Computationally Supported Inversion of Ketoreductase Stereoselectivity.

Whereas directed evolution and rational design by structural inspection are established tools for enzyme redesign, computational methods are less mature but have the potential to predict small sets of mutants with desired properties without laboratory screening of large libraries. We have explored the use of computational enzyme redesign to change the enantioselectivity of a highly thermostable alcohol dehydrogenase from Thermus thermophilus in the asymmetric reduction of ketones. The enzyme reduces acetophenone to (S)-1-phenylethanol. To invert the enantioselectivity, we used an adapted CASCO workflow which included Rosetta for enzyme design and molecular dynamics simulations for ranking. To correct for unrealistic binding modes, we used Boltzmann weighing of binding energies computed by a linear interaction energy approach. This computationally cheap method predicted four variants with inverted enantioselectivity, each with 6-8 mutations around the substrate-binding site, causing only modest reduction (2- to 7-fold) of kcat /KM values. Laboratory testing showed that three variants indeed had inverted enantioselectivity, producing (R)-alcohols with up to 99 % enantiomeric excess. The broad substrate range allowed reduction of acetophenone derivatives with full conversion to highly enantioenriched alcohols. The results demonstrate the use of computational methods to control ketoreductase stereoselectivity in asymmetric transformations with minimal experimental screening.

Catalytic and structural properties of ATP-dependent caprolactamase from Pseudomonas jessenii.

Caprolactamase is the first enzyme in the caprolactam degradation pathway of Pseudomonas jessenii. It is composed of two subunits (CapA and CapB) and sequence-related to other ATP-dependent enzymes involved in lactam hydrolysis, like 5-oxoprolinases and hydantoinases. Low sequence similarity also exists with ATP-dependent acetone- and acetophenone carboxylases. The caprolactamase was produced in Escherichia coli, isolated by His-tag affinity chromatography, and subjected to functional and structural studies. Activity toward caprolactam required ATP and was dependent on the presence of bicarbonate in the assay buffer. The hydrolysis product was identified as 6-aminocaproic acid. Quantum mechanical modeling indicated that the hydrolysis of caprolactam was highly disfavored (ΔG0 '= 23 kJ/mol), which explained the ATP dependence. A crystal structure showed that the enzyme exists as an (αß)2 tetramer and revealed an ATP-binding site in CapA and a Zn-coordinating site in CapB. Mutations in the ATP-binding site of CapA (D11A and D295A) significantly reduced product formation. Mutants with substitutions in the metal binding site of CapB (D41A, H99A, D101A, and H124A) were inactive and less thermostable than the wild-type enzyme. These residues proved to be essential for activity and on basis of the experimental findings we propose possible mechanisms for ATP-dependent lactam hydrolysis.

Stabilizing AqdC, a Pseudomonas Quinolone Signal-Cleaving Dioxygenase from Mycobacteria, by FRESCO-Based Protein Engineering.

The mycobacterial PQS dioxygenase AqdC, a cofactor-less protein with an α/ß-hydrolase fold, inactivates the virulence-associated quorum-sensing signal molecule 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) produced by the opportunistic pathogen Pseudomonas aeruginosa and is therefore a potential anti-virulence tool. We have used computational library design to predict stabilizing amino acid replacements in AqdC. While 57 out of 91 tested single substitutions throughout the protein led to stabilization, as judged by increases in Tappm of >2 °C, they all impaired catalytic activity. Combining substitutions, the proteins AqdC-G40K-A134L-G220D-Y238W and AqdC-G40K-G220D-Y238W showed extended half-lives and the best trade-off between stability and activity, with increases in Tappm of 11.8 and 6.1 °C and relative activities of 22 and 72 %, respectively, compared to AqdC. Molecular dynamics simulations and principal component analysis suggested that stabilized proteins are less flexible than AqdC, and the loss of catalytic activity likely correlates with an inability to effectively open the entrance to the active site.

CYP154C5 Regioselectivity in Steroid Hydroxylation Explored by Substrate Modifications and Protein Engineering*.

CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16α-position. Using protein engineering and substrate modifications based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternative binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that the entrance of water to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-hydroxylation was observed for wild-type CYP154C5 in the conversion of 5α-androstan-3-one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the structure-function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation.

