of these copolymers.

Single-molecule F\"orster Resonance Energy Transfer (smFRET) is ideally suited to resolve the dynamics of biomolecules. A significant challenge to date is capturing and quantifying the exchange between multiple conformational states, mainly when these dynamics occur on the sub-millisecond timescale. Many methods for the quantitative analysis are challenged if more than two states are involved, and the appropriate choice of the number of states in the kinetic network is difficult. An additional complication arises if dynamically active molecules coexist with solely static molecules in similar conformational states. To address these problems, we developed an integrative analysis framework that combines the information from FRET-lines, time-correlated single photon counting, and fluorescence correlation spectroscopy. While individually, these methodologies provide ambiguous results for quantitatively characterize the dynamics in complex kinetic networks, the global analysis enables accurate determination of the number of states, their kinetic connectivity, and the transition rate constants. To challenge the potential of smFRET to study multi-state kinetic networks, we benchmark the framework using synthetic data of three-state systems with different kinetic connectivity and interconversion rates., Comment: Main Text (31 pages, 9 figures, 3 tables) with Supporting Information (40 pages, 10 figures, 2 tables)

A series of novel aromatic primary sulfonamides decorated with diversely substituted 1,2,4-oxadiazole periphery groups has been prepared using a parallel chemistry approach. The compounds displayed a potent inhibition of cytosolic hCA II and membrane-bound hCA IX isoforms. Due to a different cellular localization of the two target enzymes, the compounds can be viewed as selective inhibition tools for either isoform, depending on the cellular permeability profile. The SAR findings revealed in this study has been well rationalized by docking simulation of the key compounds against the crystal structures of the relevant hCA isoforms.

A hypothesis that simultaneous targeting cancer-related carbonic anhydrase hCA IX and hCA XII isoforms (whose overexpression is a cancer cell’s defence mechanism against hypoxia) along with thioredoxin reductase (overexpressed in cancers as a defence against oxidative stress) may lead to synergistic antiproliferative effects was confirmed by testing combinations of the two inhibitor classes against pancreatic cancer cells (PANC-1). Combining both pharmacophoric motifs within one molecule led to a sharp increase of cytotoxicity. This preliminary observation sets the ground for a fundamentally new approach to anticancer agent design., Graphical Abstract

Testing of an expanded, 800-compound set of analogues of the earlier described Strecker-type α-aminonitriles (selected from publicly available Enamine Ltd. Screening Collection) in thermal shift assay against bovine carbonic anhydrase (bCA) led to further validation of this new class of inhibitors and identification a new, refined chemotype represented by inhibitors with 10-improved potency.

By exploiting the power of multicomponent chemistry, a relatively small, diverse set of primary sulfonamides was synthesized and screened against a panel of human carbonic anhydrases to reveal a low-nanomolar, albeit non-selective hCA IV lead inhibitor. Investigation of the docking poses of this compound identified a hydrophilic pocket unique to hCA IV and conveniently positioned near the carboxylate functionality of the initial lead. Various residues capable of forming hydrogen bonds as well as salt bridges were placed in this pocket via a carboxamides linkage, which led to drastic improvement of potency and selectivity towards hCA IV. This improvement of the desired inhibitory profile was rationalized by the new contacts as had been envisioned. These new tool compounds were shown to possess selective, dose-dependent cytotoxicity against human glioma T98G cell line. The latter showed a substantially increased hCA IV mRNA expression under hypoxic conditions.

Herein we report the synthesis of a set of thirty-four primary sulfonamides generated via formal N–H-insertion of metal carbenes into anilinic amino group of sulfanilamide and its meta-substituted analog. Obtained compounds were tested in vitro as inhibitors of four physiologically significant isoforms of the metalloenzyme human carbonic anhydrase (hCA, EC 4.2.1.1). Many of the synthesized sulfonamides displayed low nanomolar Ki values against therapeutically relevant hCA II, IX, and XII, whereas they did not potently inhibit hCA I. Provided the promising activity profiles of the substances towards tumor-associated hCA IX and XII isozymes, single-concentration MTT test was performed for the entire set. Disappointingly, most of the discovered hCA inhibitors did not significantly suppress the growth of cancer cells either in normoxia or CoCl2 induced hypoxic conditions. The only two compounds exerting profound antiproliferative effect turned out to be modest hCA inhibitors. Their out of the range activity in cells is likely attributive to the presence of Michael acceptor substructure which can potentially act either through the inhibition of Thioredoxin reductases (TrxRs, EC 1.8.1.9) or nonspecific covalent binding to cell proteins.

