Pichia pastoris and a radiolabel transport assay was established. Importantly and in contrast to mammalian cells, P. pastoris has proven useful as eukaryotic expression system to successfully express human LAT1 and LAT2 in the plasma membrane without the requirement of co-expressed trafficking chaperone 4F2hc. Our results show a novel function of the heavy chain 4F2hc that impacts transport by modulating the substrate affinity and specificity of corresponding LATs. In addition, the presented data confirm that the light chains LAT1 and LAT2 constitute the substrate-transporting subunits of the HATs, and that light chains are also functional in the absence of the ancillary protein 4F2hc.The human L-type amino acid transporters LAT1 and LAT2 mediate the transport of amino acids and amino acid derivatives across plasma membranes in a sodium-independent, obligatory antiport mode. In mammalian cells, LAT1 and LAT2 associate with the type-II membrane N-glycoprotein 4F2hc to form heteromeric amino acid transporters (HATs). The glycosylated ancillary protein 4F2hc is known to be important for successful trafficking of the unglycosylated transporters to the plasma membrane. The heavy  and light  chains belong to the solute carrier (SLC) families SLC3 and SLC7, and are covalently linked by a conserved disulfide bridge. Overexpression, absence, or malfunction of certain HATs is associated with human diseases and HATs are therefore considered therapeutic targets. Here, we present a comparative, functional characterization of the HATs 4F2hc-LAT1 and 4F2hc-LAT2, and their light chains LAT1 and LAT2. For this purpose, the HATs and the light chains were expressed in the methylotrophic yeast  We found that the heavy chain 4F2hc modulates the substrate affinity and specificity of LATs. In addition, the presented data confirm that the light chains LAT1 and LAT2 constitute the substrate-transporting components of the HATs and that these two light chains are also functional in the absence of 4F2hc. Thus, P. pastoris has proven useful as eukaryotic system to express and characterize the transport function of the human light chains LAT1 and LAT2 in the absence of co-expressed heavy chain/ancillary protein 4F2hc.The methylotrophic yeast proteins ,31 and fproteins . In an ooduction . Expressvy chain ,26,33. Ied 4F2hc . In contPichia pastoris. Western blot analysis indicated expression of the corresponding HATs and LATs  of L-leucine by homologues competition for all constructs using the obtained time points, i.e., 10 min  and 2 min (LAT2) , 11 µM (LAT1), 249 µM (4F2hc-LAT2), and 42 µM (LAT2)  . These IT2) (50s  gave firM (LAT2) . As refl., 18 µM  and 20 µ., 18 µM  for 4F2h., 18 µM . Recentl., 18 µM . In contin 4F2hc ,37.Km to compete with [3H]L-leucine uptake  for L-histidine to 23 µM using P. pastoris cells expressing human 4F2hc-LAT1  L-leucine, L-isoleucine, and L-valine were comparable C. Again,nsporter D. Only Lin 4F2hc .P. pastoris has proven useful for the here-presented comparative transporter study. In contrast to mammalian cells, the substrate-transporting subunits LAT1 and LAT2 could be successfully expressed functional in Pichia in the absence of their ancillary N-glycoprotein 4F2hc. Therefore, this eukaryotic expression system, which also allows post-translational modifications such as glycosylation and disulfide bridge formation, opens the possibility to investigate the influence of pharmacologically relevant compounds on the function of heteromeric complexes and their transporters alone.We have shown and confirmed that the light chains LAT1 and LAT2 are the substrate-transporting subunits of the corresponding HATs 4F2hc-LAT1 and 4F2hc-LAT2, and that 4F2hc is not essential for the transport activity of the corresponding LATs. LAT1 and LAT2 have relatively high specificities for L-leucine, and modest specificities for other amino acids. That the ancillary protein 4F2hc is responsible for chaperoning the trafficking of light chains  to the plasma membrane of mammalian cells is a well-accepted and described concept. Our comparative transporter study revealed a novel function of 4F2hc, i.e., upon association of this ancillary protein with LAT1 and LAT2, the substrate affinity and specificity of these light subunits is modulated by significantly broadening their substrate specificities. The methylotrophic yeast Pichia pastoris clones expressing human 4F2hc-LAT2 or LAT2 was described in detail previously L-leucine were selected for further experiments and verified for correct integration by PCR. Selected clones and untransformed P. pastoris KM71H cells were initially inoculated in 10 mL of YPD media as a seed culture in 50 mL culture tubes and grown for 24 h at 30 °C and 300 rpm in an incubator shaker . From seed cultures, 5 mL of inoculum were added to 500 mL buffered glycerol-complex medium  glycerol, 1% (w/v) bacto yeast extract (BD Biosciences), 2% (w/v) peptone , 100 mM potassium phosphate pH 6.0, 1.34% (w/v) yeast nitrogen base YNB , 4 × 10−5% (w/v) biotin) and grown overnight (12–14 h) to OD600 4–6 at 30 °C and 200 rpm using 2 L Erlenmeyer culture flasks in an incubator shaker . For protein expression, cells were pelleted by centrifugation  and resuspended in 1/5 to 1/10 of the original culture volume  in buffered methanol-complex medium  methanol instead of 1% (v/v) glycerol) to a final cell density of OD600 40. Resuspended cells (50–75 mL) were grown in 1 L Erlenmeyer culture flasks at 30 °C and 300 rpm in an incubator shaker . Conditions for induction were maintained by supplementing the expression culture with methanol after 20 and 24 h to a final concentration of 1% (v/v). Cells were harvested 28 h post induction by centrifugation . The resulting pellet was resuspended in transport buffer (150 mM choline chloride (ChCl), 1 mM MgCl2, 1 mM CaCl2, 10 mM HEPES and 10 mM Tris, pH 7.4) containing 50% (v/v) glycerol, the OD600 was adjusted to 40, and the cells were finally stored at −18 °C.Electrocompetent P. pastoris cells expressing the corresponding HAT or LAT used in the here-presented functional studies. For Western blots, ~15 mg Pichia cells expressing HATs or LATs were suspended in 1 mL of 50 mM potassium phosphate pH 7.4, 1 mM EDTA, 5% (v/v) glycerol, 2% (w/v) SDS, 5 mM oxidized glutathione and protease inhibitor . Cells were lysed at room temperature by homogenization with 20 strokes using a glass Teflon homogenizer. This lysis procedure was repeated four more times after 30 min incubation between each pass. Insolubilized cells and debris were separated by centrifugation at 12,000× g . The supernatant was mixed with 5x non-reducing sample