Computational Prediction of ω-Transaminase Specificity by a Combination of Docking and Molecular Dynamics Simulations.

ω-Transaminases (ω-TAs) catalyze the conversion of ketones to chiral amines, often with high enantioselectivity and specificity, which makes them attractive for industrial production of chiral amines. Tailoring ω-TAs to accept non-natural substrates is necessary because of their limited substrate range. We present a computational protocol for predicting the enantioselectivity and catalytic selectivity of an ω-TA from Vibrio fluvialis with different substrates and benchmark it against 62 compounds gathered from the literature. Rosetta-generated complexes containing an external aldimine intermediate of the transamination reaction are used as starting conformations for multiple short independent molecular dynamics (MD) simulations. The combination of molecular docking and MD simulations ensures sufficient and accurate sampling of the relevant conformational space. Based on the frequency of near-attack conformations observed during the MD trajectories, enantioselectivities can be quantitatively predicted. The predicted enantioselectivities are in agreement with a benchmark dataset of experimentally determined ee% values. The substrate-range predictions can be based on the docking score of the external aldimine intermediate. The low computational cost required to run the presented framework makes it feasible for use in enzyme design to screen thousands of enzyme variants.

Computational Design of Enantiocomplementary Epoxide Hydrolases for Asymmetric Synthesis of Aliphatic and Aromatic Diols.

The use of enzymes in preparative biocatalysis often requires tailoring enzyme selectivity by protein engineering. Herein we explore the use of computational library design and molecular dynamics simulations to create variants of limonene epoxide hydrolase that produce enantiomeric diols from meso-epoxides. Three substrates of different sizes were targeted: cis-2,3-butene oxide, cyclopentene oxide, and cis-stilbene oxide. Most of the 28 designs tested were active and showed the predicted enantioselectivity. Excellent enantioselectivities were obtained for the bulky substrate cis-stilbene oxide, and enantiocomplementary mutants produced (S,S)- and (R,R)-stilbene diol with >97 % enantiomeric excess. An (R,R)-selective mutant was used to prepare (R,R)-stilbene diol with high enantiopurity (98 % conversion into diol, >99 % ee). Some variants displayed higher catalytic rates (kcat ) than the original enzyme, but in most cases KM values increased as well. The results demonstrate the feasibility of computational design and screening to engineer enantioselective epoxide hydrolase variants with very limited laboratory screening.

Computational redesign of enzymes for regio- and enantioselective hydroamination.

Introduction of innovative biocatalytic processes offers great promise for applications in green chemistry. However, owing to limited catalytic performance, the enzymes harvested from nature's biodiversity often need to be improved for their desired functions by time-consuming iterative rounds of laboratory evolution. Here we describe the use of structure-based computational enzyme design to convert Bacillus sp. YM55-1 aspartase, an enzyme with a very narrow substrate scope, to a set of complementary hydroamination biocatalysts. The redesigned enzymes catalyze asymmetric addition of ammonia to substituted acrylates, affording enantiopure aliphatic, polar and aromatic ß-amino acids that are valuable building blocks for the synthesis of pharmaceuticals and bioactive compounds. Without a requirement for further optimization by laboratory evolution, the redesigned enzymes exhibit substrate tolerance up to a concentration of 300 g/L, conversion up to 99%, ß-regioselectivity >99% and product enantiomeric excess >99%. The results highlight the use of computational design to rapidly adapt an enzyme to industrially viable reactions.

Exploring the Selective Demethylation of Aryl Methyl Ethers with a Pseudomonas Rieske Monooxygenase.

Biocatalytic dealkylation of aryl methyl ethers is an attractive reaction for valorization of lignin components, as well as for deprotection of hydroxy functionalities in synthetic chemistry. We explored the demethylation of various aryl methyl ethers by using an oxidative demethylase from Pseudomonas sp. HR199. The Rieske monooxygenase VanA and its partner electron transfer protein VanB were recombinantly coexpressed in Escherichia coli and they constituted at least 25 % of the total protein content. Enzymatic transformations showed that VanB accepts NADH and NADPH as electron donors. The VanA-VanB system demethylates a number of aromatic substrates, the presence of a carboxylic acid moiety is essential, and the catalysis occurs selectively at the meta position to this carboxylic acid in the aromatic ring. The reaction is inhibited by the by-product formaldehyde. Therefore, we tested three different cascade/tandem reactions for cofactor regeneration and formaldehyde elimination; in particular, conversion was improved by addition of formaldehyde dehydrogenase and formate dehydrogenase. Finally, the biocatalyst was applied for the preparation of protocatechuic acid from vanillic acid, giving a 77 % yield of the desired product. The described reaction may find application in the conversion of lignin components into diverse hydroxyaromatic building blocks and generally offers potential for new, mild methods for efficient unmasking of phenols.

