Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Asymmetric Chemoenzymatic Synthesis of Ramatroban Using Lipases and Oxidoreductases

A chemoenzymatic asymmetric route for the preparation of enantiopure (<i>R</i>)-ramatroban has been developed for the first time. The action of lipases and oxidoreductases has been independently studied, and both were found as excellent biocatalysts for the production of adequate chiral intermediates under very mild reaction conditions. CAL-B efficiently catalyzed the resolution of (±)-2,3,4,9-tetrahydro-1<i>H</i>-carbazol-3-ol that was acylated with high stereocontrol. On the other hand, ADH-A mediated bioreduction of 4,9-dihydro-1<i>H</i>-carbazol-3­(2<i>H</i>)-one provided an alternative access to the same enantiopure alcohol previously obtained through lipase-catalyzed resolution, a useful synthetic building block in the synthesis of ramatroban. Inversion of the absolute configuration of (<i>S</i>)-2,3,4,9-tetrahydro-1<i>H</i>-carbazol-3-ol has been identified as a key point in the synthetic route, optimizing this process to avoid racemization of the azide intermediate, finally yielding (<i>R</i>)-ramatroban in enantiopure form by the formation of the corresponding amine and the convenient functionalization of both exocyclic and indole nitrogen atoms

Highly Stereoselective Chemoenzymatic Synthesis of the 3<i>H</i>-Isobenzofuran Skeleton. Access to Enantiopure 3-Methylphthalides

A straightforward synthesis of (<i>S</i>)-3-methylphthalides has been developed, with the key asymmetric step being the bioreduction of 2-acetylbenzonitriles. Enzymatic processes have been found to be highly dependent on the pH value, with acidic conditions being required to avoid undesired side reactions. Baker’s yeast was found to be the best biocatalyst acting in a highly stereoselective fashion. The simple treatment of the reaction crudes with aqueous HCl has provided access to enantiopure (<i>S</i>)-3-methylphthalides in moderate to excellent yields

One-Pot Synthesis of Enantiopure 3,4-Dihydroisocoumarins through Dynamic Reductive Kinetic Resolution Processes

A straightforward chemoenzymatic synthesis of enantiopure 4-alkyl-3-methyl-3,4-dihydroisocoumarins through a ketoreductase-catalyzed one-pot dynamic reductive kinetic resolution is reported. <i>E. coli</i>/ADH-A cells have shown outstanding diastereo- and enantioselectivity toward the bioreduction of a series of racemic ketones, with the use of anion exchange resins or triethylamine being compatible in the same aqueous reaction medium. The so-obtained enantiopure alcohols were subsequently cyclized in acid media affording the corresponding lactones in good to excellent conversions (72–96%) and excellent selectivities (dr ≥99:1 and ee >99%)

One-Pot Synthesis of Enantiopure 3,4-Dihydroisocoumarins through Dynamic Reductive Kinetic Resolution Processes

A straightforward chemoenzymatic synthesis of enantiopure 4-alkyl-3-methyl-3,4-dihydroisocoumarins through a ketoreductase-catalyzed one-pot dynamic reductive kinetic resolution is reported. <i>E. coli</i>/ADH-A cells have shown outstanding diastereo- and enantioselectivity toward the bioreduction of a series of racemic ketones, with the use of anion exchange resins or triethylamine being compatible in the same aqueous reaction medium. The so-obtained enantiopure alcohols were subsequently cyclized in acid media affording the corresponding lactones in good to excellent conversions (72–96%) and excellent selectivities (dr ≥99:1 and ee >99%)

Chemoenzymatic Asymmetric Synthesis of 1,4-Benzoxazine Derivatives: Application in the Synthesis of a Levofloxacin Precursor

A versatile and general route has been developed for the asymmetric synthesis of a wide family of 3-methyl-3,4-dihydro-2<i>H</i>-benzo­[<i>b</i>]­[1,4]­oxazines bearing different pattern substitutions in the aromatic ring. Whereas hydrolases were not suitable for resolution of these racemic cyclic nitrogenated amines, alternative chemoenzymatic strategies were designed through independent pathways leading to both amine antipodes. On one hand, bioreduction of 1-(2-nitrophenoxy)­propan-2-ones allowed the recovery of the enantiopure (<i>S</i>)-alcohols in high yields using the alcohol dehydrogenase from <i>Rhodococcus ruber</i> (ADH-A), whereas evo-1.1.200 ADH led to their counterpart (<i>R</i>)-enantiomers also with complete selectivity and quantitative conversion. Alternatively, lipase-catalyzed acetylation of these racemic alcohols, and the complementary hydrolysis of the acetate analogues, gave access to the corresponding optically enriched products with high stereodiscrimination. Particularly attractive was the design of a chemoenzymatic strategy in six steps for the production of (<i>S</i>)-(−)-7,8-difluoro-3-methyl-3,4-dihydro-2<i>H</i>-benzo-[<i>b</i>]­[1,4]­oxazine, which is a key precursor of the antimicrobial agent Levofloxacin

Stereoselective Synthesis of 2,3-Disubstituted Indoline Diastereoisomers by Chemoenzymatic Processes

Racemic indolines including a variety of structural motifs such as C-2 and C-3 substitutions (alkyl or aryl), <i>cis</i>/<i>trans</i> relative stereochemistry and functionalization of the aromatic ring (fluoro, methyl or methoxy groups) have been efficiently prepared through Fischer indolization and subsequent diastereoselective reduction of the unprotected indoles. Combination of <i>Candida antarctica</i> lipase type A and allyl 3-methoxyphenyl carbonate has been identified as the best tandem for their kinetic resolutions, observing excellent stereodiscriminations for most of the tested indolines

Biocatalytic Transamination for the Asymmetric Synthesis of Pyridylalkylamines. Structural and Activity Features in the Reactivity of Transaminases

A set of transaminases has been investigated for the biocatalytic amination of 1-(4-chloropyridin-2-yl)­alkan-1-ones. The influence of the chain length of the <i>n</i>-1-alkanone at the C-2 position of the pyridine has been studied in the reaction with different (<i>R</i>)- and (<i>S</i>)-selective transaminases. Thus, enantiopure amines were isolated with high purity starting from a wide selection of prochiral ketones. On the one hand, excellent yields (from 97 to >99% conversion, up to 93% isolated yield) and stereoselectivity values (>99% ee for both amine enantiomers) were found for <i>n</i>-1-alkanone linear short chain substituents such as ethanone or propanone. On the other hand, more hindered substrates were accepted only when using evolved enzymes such as an evolved variant of (<i>R</i>)-<i>Arthrobacter</i> (ArRmut11-TA). An initial common structural feature was the presence of a chlorine atom on the C-4 position of the pyridine core, which was found to increase the reactivity of the starting ketone, giving extra versatility for the introduction of other chemical functionalities toward more complex and applicable organic molecules. In order to gain a deeper understanding about the substrate specificity of different transaminases, additional structural features were considered by variation of the acetyl group position on the pyridine ring and the use of related acetophenone derivatives