buffer  glycerol, 2% (w/v) SDS, 0.01% (w/v) bromophenol blue) to a final concentration of 2.5x non-reducing sample buffer and separated on a 10% SDS-PAGE gel. The His-tagged transporter LAT1 was detected using a mouse anti-His5 primary antibody  at a dilution of 1:3000 and a goat anti mouse IgG (H+L) horse radish peroxidase (HRP) conjugated secondary antibody  at a dilution of 1:3000. Strep-tagged transporters  were detected with HRP-conjugated streptavidin  at a dilution of 1:30,000.Immunoblotting experiments were performed using P. pastoris cells at OD600 40 expressing the corresponding transporter were thawed, diluted 1:50 in transport buffer, and pelleted by centrifugation . Subsequently, the pellet was washed by resuspending in 50 mL transport buffer and pelleted again. The washing step was repeated twice. Finally, the cell pellet was resuspended in 4 mL of transport buffer and incubated for 20 min at 30 °C under agitation . The density of the yeast suspension was adjusted with transport buffer to OD600 25  and 7.5 (LAT2). All transport experiments were performed in a reaction volume of 100 µL. For time course experiments, the reaction mixture contained 40 µL cell suspension and 60 µL substrate master mix ) to a specific activity of 20 Ci/mmol resulting in a final L-leucine concentration of 100 nM. For the determination of the Michaelis–Menten constant (Km), the reaction mixture contained 40 µL cell suspension and 60 µL of L-leucine solution yielding final concentrations ranging from 1–3000 µM (4F2hc-LAT2 and LAT2) and 1–1000 µM (4F2hc-LAT1 and LAT1), which were spiked with [3H]L-leucine to a specific activity of 0.033 Ci/mmol. For L-leucine and L-histidine IC50 experiments, the reaction mixture contained 40 µL cell suspension, 50 µL of competitor solution at different concentrations, i.e., 0.01–8000 µM , 0.01–10,000 µM  and 1–10,000 µM , and 10 µL substrate master mix ) to a specific activity of 20 Ci/mmol resulting in a final L-leucine concentration of 100 nM. For substrate profiling , the reaction mixture contained 40 µL cell suspension, 50 µL of competitor solution , and 10 µL substrate master mix ) to a specific activity of 20 Ci/mmol resulting in a final L-leucine concentration of 100 nM. Competitors were prepared in 10% (v/v) DMSO yielding a final concentration of 0.5% in the assay. Control samples contained the same concentration of DMSO. Final OD600 values in uptake experiments were 10 for 4F2hc-LAT1, 4F2hc-LAT2, and LAT1, and 3 for LAT2. All transport reactions were done in 2 mL reaction tubes (Eppendorf) at 25 °C under agitation . Transport was terminated after 10 min for 4F2hc-LAT1, 4F2hc-LAT2, or LAT, and 2 min for LAT2 by addition of 600 µL of pre-chilled transport buffer. Cells were rapidly separated from the buffer by transferring the stopped reactions on a 96-well 0.66 mm glass fiber filter plate  and vacuum filtration. Each well was washed with 2 mL of ice-cold transport buffer to remove free radioligand. The plate was then dried overnight at 37 °C and the backside was sealed with back seal . The trapped radioligand was released by addition of 200 µL scintillation cocktail  to each well and the plate topside was sealed with TopsealTM-A Plus (PerkinElmer), followed by incubation for 30 min at 25 °C and 1000 rpm . Counts were measured in each well for 2 min with a scintillation counter .For transport experiments, 3 mL P. pastoris cells was subtracted from the transporter signal. Michaelis–Menten saturation curves were fitted into data points of independent experiments. Data points were then individually normalized using the corresponding Vmax values . Data points from corresponding concentrations were averaged and SD obtained. Finally, Michaelis–Menten saturation curves were fitted to the averaged data yielding Km values. To determine the half maximal inhibitory concentration (IC50) values of heterologous  L-leucine transport competition, a sigmoidal model curve was fitted to the net transport signals of independent experiments. Every experimental data point was individually normalized using the corresponding upper plateau values . Data points from corresponding concentrations were averaged and a sigmoidal model curve was fitted to the data in order to obtain the IC50 value. Prism6 (GraphPad Software) was used for data analysis.Experimental data points were performed at least in triplicate. For data analysis, the signal of the untransformed


To generate mAbs, mice were immunized with LNFPIII-DEX (P3DEX) plus CpGs in VacSIM®, a novel vaccine/drug delivery platform. Mice were boosted with LNFPIII-HSA (P3HSA) plus CpGs in Incomplete Freund’s Adjuvant (IFA). Splenocytes from immunized mice were used to generate hybridomas and were screened against LNFPIII conjugates via enzyme-linked immunosorbent assay (ELISA). Three positive hybridomas were expanded, and one hybridoma, producing IgG and IgM antibodies, was cloned via flow cytometry. Clone F1P2H4D8D5 was selected because it produced IgG1 mAbs, but rescreening unexpectedly showed binding to both LNFPIII and lacto-N-neotetraose (LNnT) conjugates. To further assess the specificity of the mAb, we screened it on two glycan microarrays and found no significant binding. This finding suggests that the mAb binds to the acetylphenylenediamine (APD) linker-spacer structure of the conjugate. We present the results herein, suggesting that our new mAb could be a useful probe for conjugates using similar linker spacer structures.Monoclonal antibodies (mAbs) that recognize glycans are useful tools to assess carbohydrates’ structure and function. We sought to produce IgG mAbs to the human milk oligosaccharide (HMO), lacto-N-fucopentaose III (LNFPIII). LNFPIII contains the Lewis Glycans are considered core biological building blocks. Glycans are ubiquitous in nature and exert their effects via their own properties or via the modification of proteins and lipids. Their presence on cell surfaces provide strength and protection, and they also serve as ligands for receptors  to modulate signaling . GlycansThe field of glycobiology lacks well-developed tools that facilitate a functional analysis. In terms of structure, glycans are more complex than nucleic acids and proteins because of the assortment of known monosaccharides and their ability to be linked in various numbers and fashions  . In thisx trisaccharide, α-L-Fuc-(1→3)-[β-D-Gal-(1→4)]-D-GlcNAc, present on the surface of schistosome eggs, adult tissues, and cancerous tumors [x antigen. mAbs against the Lewisx antigen are well-reported in the literature, but mAbs recognizing HMO structures are rare [x must be presented on adjacent molecules