Efficient Enzymatic Cyclization of Disulfide-Rich Peptides by Using Peptide Ligases.

Disulfide-rich macrocyclic peptides-cyclotides, for example-represent a promising class of molecules with potential therapeutic use. Despite their potential their efficient synthesis at large scale still represents a major challenge. Here we report new chemoenzymatic strategies using peptide ligase variants-inter alia, omniligase-1-for the efficient and scalable one-pot cyclization and folding of the native cyclotides MCoTI-II, kalata B1 and variants thereof, as well as of the θ-defensin RTD-1. The synthesis of the kB1 variant T20K was successfully demonstrated at multi-gram scale. The existence of several ligation sites for each macrocycle makes this approach highly flexible and facilitates both the larger-scale manufacture and the engineering of bioactive, grafted cyclotide variants, therefore clearly offering a valuable and powerful extension of the existing toolbox of enzymes for peptide head-to-tail cyclization.

Exploring PTDH-P450BM3 Variants for the Synthesis of Drug Metabolites.

The conversion of a series of pharmaceutical compounds was examined with three variants of cytochrome P450BM3 fused to phosphite dehydrogenase (PTDH) to enable cofactor recycling. Conditions for enzyme production were optimized, and the purified PTDH-P450BM3 variants were tested against 32 commercial drugs by using rapid UPLC-MS analysis. The sets of mutations (R47L/F87V/L188Q and R47L/F87V/L188Q/E267V/G415S) improved conversion for all compounds, and a variety of products were detected. Product analysis showed that reaction types included C-hydroxylation, N-oxidation, demethylation, and aromatization. Interestingly, enzymatic aromatization could occur independent of the addition of reducing coenzyme. These results identified new conversions catalyzed by P450BM3 variants and showed that a small set of mutations in the oxygenase domain could broaden both substrate range and reaction type.

Design of a substrate-tailored peptiligase variant for the efficient synthesis of thymosin-α1.

The synthesis of thymosin-α1, an acetylated 28 amino acid long therapeutic peptide, via conventional chemical methods is exceptionally challenging. The enzymatic coupling of unprotected peptide segments in water offers great potential for a more efficient synthesis of peptides that are difficult to synthesize. Based on the design of a highly engineered peptide ligase, we developed a fully convergent chemo-enzymatic peptide synthesis (CEPS) process for the production of thymosin-α1via a 14-mer + 14-mer segment condensation strategy. Using structure-inspired enzyme engineering, the thiol-subtilisin variant peptiligase was tailored to recognize the respective 14-mer thymosin-α1 segments in order to create a clearly improved biocatalyst, termed thymoligase. Thymoligase catalyzes peptide bond formation between both segments with a very high efficiency (>94% yield) and is expected to be well applicable to many other ligations in which residues with similar characteristics (e.g. Arg and Glu) are present in the respective positions P1 and P1'. The crystal structure of thymoligase was determined and shown to be in good agreement with the model used for the engineering studies. The combination of the solid phase peptide synthesis (SPPS) of the 14-mer segments and their thymoligase-catalyzed ligation on a gram scale resulted in a significantly increased, two-fold higher overall yield (55%) of thymosin-α1 compared to those typical of existing industrial processes.

Characterization of the caprolactam degradation pathway in Pseudomonas jessenii using mass spectrometry-based proteomics.