The tumour-associated, cell membrane-bound isoforms IX and XII of human carbonic anhydrase (CA, EC 4.2.1.1) are overexpressed in cancer cells contributing to the hypoxic tumour pH/metabolism regulating machinery and as thus, can serve as markers of malignant neoplastic tissue. Inhibitors of CAs can be employed both for the treatment of hypoxic tumours and in the design of radiotracers for positron emission tomography and imaging of such cancers. The present review provides a comprehensive summary of the progress achieved to-date in the field of developing PET-tracers based on monoclonal antibodies, biomolecules, and small-molecule ligands of CA IX and XII.

A series of novel benzene sulfonamides (previously evaluated as selective cyclooxygenase-2 inhibitors) has been profiled against human carbonic anhydrases I, II, IV and VII in an attempt to observe the manifestation of the well established “tail” approach for designing potent, isoform-selective inhibitors of carbonic anhydrases (CAs, EC 4.2.1.1). The compounds displayed an excellent (pKi 7–8) inhibitory profile against CA II (a cytosolic anti-glaucoma and anti-edema biological target) and CA VII (also a cytosolic target believed to be involved in epilepsy and neuropathic pain) and a marked (1–2 orders of magnitude) selectivity against cytosolic isoform CA I and membrane-bound isoform CA IV. The separation of the CA II and CA IV (both of which are catalytically active isoforms, highly sensitive to sulfonamide-type inhibitors) is particularly remarkable and is adding significantly to the global body of data on the chemical biology of carbonic anhydrases.

An expanded set of pyridazine-containing benzene sulfonamides was investigated for inhibition of four human carbonic anhydrase isoforms, which revealed a pronounced inhibition trend toward hCA IX, a cancer-related, membrane-bound isoform of the enzyme. Comparison of antiproliferative effects of these compounds against cancer (PANC-1) and normal (ARPE-19) cells at 50 μM concentration narrowed the selection of compounds to the eight which displayed selective growth inhibition toward the cancer cells. More detailed investigation in concentration-dependent mode against normal (ARPE-19) and two cancer cell lines (PANC-1 and SK-MEL-2) identified two lead compounds one of which displayed a notable cytotoxicity toward pancreatic cancer cells while the other targeted the melanoma cells. These findings significantly expand the knowledge base concerning the hCA IX inhibitors whose inhibitory potency against a recombinant enzyme translates into selective anticancer activity under hypoxic conditions which are aimed to model the environment of a growing tumor.

An expanded set of diversely substituted 1,2,4-oxadiazole-containing primary aromatic sulfonamides was synthesized and tested for inhibition of human carbonic anhydrase I, II, IX and XII isoforms. The initial biochemical profiling revealed a significantly more potent inhibition of cancer-related, membrane-bound isoform hCA IX (reaching into submicromolar range), on top of potent inhibition of hCA XII that is another cancer target. The observed structure-activity relationships have been rationalized by molecular modeling. Comparative single-concentration profiling of the carbonic anhydrase inhibitors synthesized for antiproliferative effects against normal (ARPE-19) and cancer (PANC-1) cell lines under chemically induced hypoxia conditions revealed several candidate compounds selectively targeting cancer cells. More in-depth characterization of these leads revealed two structurally related compounds that showed promising selective cytotoxicity against pancreatic cancer (PANC-1) and melanoma (SK-MEL-2) cell lines.

Previously unknown N-aminosaccharin was prepared in good yield via the one-step direct amination of saccharin sodium salt with hydroxylamine-O-mesitylenesulfonic acid (MSH) and its reactivity investigated. N-aminosaccharin and its derivatives were tested against hCA isoforms and the parent compound was identified to be a selective, low micromolar inhibitor (Ki = 8.8 μM) of hCA I. These findings provide a ligand-efficient starting point for the design of potent hCA I inhibitors – a promising drug target for retinal/cerebral edema treatment.

A previously reported condensation reaction of ambiphilic 2-(1H-pyrazol-5-yl)phenols with various o-chloro-substituted nitroaromatic synthons to provide medicinally important tetracyclic pyrazolo[1,5-d][1,4]oxazepines was extended to more readily available and convenient-to-use 1,2-dihaloazines. Although 4,5-dichloropyridazin-3(2H)-ones and 2,3-dichloropyrazine are popular bis(electrophilic) partners in various SNAr-type condensations and also were efficiently used in the reaction reported herein, the facility with which various 3-bromo-2-chloropyridines underwent the same cyclization was unexpected. These reactions are presumed to occur as sequential intermolecular SNAr/Smiles rearrangement/intramolecular SNAr tandem reactions and provide a rare example of the transition-metal-free substitution of a bromine atom at the 3-position of a pyridine ring.