or in a multimeric form to bind to or activate cells [In contrast to lectins, GBPs bind to specific determinants and do not discriminate between O-glycans, N-glycans, or glycolipids . Severals tumors ,15,16,17s tumors . Herein,are rare ,17,19. Pte cells . HMOs, ste cells .Here, we report the development and characterization of a novel IgG mAb (F1P2H4D8D5) generated from the splenocytes of mice immunized with LNFPIII glycan conjugates. LNFPIII conjugates are composed of 10–12 molecules of LNFPIII conjugated to a 40 kDa dextran (P3DEX) or to human serum albumin (P3HSA) via an acetylphenylenediamine (APD) linker. The combined use of a carrier (DEX or HSA) and this linker method increases the concentration of LNFPIII in the conjugates and allows each molecule to rotate in space to bind to cellular receptors. Unlike free LNFPIII, these LNFPIII conjugates have been shown to act on B cells, macrophages, dendritic cells, hepatocytes, and adipocytes in vivo ,24,25,26The initial screening of mAb F1P2H4D8D5 showed binding to LNFPIII conjugates and to conjugates containing the structurally similar tetrasaccharide, LNnT. LNnT conjugates are also conjugated to a 40 kDa dextran (NTDEX) or to human serum albumin (NTHSA). The results of additional screens suggested that the mAb might be binding to the linker spacer of the conjugate and this possibility was partially confirmed when the mAb failed to bind to any glycan structures on both human milk and general glycan structure microarrays. Since APD linkers are used to produce a wide array of conjugates, an anti-APD mAb is valuable to the research community as a probe for any structures produced using the same linker ,28. The LNFPIII was synthesized by Dr. George Wang . LNnT was synthesized by Neose Technologies, Inc. LNFPIII and LNnT were sent to Dr. Thomas Norberg  for conjugation to carriers, dextran from Leuconostoc mesenteroides , or human serum albumin  using proprietary APD linker-spacers. On average, each conjugate had 10–12 HMO monomers per 40 kDa dextran or HSA.g for 1 h at 4 °C, and the supernatant was collected as SEA. Protein was quantified via the Pierce™ BCA Protein Assay Kit  and stored at −80 °C until use.Soluble schistosome egg antigen (SEA) was prepared as described previously . Brieflyg for 1 h at 4 °C. Deoxycholate was removed from SMP by desalting on Pierce™ Polyacrylamide Spin Desalting Columns, 7K MWCO, 0.7 mL . Protein was quantified via the Pierce™ BCA Protein Assay Kit  and stored at −80 °C until use.Schistosomula membrane protein (SMP) was prepared as described previously . In briew/v VacSIM™ (Vaccine Self-Assembling Immune Matrix) [Glycoconjugates were dissolved in 0.9% sterile saline and stored at −80 °C until use. 200 µg of P3DEX plus 50 µg of CpG ODN 1826  were added to 1%  Matrix) , then in® CRL-1581™) were grown to 100% confluence in DMEM containing 20% FBS + 8 mM L-glutamine + 100 µg/mL penn/strep at 37 °C in 5% CO2. Splenocytes were mixed with Sp2/0-Ag14 cells at a ratio of 8:1 splenocytes per Sp2/0-Ag14 cell and then pelleted by centrifugation. The supernatant was removed, and cells were dislodged by thwacking the tube. Cell fusion was induced by adding 0.7 mL of 50% polyethylene glycol  for 1 min at 37 °C. Cells were pelleted, the supernatant was removed, and cells were gently resuspended by slow addition of 7 mL of warm (37 °C) DMEM. Then, cells were diluted with an additional 43 mL of hybridoma HAT media (DMEM supplemented with Gibco™ HAT Supplement (50X) ) + 15% FBS + 1% oxaloacetate-pyruvate-insulin (OPI)  + 100 µg/mL penn/strep and plated into 96-well plates at 100 µL/well. 7D post-fusion, 100 µL/well of hybridoma HT media (DMEM) supplemented with hypoxanthine-thymidine (HT) + 15% FBS + 1% OPI + 100 µg/mL penn/strep was added. Plates were screened for hybridoma growth and the cell supernatants were tested by ELISA against P3DEX. Hybridomas positive for P3DEX and IgG, but not HSA and DEX, were cloned via flow sorting on a MoFlo Astrios Cell Sorter. In brief, cells were stained using a LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit , resuspended in hybridoma HT media, and sorted into a single cell per well. The determination of Ig subtypes was performed with the SBA Clonotyping System-HRP  via indirect ELISA, in order to determine the specific immunoglobulin subclass. Positive hybridomas were propagated and stored in liquid nitrogen.Mice were sacrificed on D45 for spleen removal and the preparation of splenocytes. Red blood cells (RBCs) were lysed by adding 2 mL of RBC Lysis Buffer  and incubating for 3 min, followed by two washes in HyClone™ Dulbecco’s Modified Eagles Medium (DMEM) . Sp2/0-Ag14 cells  were coated at 100 µL/well with one of the following: (1) 5 μg/mL DEX, (2) 5 μg/mL HSA, (3) 50 μg/mL P3DEX, (4) 100 μg/mL P3HSA, (5) 50 μg/mL NTDEX, (6) 100 μg/mL NTHSA, (7) 5 μg/mL SEA, or (8) 5 μg/mL SMP in 0.05 M carbonate bicarbonate buffer, pH 9.6, and incubated for 16 h at 4 °C. Plates were then washed 6× in PBS with 0.05% Tween-20 (PBST) and blocked via the addition of 300 µL/well of 2.5% milk in PBST at room temperature (RT) for 2 h. After additional washing, 100 µL/well of mice serum (1:100 in PBS) or hybridoma supernatant was added to the plate and incubated at RT for 2 h. Plates were washed and horseradish peroxidase-conjugated anti-mouse IgG (H+L) and IgM  or a panel of isotyping antibodies  were added as required to the wells at a dilution of 1:4000 in PBST at RT for 1 h. Plates were washed four times, and color was developed using 3,3′,5,5′-tetramethylbenzidine . ELISA reactions were stopped after 10 min of incubation in the dark at RT with 50 µL of sulfuric acid. The optical density (OD) was determined using the SPECTROstar Nano Microplate Reader at 450/570 nm.2. Supernatants were recovered at 100% confluence and stored at 4 °C until purification. In order to isolate IgG antibodies, hybridoma culture supernatants were purified via ammonium sulfate precipitation according to Fishman and Berg (2018), with some modifications [g for 30 min at 4 °C. Supernatants were then slowly mixed with saturated ammonium sulfate for a final w/v concentration of 25% and incubated at 4 °C overnight. The solution was then centrifuged again at 3000× g for 30 min at 4 °C. Saturated ammonium sulfate was then added to reach a final w/v concentration of 50% and was incubated at 4 °C overnight. The solution then underwent a third round of centrifugation at 3000× g for 30 min at 4 °C. The supernatant was discarded, and the remaining pellet was resuspended in 30% of the original volume of 1× PBS. The sample was dialyzed against three changes of 1× PBS using 10 K Slide-A-Lyzer™ cassettes . The protein concentration was determined using a Nanodrop 2000 spectrophotometer. Following ammonium sulfate precipitation, eluted proteins were further purified using HiTrap protein G columns  according to the manufacturer’s protocol. After protein quantification, 2.5 or 5 µg of purified antibody was loaded into two identical 4–20% SDS-PAGE gels. Gels were run for 2 h at 50 V, 3.00 A, 300 W using the PowerPac™ HC High-Current Power Supply . One gel was stained with Coomassie Brilliant Blue R-250 , and the other was transferred to a PVDF membrane after electrophoresis using the Trans-Blot Turbo RTA Mini 0.45 µm LF PVDF Transfer Kit . Gels were transferred using the Trans-Blot Turbo Transfer System  programmed for mixed molecular weight proteins at 2.5 A and 25 V for 7 min. The membrane was blocked at 4 °C for 16 h in 5% milk in tris-buffered saline plus 0.05% Tween (TBST). Subsequently, the membrane was incubated with peroxidase-conjugated anti-mouse IgG and IgM diluted in 3% milk in TBST at RT for 1 h. The membrane was washed 5× and revealed using the ECL Plus Western Blotting Detection System , and images were captured using chemiluminscence detection on a Bio-Rad imager.We selected a hybridoma clone secreting IgG1: mAb F1P2H4D8D5 for further analysis. Hybridomas were grown to 80% confluency in DMEM containing 20% FBS + 8 mM L-glutamine + 100 µg/mL penn/strep at 37 °C in 5% COications . In brie® TIB-71™) cells were grown in DMEM plus 10% FBS, 4 mM L-glutamine, and 100 U/mL + 100 µg/mL penn/strep. For the experiments, 3 × 105 cells/well were plated in a 6-well plate. Once 70–80% confluent, cells were stimulated with (1) sterile saline, (2) 50 µg/mL DEX, (3) 50 µg/mL HSA, (4) 50 µg/mL P3DEX, (5) 50 µg/mL NTDEX, (6) 50 µg/mL P3HSA, or (7) 50 µg/mL NTHSA. Cells were stimulated for 60 s, washed, and lysed using NP-40 buffer . Protein was quantified using a Nanodrop, and lysates were stored at −80 °C until use.RAW 264.7  and transferred to a PVDF membrane, as described in https://ncfg.hms.harvard.edu/microarrays and are described in [mAbs were characterized in terms of: (1) specificity to different antigen targets in ELISAs and Western blots, and (2) a microarray analysis conducted at Harvard’s Center for Functional Glycomics (NCFG). ELISAs were performed according to ribed in .w/v [p < 0.001) compared to the pre-prime levels  contained hybridomas. The screening of hybridomas generated from one mouse yielded three hybridomas (3/75) producing antibodies to LNFPIII conjugates. We isotyped these hybridomas and determined that F1P2C8 was IgG subclass, while F1P2D8 and F1P2H4 were both IgG and IgM . F1P2H4 Following ammonium sulfate precipitation and protein G column purification, supernatants from clone F1P2H4D8D5 were run via SDS-PAGE electrophoresis. Next, we performed another screen of the purified mAb via ELISA against each of the sugar conjugates, the carrier structures (DEX and HSA), as well as against schisosome egg antigen (SEA) and schistosomula membrane protein (SMP). In this ELISA, our positive control mAb E.5 recognized P3DEX, P3HSA, SEA, and SMP (as expected) and did not bind to NTDEX or the carriers. The sole difference between LNFPIII and LNnT is the presence/absence of an α1,3-linked fucose residue, as depicted in the chemical structures shown in In x trisaccharide. Again, we were surprised to see that mAb F1P2H4D8D5 did not bind to any of the glycans (1–159) present on the array. This result supported our hypothesis that mAb F1P2H4D8D5 likely binds to the APD linker, since it did not recognize DEX (ELISA), HSA (ELISA/Western blot), or the sugar monomer/structures .To further characterize mAb F1P2H4D8D5 and determine if it recognized any common glycan structures, we sent aliquots of purified mAb to Harvard’s National Center for Functional Glycomics to be tested for binding on a Human Milk Glycan Microarray . Surprisx antibodies are IgM and hybridoma F1P2D8 contained IgM mAbs, this hybridoma is more likely to be specific for LNFPIII (We attempted to produce mAbs binding to LNFPIII by immunizing mice with LNFPIII conjugates. We detected anti-LNFPIII conjugate antibodies in the sera of immunized mice after the prime. We boosted with the aim of generating IgG antibodies . Initial LNFPIII . This co LNFPIII . Future x. Taken together, these results further led to the conclusion that F1P2H4D8D5 binds to the linker spacer of the glycan conjugates.The lack of available tools in glycan biology presents some important limitations. Based on our findings, we deduce that F1P2H4D8D5 binds to the linker spacer of the conjugate because it does not recognize the 40 kDa dextran or HSA carrier a,b or anx trisaccharide, there are few reports of antibodies specific to the HMOs, LNFPIII, or LNnT.Overall, this is important because mAb F1P2H4D8D5 recognized LNFPIII and LNnT conjugates internalized by RAW 264.7 cells. This mAb can be used as an immunological probe for ELISA, Western blot, immunoprecipitation, etc. to recognize conjugates that use this linker-spacer technology. Furthermore, it is important because the in vivo generation of antibodies to drug conjugates can result in drug clearance prior to therapeutic effects. While it is known that mAbs can be generated to the LewisIt is difficult to generate mAbs to carbohydrate moieties. We sought to produce mAbs to the HMO LNFPIII, but appear to have generated mAbs to the APD linker of LNFPIII and LNnT conjugates. We immunized with these conjugates, given that LNFPIII and LNnT do not appear to be antigenic in their free form. The conjugation to this APD linker allows the glycoconjugate to cross-link multiple receptors and bind multiple cell types ,34,35,36