Some bacterial cultures are capable of growth on caprolactam as sole carbon and nitrogen source, but the enzymes of the catabolic pathway have not been described. We isolated a caprolactam-degrading strain of Pseudomonas jessenii from soil and identified proteins and genes putatively involved in caprolactam metabolism using quantitative mass spectrometry-based proteomics. This led to the discovery of a caprolactamase and an aminotransferase that are involved in the initial steps of caprolactam conversion. Additionally, various proteins were identified that likely are involved in later steps of the pathway. The caprolactamase consists of two subunits and demonstrated high sequence identity to the 5-oxoprolinases. Escherichia coli cells expressing this caprolactamase did not convert 5-oxoproline but were able to hydrolyze caprolactam to form 6-aminocaproic acid in an ATP-dependent manner. Characterization of the aminotransferase revealed that the enzyme deaminates 6-aminocaproic acid to produce 6-oxohexanoate with pyruvate as amino acceptor. The amino acid sequence of the aminotransferase showed high similarity to subgroup II ω-aminotransferases of the PLP-fold type I proteins. Finally, analyses of the genome sequence revealed the presence of a caprolactam catabolism gene cluster comprising a set of genes involved in the conversion of caprolactam to adipate.

Association between psychotropic medications and presence of sleep bruxism: A systematic review.

The purpose of this study was to systematically review the literature for studies that investigated the association between use of psychotropic medications and presence of sleep bruxism (SB). Observational studies were selected in a two-phase process. Searches were performed on six electronic databases, and a grey literature search was conducted on three databases. SB diagnosis was based on questionnaires or clinical examinations; no polysomnography examinations were performed. Risk of bias was assessed using the Joanna Briggs Institute Critical Appraisal Checklist for Analytical Cross-Sectional Studies. Overall quality of evidence was evaluated according to the Grading of Recommendations Assessment, Development and Evaluation criteria. Five analytical cross-sectional studies were included, evaluating antidepressants, anticonvulsants and psychostimulants. One study was judged as low risk of bias, three as moderate risk and one high risk. Antidepressants were evaluated in adult populations only; duloxetine (Odds Ratio [OR] = 2.16; 95% Confidence Interval [95% CI] = 1.12-4.17), paroxetine (OR = 3.63; 95% CI = 2.15-6.13) and venlafaxine (OR = 2.28; 95% CI = 1.34-3.86) were positively associated with SB risk. No increased odds of SB were observed considering use of citalopram, escitalopram, fluoxetine, mirtazapine and sertraline. With regard to anticonvulsants, only barbiturates were associated with SB in children (OR = 14.70; 95% CI = 1.85-116.90), while no increased odds were observed for benzodiazepine, carbamazepine and valproate. The only psychostimulant evaluated was methylphenidate, and an association with SB was observed in adolescents (OR = 1.67; 95% CI = 1.03-2.68). Findings from this SR suggested that medications such as duloxetine, paroxetine, venlafaxine, barbiturates and methylphenidate might be associated with SB; however, overall quality of evidence was considered very low, and therefore, caution is recommended.

[Epi-Bowman Keratectomy: Clinical Evaluation of a New Method of Surface Ablation]. / Epi-Bowman-Keratektomie: klinische Beurteilung einer neuen Variante der Surface Ablation.

PURPOSE: A new device for epithelial abrasion before excimer laser surface ablation or corneal cross-linking (CXL) has recently been introduced (Epi-Clear™, Orca Surgical, Kiryat-Shmona, Israel). We have reviewed the literature on the clinical results, potential benefits and drawbacks of this instrument, compared to other methods of epithelial removal. METHOD: Literature search for "Epi-Bowman Keratectomy", "Epi-clear", and "Epikeratome" yielded 1 peer-review publication, 1 non-peer-review publication, 18 posters and presentations at international conferences (European Society of Cataract and Refractive Surgeons [ESCRS] and American Society of Cataract and Refractive Surgery [ASCRS]) on the use of the Epi-Clear™ device before surface ablation, 2 posters on the use of Epi-Clear before corneal crosslinking and 1 presentation on the experimental use of Epi-Clear for removal of a pterygium. RESULTS: Comparison of laser ablation after epithelial removal with the Epi-Clear device (Epi-Bowman Keratectomy™, EBK™) to other established methods of surface ablation, i.e. alcohol-assisted PRK or PRK with a metallic scraper, EBK, suggests that the results are generally similar. Pain perception, haze formation, and epithelial healing are reported to be better than with conventional surface ablation methods. Studies evaluating the use of the Epi-Clear device before CXL report that the healing time is significantly reduced and that less pain is perceived. CONCLUSION: The Epi-Clear device seems to be a promising new option for epithelial removal before refractive laser ablation, although a convincing explanation for its potential superiority is still missing. In contrast, when the Epi-Clear device is used before CXL, then the Bowman's layer remains intact; this may provide an adequate explanation for the reported benefits of this application. However, currently available studies are of low level of evidence, so that more prospective randomised trials are needed for a robust evaluation of this treatment.