4-Chloro-3-nitrobenzenesulfonamide reacted cleanly at room-temperature with a range of bis-electrophilic phenols bearing an NH-acidic functionality (secondary carboxamide or pyrazole) in the ortho-position. This produced a novel class of [1,4]oxazepine-based primary sulfonamides which exhibited strong inhibition of therapeutically relevant human carbonic anhydrases. 2-Chloronitrobenzene did not enter a similar cyclocondensation process, even under prolonged heating. Thus, the primary sulfonamide functionality plays a dual role by enabling the [1,4]oxazepine ring construction and acting as a enzyme prosthetic zinc-binding group when the resulting [1,4]oxazepine sulfonamides are employed as carbonic anhydrase inhibitors.

A streamlined synthetic methodology towards novel tetracyclic 1,4-oxazepines from readily available precursors is described. The compounds, designed as more soluble version of the earlier described, poorly soluble dibenzo[b,f][1,4]oxazepines, were obtained in high yields and as a single regioisomer as a result of three tandem chemical events—nucleophilic aromatic substitution, Smiles rearrangement and denitrocyclization.

Four inhibitors of human carbonic anhydrase II (hCA II) were designed based on the previously reported subnanomolar 1,3-oxazole-based sulfonamide inhibitors of the enzyme to incorporate primary and secondary amine functionality in the carboxamide side chain. The new hydrophilic compounds were found to inhibit the target isoform in sub-nanomolar to low nanomolar range with a good degree of selectivity to several other hCA isoforms. The hydrophilic character of these compounds is advantageous for intraocular residence time but not for corneal permeability which generally requires that a drug be sufficiently lipophilic. Two of the four compounds investigated, however, were found to exert comparable efficacy as 1% eye drops in PBS to that of the clinically used 2% dorzolamide (Trusopt®) eye drops. This indicated that the absorption of the compounds may occur via alternative route across conjunctiva and sclera.

Replacement of amide moiety with the 1,2,4-oxadiazole core in the scaffold of recently reported efflux pump inhibitors afforded a novel series of oxadiazole/2-imidazoline hybrids. The latter compounds exhibited promising antibacterial activity on both Gram-positive (Staphylococcus aureus, Bacillus subtilis) and Gram-negative (Escherichia coli, Pseudomonas fluorescens) strains. Furthermore, selected compounds markedly inhibited the growth of certain drug-resistant bacteria. Additionally, the study revealed the antiproliferative activity of several antibacterial frontrunners against pancreas ductal adenocarcinoma (PANC-1) cell line, as well as their type-selective monoamine oxidase (MAO) inhibitory profile.

DNA polymerases are responsible for the accurate replication of DNA. Kinetic, single-molecule, and x-ray studies show that multiple conformational states are important for DNA polymerase fidelity. Using high precision FRET measurements, we show that Klentaq1 (the Klenow fragment of Thermus aquaticus DNA polymerase 1) is in equilibrium between three structurally distinct states. In the absence of nucleotide, the enzyme is mostly open, whereas in the presence of DNA and a correctly base-pairing dNTP, it re-equilibrates to a closed state. In the presence of a dNTP alone, with DNA and an incorrect dNTP, or in elevated MgCl2 concentrations, an intermediate state termed the “nucleotide-binding” state predominates. Photon distribution and hidden Markov modeling revealed fast dynamic and slow conformational processes occurring between all three states in a complex energy landscape suggesting a mechanism in which dNTP delivery is mediated by the nucleotide-binding state. After nucleotide binding, correct dNTPs are transported to the closed state, whereas incorrect dNTPs are delivered to the open state.

A substantial improvement of potency and selectivity of imidazoline-based inhibitors of hCA VII (a promising target for the treatment of seizures and neuropathic pain) was achieved by simply switching the position of the benzenesulfonamide moiety from N1 (as in the earlier reported series) to C2. Selectivity indices vs the off-target isoforms (hCA I, I, I and IV) greater than 100 were reached, which is exceedingly rare for hCA VII inhibitors. The drastic profile improvement of the new series has been rationalized by an additional hydrogen bonding with the nonconserved Q69 residue in the active site of hCA VII (absent in the other three isoforms studied), which also results in a favorable accommodation of the inhibitor’s lipophilic periphery in the nearby hydrophobic pocket. The robustness of the docking simulations was tested and confirmed by molecular dynamics simulations.