Single-chain variable fragments (scFvs) are small-sized artificial constructs composed of the immunoglobulin heavy and light chain variable regions connected by a peptide linker. We have previously described an anti-fibroblast growth factor 2 (FGF2) immunoglobulin G (IgG) monoclonal antibody (mAb), named 3F12E7, with notable antitumor potential revealed by preclinical assays. FGF2 is a known angiogenesis-associated molecule implicated in tumor progression. In this report, we describe a recombinant scFv format for the 3F12E7 mAb. The results demonstrate that the generated 3F12E7 scFv, although prone to aggregation, comprises an active anti-FGF2 product that contains monomers and small oligomers. Functionally, the 3F12E7 scFv preparations specifically recognize FGF2 and inhibit tumor growth similar to the corresponding full-length IgG counterpart in an experimental model. In silico molecular analysis provided insights into the aggregation propensity and the antigen-recognition by scFv units. Antigen-binding determinants were predicted outside the most aggregation-prone hotspots. Overall, our experimental and prediction dataset describes an scFv scaffold for the 3F12E7 mAb and also provides insights to further engineer non-aggregated anti-FGF2 scFv-based tools for therapeutic and research purposes. ScFvs consist of artificial constructs composed of the variable regions of the heavy (VH) and light (VL) immunoglobulin chains connected by a flexible peptide linker in a way to retain the antigen-binding properties of the corresponding IgG antibody2. The scFvs are often produced with high yield using bacterial expression systems3, which comes as an advantage over the production of full-sized IgGs by hybridoma cells. Also, the expected small molecular size for such recombinant constructions may imply higher diffusion inside tumors and blood clearance3. Several scFvs have been described to target tumors, with some of them already approved for clinical use5.Advances in recombinant antibody technology led to the development of a large variety of engineered monoclonal antibody (mAb) constructions differing in pharmacokinetic and binding properties for research, diagnostic, and therapeutic applications. Among the recombinant antibody formats, there is the single-chain variable fragment (scFv)6, based on the conventional hybridoma technology7. FGF2 is a key angiogenesis-related factor implicated in the development and progression of several tumors10. The biological effects of FGF2 are exerted through the formation of ternary FGF2-heparin-FGF receptor complexes in several cells in the tumor microenvironment, including endothelial and neoplastic cells12.To target tumor growth, our research group previously generated an immunoglobulin G (IgG) monoclonal antibody (mAb) directed against the fibroblast growth factor 2 (FGF2)6. However, despite the promising data, this full-length IgG mAb failed to inhibit the growth of already established tumors, which encouraged us to design new small-sized agents to target FGF2.Our anti-FGF2 IgG mAb, named 3F12E7, showed promising results in experimental approaches. It was demonstrated to successfully reduce tumor blood vessel density and to inhibit tumor growth and metastasis, as previously describedH and VL domains of the 3F12E7 mAb. The functional activity of the 3F12E7 scFv was evaluated considering its aggregation and binding properties. Our results revealed a soluble anti-FGF2 product that, although prone to aggregation, contains monomers and small oligomers. The obtained scFv succeeded in specifically recognizing FGF2 in tumor extracts and inhibiting experimental tumor growth similar to the corresponding full-length IgG antibody counterpart. Also, in-silico predictions provide useful insights to understand the antigen-recognition by scFv units and to further engineer non-aggregated scFv-based tools for therapeutic and experimental applications.In this report, we describe an scFv construct generated by the fusion of the VH) and light (VL) variable chains were amplified from the cDNA of 3F12E7 hybridoma cells 3] peptide as a linker  pLysS cells. The bacterial expression vector is schematically indicated in Fig. To proceed with the study and obtain a purified scFv, we opted to clone the 3F12E7 scFv gene into pET26b vector, which was further transformed to The binding of the affinity-purified 3F12E7 scFv to FGF2 was confirmed by ELISA analysis Fig. a.Figure The antitumor effect of 3F12E7 anti-FGF2 scFv was evaluated in the B16-F10 experimental tumor model. Tumor-bearing mice received 3F12E7 mAb in its full-length or scFv format (or IgG control) every two days. Tumor growth curves and the tumor mass on day 12 are provided in Fig. 14, which accentuates the property of particular scFv constructs to aggregate. Considering the protein primary structure, different web-based servers  were used to predict the aggregation-prone segments of the 3F12E7 scFv. Figure H and VL domains. CDRs were determined using the Paratome web server.Production of functional scFv in bacteria is known to be challenging due to the frequent protein accumulation within inclusion bodies and the formation of kinetically trapped misfolded unitsThe aggregation state of the 3F12E7 scFv was assessed by native gel electrophoresis (BN-PAGE) and dynamic light scattering (DLS) analysis. BN-PAGE data indicate monomer and small oligomers in the scFv preparation. The representative BN-PAGE profile in Fig. Size-exclusion chromatographic (SEC) analysis of soluble 3F12E7 scFv samples (on a PD-10 column) revealed that the initial eluted fractions with 280-nm absorbance have different FGF2 binding properties, as indicated in Fig. The 3F12E7 scFv was found specific to FGF2. Immunoblotting assays performed with B16-F10 tumor extracts showed that biotinylated 3F12E7 scFv exhibited the same protein labeling pattern as that of the full-length IgG counterpart. An ~ 18-kDa band is detected in blots incubated with both formats of the 3F12E7 mAb Fig. b. The biIn silico molecular analyses provided insights into the interaction between the 3F12E7 scFv and its ligand, FGF2 (PDB ID: 1bfg), using for that a homology model of the scFv assembly in a monomeric closed state. Docking studies were performed with the Rosetta server and metrics of the top 5-scored decoys are presented in Fig. H-CDR2 and VL-CDR3, as indicated in Fig. The analysis of the 3F12E7 scFv monomer-FGF2 putative interface and the 3D cartoon representation of the docked complex are depicted in Fig. 6. Our molecular construct follows a traditional scFv scheme comprising the VH and VL segments of the IgG counterpart. These domains were joined by a 15-residue glycine-serine linker, used in many of the reported scFvs18, leading to an artificial VH-VL arrangement – named 3F12E7 scFv – whose monomer molecular mass is around 35 kDa.Herein we report the construction and the initial characterization of an scFv format of the already described anti-FGF2 3F12E7 mAb20. These findings are similar to those achieved with the corresponding full-length IgG mAb and come from a product containing scFv monomer and small oligomers, as assessed by native electrophoresis (BN-PAGE). But regarding the 3F12E7 scFv colloidal profile, what attracts attention is the high soluble aggregate content. Based on dynamic light scattering (DLS) measurements, some of the soluble scFv particles surpasses 100 nm in hydrodynamic diameter, which is several times greater than that detected for a full-length IgG antibody.The 3F12E7 scFv was characterized from both functional and colloidal