Metal Dependence of the Xylose Isomerase from Piromyces sp. E2 Explored by Activity Profiling and Protein Crystallography.

Xylose isomerase from Piromyces sp. E2 (PirXI) can be used to equip Saccharomyces cerevisiae with the capacity to ferment xylose to ethanol. The biochemical properties and structure of the enzyme have not been described even though its metal content, catalytic parameters, and expression level are critical for rapid xylose utilization. We have isolated the enzyme after high-level expression in Escherichia coli, analyzed the metal dependence of its catalytic properties, and determined 12 crystal structures in the presence of different metals, substrates, and substrate analogues. The activity assays revealed that various bivalent metals can activate PirXI for xylose isomerization. Among these metals, Mn2+ is the most favorable for catalytic activity. Furthermore, the enzyme shows the highest affinity for Mn2+, which was established by measuring the activation constants (Kact) for different metals. Metal analysis of the purified enzyme showed that in vivo the enzyme binds a mixture of metals that is determined by metal availability as well as affinity, indicating that the native metal composition can influence activity. The crystal structures show the presence of an active site similar to that of other xylose isomerases, with a d-xylose binding site containing two tryptophans and a catalytic histidine, as well as two metal binding sites that are formed by carboxylate groups of conserved aspartates and glutamates. The binding positions and conformations of the metal-coordinating residues varied slightly for different metals, which is hypothesized to contribute to the observed metal dependence of the isomerase activity.

P450BM3 fused to phosphite dehydrogenase allows phosphite-driven selective oxidations.

To facilitate the wider application of the NADPH-dependent P450BM3, we fused the monooxygenase with a phosphite dehydrogenase (PTDH). The resulting monooxygenase-dehydrogenase fusion enzyme acts as a self-sufficient bifunctional catalyst, accepting phosphite as a cheap electron donor for the regeneration of NADPH.The well-expressed fusion enzyme was purified and analyzed in comparison to the parent enzymes. Using lauric acid as substrate for P450BM3, it was found that the fusion enzyme had similar substrate affinity and hydroxylation selectivity while it displayed a significantly higher activity than the non-fused monooxygenase. Phosphite-driven conversions of lauric acid at restricted NADPH concentrations confirmed multiple turnovers of the cofactor. Interestingly, both the fusion enzyme and the native P450BM3 displayed enzyme concentration dependent activity and the fused enzyme reached optimal activity at a lower enzyme concentration. This suggests that the fusion enzyme has an improved tendency to form functional oligomers.To explore the constructed phosphite-driven P450BM3 as a biocatalyst, conversions of the drug compounds omeprazole and rosiglitazone were performed. PTDH-P450BM3 driven by phosphite was found to be more efficient in terms of total turnover when compared with P450BM3 driven by NADPH. The results suggest that PTDH-P450BM3 is an attractive system for use in biocatalytic and drug metabolism studies.

[Experience with Introduction of SMILE: Learning Phase of our First 200 Treatments]. / Erfahrung bei der Einführung von SMILE: Lernkurve der ersten 200 Behandlungen.