A diverse set of mono- and bis-sulfonamide was obtained via a direct, chemoselective sulfochlorination of readily available yet hitherto unexplored N-arylpyrazole template. Biochemical profiling of compounds thus obtained against a panel of human carbonic anhydrases (hCA I, hCA II, hCA IV and hCA VII) revealed a number of leads that are promising from the isoform selectivity prospective and exhibit potent inhibition profile (from nanomolar to micromolar range). The observed SAR trends have been rationalized by in silico docking of selected compounds into the active site of all four isoforms. The results reported in this paper clearly attest to the power of direct sulfochlorination as the means to create carbonic anhydrase focused sets in order to identify isoform selective inhibitors of closely related enzymes., Graphical Abstract

Three distinct series of isoxazole-based primary mono- and bis-sulfonamides have been synthesized via direct sulfochlorination, each of them delivering nanomolar inhibitors of human carbonic anhydrase. Certain pronounced SAR trends have been established and rationalized by in silico docking. These findings expand the structure-activity knowledge base for heterocycle-containing sulfonamide carbonic anhydrase inhibitors and further validate the power of direct electrophilic sulfochlorination as a means of introducing the pharmacophoric primary sulfonamide group into structurally diverse aromatic precursors.

Carbonic anhydrase inhibitors (CAIs) are of growing interest since various isoforms of the enzyme are identified as promising drug targets for treatment of disease. The principal drawback of the clinically used CAIs is the lack of isoform selectivity, which may lead to observable side effects. Studies aiming at the design of isoform-selective CAIs entail generation and biological testing of arrays of compounds, which is a resource- and time-consuming process. Employment of multicomponent reactions is an efficient synthetic strategy in terms of gaining convenient and speedy access to a range of scaffolds with a high degree of molecular diversity. However, this powerful tool appears to be underutilized for the discovery of novel CAIs. A number of studies employing multicomponent reactions in CAI synthesis have been reported in literature. Some of these reports provide inspiring examples of successful use of multicomponent chemistry to construct novel potent and often isoform-selective inhibitors. On critical reading of several publications, however, it becomes apparent that for some chemical series designed as CAIs, the desired inhibitory properties are only assumed and never tested for. In these cases, the biological profile is reported based on the results of phenotypical cellular assays, with no correlation with the intended on-target activity. Present review aims at critically assessing the current literature on the multicomponent chemistry in the CAI design.

Forster resonance energy transfer (FRET) due to its sensitivity of distance has been widely used to investigate the structure and interaction of biomolecules. Multiparameter fluorescence image spectroscopy (MFIS) provides particular advantages to FRET imaging because all the fluorescence parameters are monitored simultaneously with picosecond accuracy, which allows for a comprehensive analysis on biological systems. Traditionally, a reduction in average donor lifetime or an increase of average FRET efficiency was used as an indicator for molecular interaction. However, such changes observed in FRET-imaging can have two reasons: (1) the conformational change or (2) change in fraction of FRET-active species. To resolve this ambiguity, we introduce a new sub-ensemble analysis method to directly visualize and quantitatively analyze both factors. Characterization of true FRET efficiency enabled us to detect even subtle FRET variations and provided crucial information about the structural properties of molecular complexes. Furthermore, from determined fraction of FRET-active species, utilizing the intrinsic cell-to-cell variations of protein concentration, we show that dissociation constant (KD) of membrane-receptor interactions can be characterized in living cells.

Condensation of 2-(1H-pyrazol-5-yl)phenols with 1-chloro-2-nitrobenzenes under basic conditions in N,N-dimethyl­formamide results in a tandem, atom-economical, aromatic nucleophilic substitution–Smiles rearrangement–denitrocyclization process to provide pyrazolo-fused dibenzo[b,f][1,4]oxazepines as a single regioisomer.

An analysis method of lifetime, polarization and spectrally filtered fluorescence correlation spectroscopy, referred to as filtered FCS (fFCS), is introduced. It uses, but is not limited to, multiparameter fluorescence detection to differentiate between molecular species with respect to their fluorescence lifetime, polarization and spectral information. Like the recently introduced fluorescence lifetime correlation spectroscopy (FLCS) [Chem. Phys. Lett. 2002, 353, 439-445], fFCS is based on pulsed laser excitation. However, it uses the species-specific polarization and spectrally resolved fluorescence decays to generate filters. We determined the most efficient method to generate global filters taking into account the anisotropy information. Thus, fFCS is able to distinguish species, even if they have very close or the same fluorescence lifetime, given differences in other fluorescence parameters. fFCS can be applied as a tool to compute species-specific auto- (SACF) and cross- correlation (SCCF) functions from a mixture of different species for accurate and quantitative analysis of their concentration, diffusion and kinetic properties. The computed correlation curves are also free from artifacts caused by unspecific background signal. We tested this methodology by simulating the extreme case of ligand-receptor binding processes monitored only by differences in fluorescence anisotropy. Furthermore, we apply fFCS to an experimental single-molecule FRET study of an open-to-closed conformational transition of the protein Syntaxin-1. In conclusion, fFCS and the global analysis of the SACFs and SCCF is a key tool to investigate binding processes and conformational dynamics of biomolecules in a nanosecond-to-millisecond time range as well as to unravel the involved molecular states.