perspectives. The provided experimental results demonstrate the scFv effectiveness in binding FGF2 and inhibiting the growth of B16-F10 experimental tumors, known to be dependent on the FGF2-mediated signaling14. This may be significantly boosted by the particular propensity of the designed scFv to aggregate. Our in-silico analyses pointed several aggregation hotspots within the 3F12E7 scFv amino acid sequence. This feature should not be considered exclusive to our construction, since high-molecular-mass multimers have already been found in previously described scFv preparations23.The 3F12E7 scFv aggregation potentially reflects aspects related to the employed protein expression and refolding strategy. Large-scale production of scFvs in bacterial expression systems, although practical and time efficient, often leads to a product containing aggregates24. Notwithstanding that, the 3F12E7 scFv revealed specific to FGF2, as assessed by immunoblotting assays. Also, SEC analysis of such soluble scFv product showed that even the initial protein elution fractions, supposedly enriched by large aggregate entities, bind to FGF2 without any important cross-reactivity to albumin. It should be further noted that the antigen-binding profile of these SEC fractions provides evidence that 3F12E7 scFv multimeric species have distinct anti-FGF2 properties. Two nonconsecutive fractions are underscored by their relatively high reactivity to FGF2, compared to the other evaluated samples. It is possible that such characteristic comes from a particular oligomeric content exhibiting multivalence, with higher avidity over the monomeric counterpart, which is consistent with the already described for other scFvs26.An scFv product composed of large soluble aggregate particles naturally raises concerns about its biological applicability. As an additional complicating factor, the high protein concentration formulations typically used for in vivo applications may favor the formation of aggregates27.Although misfolded forms are typically not active and prone to irreversible aggregation, we cannot discard that denatured antibody complexes may retain a fraction of functionally active fragments. Following SEC separation of biotinylated 3F12E7 scFv, an analysis of the initial protein eluates through BN-PAGE points out strong monomer-related bands in all these fractions. It is possible that monomers weakly anchored to denatured core units were released during electrophoretic separation, contributing in part to the obtained result. Also, we cannot rule out that the biotin labeling reaction, performed after the protein purification and refolding steps, could have impaired, in some way, the stability of the scFv complexes. What our findings collectively demonstrate, though, is that, despite being densely populated by soluble aggregated components, both the unlabeled and the biotinylated scFv materials are anti-FGF2 active and comprise a structurally and functionally heterogeneous molecular repertoire that includes monomeric and small oligomeric conformers. It should be noted that, although DLS data do not reveal particles compatible with small scFv units, this may be due to the light scattering by large structures. According to Rayleigh’s approximation, the intensity of scattering is proportional to the sixth power of a particle diameter12. It is worth noting that our computational predictions are in line with the previous finding that the full-length IgG format of the 3F12E7 mAb inhibits the binding of FGF2 to FGFR6. Also, it is interesting to note that, consistent with previous reports29, most of the 3F12E7 scFv aggregation-prone motifs were predicted within CDR loops. Despite that, the CDR1 domain, predicted to concentrate most of the aggregation-prone residues in both heavy- and light-chain antibody segments, potentially does not have a critical role in FGF2 binding. In-silico analysis of the putative scFv-FGF2 interface revealed that most of the antibody determinants involved in antigen recognition are located within the heavy-chain CDR2 and the light-chain CDR3, whose residues are less prone to aggregation.Our exploratory in-silico analyses additionally provide mechanistic insights into the antigen–antibody interaction. The 3F12E7 scFv was predicted to contact non-linear stretches within the FGF2 surface, which englobes the heparin and the receptor-binding sites. The inhibition of such FGF2 function-related domains by the scFv construct possibly contributed to the detected in vitro and in vivo effects. It is known that the interaction of FGF2 to both its cognate receptor and heparin accounts for the FGF2-mediated signal transduction3, the demonstrated specificity to FGF2 put the employed scFv design as a potential template to be further used to engineer a monomeric product for therapeutic purposes and also for addressing the significance of FGF2 inhibition on tumor growth.Overall, our results describe and discuss a promising scFv scaffold of the 3F12E7 mAb. While the detected colloidal features are away from the desired for in vivo interventions6 and 1F5H230 hybridoma cells were cultured in the same medium with the addition of 50 μM 2-mercaptoethanol. All cells were maintained at 37 °C in 5% CO2 and were checked for Mycoplasma contamination.Human Umbilical Vein Endothelial Cell (HUVEC), HEK293T, and B16-F10 cells, all of them obtained from ATCC (USA), were cultured in RPMI-1640 medium  with 10% fetal bovine serum . 3F12E7H and VL DNA fragments were amplified by PCR using 1 µM primers, 40 U/mL Vent polymerase (NEB BioLab), and 0.2 mM dNTP, as described previously32. For VH region, the forward primer is VH1FOR-2 (primer group A) and the back primer, VH1BACK (primer group B). For VL chain, a mix of four different primers  (primer group C) to the J gene segments encoding most of the mouse V kappa families was used as forward primer. VK2BACK (primer group D), common to the four kappa light chains, was used as back primer32. Primer sequences are listed in Table Total RNA of 3F12E7 hybridoma cells was isolated with TRIzol  and the cDNA was synthesized using a cDNA reverse transcription kit . The VH (with 340 bp) and VL (with 325 bp) region were separated on 1% agarose gel, purified with GeneClean kit , and further analyzed by an automated DNA sequencer . Paratome web server (available at http://www.ofranlab.org/paratome/) was used to predict the complementary-determining regions (CDRs) of 3F12E7 scFv, based on the primary sequence of the heavy and light variable chains33.PCR was run at 94 °C/3 min, followed by 30 cycles of 94 °C/1 min (denaturation), 56 °C/1 min , and 72 °C/1 min (elongation), with a final 5 min reaction at 72 °C. The amplified products from the VH-linker-VL format, as described previously13, using peptide [(Gly4Ser)3] peptide as a linker. The 3F12E7 scFv gene was acquired from GenScript gene synthesis service (USA) into the pcDNA3.1 expression vector. This construct, named pcDNA3.1-3F12E7 scFv, contains the scFv-coding sequence inserted between HindIII and XbaI restriction sites. The plasmid vector was digested with HindIII and XbaI and the DNA encoding scFv was confirmed by DNA sequencing. The PCR amplification was carried out in the same conditions described above.The 3F12E7 scFv was constructed in a VNcol and XhoI restriction sites, using primers E and F  pLysS (Novagen), used as an expression host. A single colony of transformed E. coli cells was incubated overnight at 37 ºC in 10 mL of Luria–Bertani (LB) medium containing kanamycin (50 µg/mL) and chloramphenicol (50 µg/mL), with shaking at 150 rpm. Pre-culture was diluted 200 × in 1.5 L of fresh LB medium and incubated until the culture density reaches an OD600 between 0.6 and 0.8. The expression of 3F12E7 scFv was induced by 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 4 h. Then, bacterial cells were harvested by centrifugation at 6000 rpm for 10 min and the cell pellet was further used for scFv purification.The pET26b-3F12E7 scFv vector was transformed into Bacterial cells expressing 3F12E7 scFv were suspended in TB buffer  and disrupted by using French Pressure Cell Press (1.6 MPa). The resulting lysate was centrifuged at 10,000 rpm for 10 min. The insoluble fraction was washed twice with TB buffer, incubated with solubilization buffer  for 2 h, and then centrifuged at 10,000 rpm. The supernatant was loaded, at a flow rate of 2.5 mL/min, on a Ni-Sepharose high-performance column  equilibrated with solubilization buffer and attached to an FPLC system . Purified scFv was recovered with 200 mM imidazole in elution buffer  and was further analyzed by SDS-PAGE. Protein refolding procedure was performed by 48-h dialysis against water at 4 °C, with water change every 12 h. Samples were filtered through a 0.22-µm filter prior to their in vitro and in vivo application.6 and CEA-binding 1F5H230 full-length IgG1 mAbs were purified on a protein G Sepharose  column. 1F5H2 mAb was used as irrelevant antibody in all performed experiments. Antibody concentration was determined by measuring the sample absorbance at 280 nm .Anti-FGF2 3F12E73F12E7 scFv binding to FGF2 was initially verified by sandwich ELISA. Briefly, 96-well plates were coated overnight at 4 °C with 5 µg/mL of (i) 3F12E7 scFv; (ii) 3F12E7 full-length IgG mAb (positive control); or (iii) irrelevant full-length IgG (negative control). The wells were blocked with 1% bovine serum albumin  in phosphate-buffered saline (PBS) and, then, incubated (or not) with 50 ng/mL recombinant FGF2 . Subsequently, wells were incubated with polyclonal anti-FGF2 antibody  and then with biotin-conjugated secondary antibody , both diluted in 0.1% BSA containing 0.1% Tween-20  (PBST).The binding of size exclusion chromatographic elution fractions of 3F12E7 scFv to FGF2 was detected by direct ELISA. For that, wells were coated overnight at 4 °C with 3F12E7 scFv (5 µg/mL). After blocking with 1% BSA in PBS, wells were incubated with FGF2  labeled to biotin. FGF2 biotinylation was performed using the EZ-Link Sulfo-NHS-Biotin kit .o-phenylenediamine  substrate. Absorbance values were read at 490 nm.In all cases, the wells were washed three times with PBST between each step. The reactions were revealed with horseradish peroxidase (HRP)-streptavidin  and 3F12E7 scFv immunoreactivity to murine tumors was evaluated on B16-F10 melanoma protein extracts separated on 15% SDS–polyacrylamide gel and further transferred to PVDF membrane . After blocking, blots were probed overnight at 4 °C with 2 µg/mL of the following biotin-labeled mAbs: (i) 3F12E7 scFv; (ii) 3F12E7 full-length IgG; or (iii) irrelevant full-length IgG. Antibody biotinylation was carried out using the EZ-Link Sulfo-NHS-Biotin kit . Membrane-bound antibodies were detected with HRP-streptavidin .ERK1/2 phosphorylation was assessed in extracts of HUVEC incubated for 48 h with 10 or 50 µg/mL 3F12E7 scFv. 3F12E7 full-length IgG and irrelevant full-length IgG mAbs (10 or 50 µg/mL) were used as controls. For that, protein cell lysates were resolved on 10% SDS–polyacrylamide gels, transferred to PVDF membranes and the blots were probed overnight at 4 °C with 1:2000-diluted anti-phospho-p44/42 MAPK  and anti-p44/42 MAPK  antibodies. β-actin  was used as loading control. Then, the membranes were washed with TBST and incubated with HRP-conjugated secondary antibody  for 1 h at room temperature.In all cases, 5% BSA in PBST was used as blocking buffer. Protein extracts were obtained with lysis buffer  and the protein content was determined by Bradford assay. Blots were revealed with ECL reagent  and chemiluminescence was captured using an Alliance imaging system . Bands were quantified using ImageJ software .34. DLS of each antibody was measured in triplicates.DLS studies were performed with Zetasizer Nano ZS equipment  using quartz cuvettes. For that, the antibodies were diluted in PBS to a final concentration of 50 µg/mL. The light scattering measurements were detected at 660 nm and data were collected at 25 °C using a solvent refractive index of 1.3333 cells/100 μL/well) were plated on 96-well plates and, 24 h later, incubated with 10 µg/mL 3F12E7 scFv. 3F12E7 full-length IgG and irrelevant full-length IgG mAbs (10 µg/mL) were used as controls. After 72 h, cells were harvested and counted using a hemocytometer. The number of viable cells was determined by the trypan blue exclusion. The experiment was performed in quadruplicate and repeated three times.HUVECs (1.5 × 105 cells/500 μL/well) were cultured on 24-well plates until confluence. Then, a scratch was made through the cell monolayer using a 200 µL pipette tip. Cells were washed twice with PBS and incubated with 10 µg/mL 3F12E7 scFv. 3F12E7 full-length IgG and irrelevant full-length IgG mAbs (10 µg/mL) were used as controls. The scratch area was photographed over 48 h using AxioObserver Z1 imaging system , and the percentage of the initial scratch area was measured using ImageJ software . Experiment performed in triplicates.HUVECs , were performed according to the guidelines of the National Institutes of Health (NIH).C57BL/6 male mice (7–8-week-old) were obtained from the animal facility of “Centro de Desenvolvimento de Modelos Experimentais para Medicina e Biologia” . Animals were maintained with water ad libitum