Background Small Incision Lenticule Extraction (SMILE) is a relatively new surgical method to correct myopia and myopic astigmatism. Although considered to be safe and effective, it is not yet popular. Potential reasons for its limited use could be that surgeons fear inferior results and a higher complication rate in the learning phase. Therefore we present our experiences with the treatment of our first 200 eyes, with special attention to refraction results, complications, and learning curve. Patients and Methods 200 myopic or myopic astigmatic eyes of 102 patients were treated with SMILE. The analysis was separated into two phases: Phase I (first 100 treatments) with large variation of laser parameters and surgical technique, phase II (second 100 treatments) with mostly constant laser parameters and identical surgical technique. Results Phase I showed the following 3-month results after a mean attempted correction of - 5.70 ± 2.54 D (range: - 1.25 to - 10.00 D): spherical equivalent - 0.03 ± 0.46 D, astigmatism - 0.34 ± 0.35 D, uncorrected distance visual acuity 1.02, corrected distance visual acuity (CDVA) 1.22. Postoperative and preoperative CDVA did not differ (p = 0.71). Mean refraction reached the targeted value after 5 days. Efficacy index was 0.55 ± 0.19 at day 1, 0.67 ± 0.22 at day 5, and 0.87 ± 0.23 at 3 months. In Phase II, the efficacy index improved to 0.63 ± 0.25 (p = 0.039) at day 1 and to 0.75 ± 0.26 (p = 0,02) at day 5. The most frequent complications were intraoperative abrasions and epithelial cells in the interface. Almost all complications occurred less frequently in Phase II. Conclusion Our first SMILE treatments showed very good refractive predictability, very good safety and rapid visual recovery. Results after high myopic corrections were equivalent to or better than our first Femto-LASIK treatments. We had a much higher complication rate than with our LASIK treatments - that have been established over several years. All complications could be managed without loss of visual acuity. Visual recovery was faster and complications less frequent with experience gained. SMILE has replaced LASIK as our preferred treatment modality for the correction of myopic astigmatic eyes.

Enzymatic network for production of ether amines from alcohols.

We constructed an enzymatic network composed of three different enzymes for the synthesis of valuable ether amines. The enzymatic reactions are interconnected to catalyze the oxidation and subsequent transamination of the substrate and to provide cofactor recycling. This allows production of the desired ether amines from the corresponding ether alcohols with inorganic ammonium as the only additional substrate. To examine conversion, individual and overall reaction equilibria were established. Using these data, it was found that the experimentally observed conversions of up to 60% observed for reactions containing 10 mM alcohol and up to 280 mM ammonia corresponded well to predicted conversions. The results indicate that efficient amination can be driven by high concentrations of ammonia and may require improving enzyme robustness for scale-up. Biotechnol. Bioeng. 2016;113: 1853-1861. © 2016 Wiley Periodicals, Inc.

X-ray crystallographic validation of structure predictions used in computational design for protein stabilization.

Protein engineering aimed at enhancing enzyme stability is increasingly supported by computational methods for calculation of mutant folding energies and for the design of disulfide bonds. To examine the accuracy of mutant structure predictions underlying these computational methods, crystal structures of thermostable limonene epoxide hydrolase variants obtained by computational library design were determined. Four different predicted effects indeed contributed to the obtained stabilization: (i) enhanced interactions between a flexible loop close to the N-terminus and the rest of the protein; (ii) improved interactions at the dimer interface; (iii) removal of unsatisfied hydrogen bonding groups; and (iv) introduction of additional positively charged groups at the surface. The structures of an eightfold and an elevenfold mutant showed that most mutations introduced the intended stabilizing interactions, and side-chain conformations were correctly predicted for 72-88% of the point mutations. However, mutations that introduced a disulfide bond in a flexible region had a larger influence on the backbone conformation than predicted. The enzyme active sites were unaltered, in agreement with the observed preservation of catalytic activities. The structures also revealed how a c-Myc tag, which was introduced for facile detection and purification, can reduce access to the active site and thereby lower the catalytic activity. Finally, sequence analysis showed that comprehensive mutant energy calculations discovered stabilizing mutations that are not proposed by the consensus or B-FIT methods.

Dual-fluorescence pH probe for bio-labelling.

Although seminaphtorhodafluor (SNARF) dyes are already widely used to measure pH in cells and at biofilms, their synthesis has low yield and results in an unspecific position of a carboxy-group. The separation of 5'- and 6'-carboxy-SNARF reveals a pKa difference of 0.15, calling into question pH measurements with the (commercially available) mixture. Here we replace the bulky external dicarboxyphenyl ring with a propionate group and evaluate the spectral properties of the new derivative. Proceeding to the ethyl-iodoacetamide, covalent linkage to cysteine protein sites is achieved efficiently as shown with a cyanobacterial phytochrome, extending the scarce application of SNARF in bio-labelling in the current literature. Application in fluorescence lifetime imaging is demonstrated both with the lifetime-based and ratiometric-yield method.