in a temperature- and humidity-controlled room (12:12-h light:dark cycle). At the end of the experiments, mice were euthanized with anesthetic overdose (ketamine and xylazine), according to the AVMA Guidelines for the Euthanasia of Animals5 B16-F10 cells in the left flank and, four days later, started receiving intraperitoneally 2 mg/kg 3F12E7 scFv. As control groups, mice received 2 mg/kg of 3F12E7 full-length IgG mAb or irrelevant full-length IgG. Treatment was performed once every two days, for 10 days. N = 7 mice/group. Tumor volume was daily monitored and calculated as follows: tumor volume [mm3] = 0.52 × (tumor length) [mm] × (tumor perpendicular width)2 [mm2].The antitumor effect of anti-FGF2 3F12E7 scFv was evaluated using an experimental melanoma model. Mice were subcutaneously implanted with 5 × 1036, Aggrescan3D37, Waltz38, FoldAmyloid39, and Tango40. The FASTA format sequence of 3F12E7 scFv was used as the input for Aggrescan, Waltz, FoldAmyloid, and Tango. For Aggrescan3D, it was used the scFv 3D structure generated by SWISS-MODEL (http://swissmodel.expasy.org). The input parameters for each predictor are listed in Table The aggregation propensity of 3F12E7 scFv was predicted using the following tools: Aggrescan41. Antibody samples were mixed with an equal volume of sample buffer containing 62.5 mM Tris–HCl, pH 6.8, 40% glycerol, and 1% Coomassie Brilliant Blue G-250 and analyzed using a 4–15% polyacrylamide gel. The cathode buffer comprised 15 mM Bis–Tris, pH 7.0, 50 mM tricine, and 0.02% Coomassie Brilliant Blue G-250, and the anode buffer contained 50 mM Bis–Tris, pH 7.0. Densitometry analysis of protein bands was performed using ImageJ software .Blue native polyacrylamide gel electrophoresis (BN − PAGE) was performed as described42. Antibody-ligand docking was performed by the Rosetta server and the quality of output models was assessed using CAPRI criteria, based on the interface root mean square deviation (Irms), the interaction score (I_sc), and the fraction of native contacts (Fnat). The best pose with Irms ≤ 1 Å, I_sc ≤ − 5, and Fnat ≥ 0.5 was selected and visualized using PYMOL (v1.7). The 3F12E7 scFv residues interacting with the FGF2 segment (104–146 amino acid residues) used for the generation of the original 3F12E7 full-length IgG mAb were determined with LigPlot+ (ver. 1.4) package, using DIMPLOT module and default parameters43.Inter-molecular interaction between 3F12E7 scFv monomer and FGF2 (PDB ID: 1bfg) was analyzed by molecular docking studies. For that, a 3D structural model of 3F12E7 scFv in a monomeric close state was generated using the SWISS-MODEL homology servert-test or by one-way ANOVA followed by Bonferroni’s post-test. P < 0.05 was considered significant.Data are expressed as mean ± s.d. of at least three independent experiments. Statistical differences between groups were evaluated by Student’s Supplementary Information.


However, these elaborate transitions and the evaluation rules towards air-stable P2-NaxTmO2 have not yet been clearly elucidated. Herein, taking P2-Na0.67MnO2 and P2-Na0.67Ni0.33Mn0.67O2 as key examples, we unveil the comprehensive structural/chemical degradation mechanisms of P2-NaxTmO2 in different ambient atmospheres by using various microscopic/spectroscopic characterizations and first-principle calculations. The extent of bulk structural/chemical transformation of P2-NaxTmO2 is determined by the amount of extracted Na+, which is mainly compensated by Na+/H+ exchange. By expanding our study to a series of Mn-based oxides, we reveal that the air-stability of P2-NaxTmO2 is highly related to their oxidation features in the first charge process and further propose a practical evaluating rule associated with redox couples for air-stable NaxTmO2 cathodes.Air-stability is one of the most important considerations for the practical application of electrode materials in energy-harvesting/storage devices, ranging from solar cells to rechargeable batteries. The promising P2-layered sodium transition metal oxides (P2-Na Air-stability is a critical challenge faced by layered sodium transition metal oxide cathodes. Here, the authors depict a general and in-depth model of the structural/chemical evolution of P2-type layered oxides in air and propose an evaluation rule for the air-stability of layered sodium cathodes. Air-stability is undeniably one of the key issues that researchers should consider, because any air-instable compounds must be prepared, stored and assembled in dry or even inert atmospheres, leading to an increase in expense and can even jeopardize whether they are successfully commercialized.Practical application is always the ultimate goal for state-of-the-art technologies and devices, such as perovskite solar cells, thin-film transistors, and electrochemical batteries4. Among various sodium storage cathodes, layered sodium transition metal oxides (NaxTmO2) have gained significant attention owing to their great variety of compositions, ease of scalable preparation and high reversible specific capacity5. In the past forty years7, the NaxTmO2 family has been greatly enriched not only thanks to the tremendous efforts toward superior cathodes with higher energy density and more stable structures10, but has also benefited from the development of electrolytes and characterization techniques14. Nonetheless, NaxTmO2 electrodes are still haunted by three major challenges, i.e. irreversible phase transitions during cycling, insufficient electrochemical performances and air/moisture instability15. The undesired structural transformations in charge/discharge processes, such as P2-O2 in Na0.67Ni0.33Mn0.67O220, P2-P2’ in Na0.67MnO223 and O3-P3 in NaNi0.5Mn0.5O224, can be suppressed or delayed by element substitution, thus resulting in improved cycling stability and rate capability. The contact between air-instable NaxTmO2 and moisture-air usually produces cracks25, electrical insulation species26, and hydration phases27, which result in a shorter lifetime and poorer rate capability of the exposed layered oxides30. Therefore, air-stability is considered an important factor to evaluate a qualified NaxTmO2 electrode33.Sodium ion batteries (SIBs) promise the potential for large-scale grid storage, due to the high abundance and wide distribution of Na sources as compared to their lithium counterparts0.7Mn0.15Co0.15O2 reacts with H2O and CO2, generating NaOH, Na2CO3, Na2CO3∙H2O and NiO on the particle surfaces and leading to declined electrochemical performances26. However, these degradation (Ni loss) reactions have not yet been extensively observed in Ni-poor/Ni-free NaxTmO2 materials, whether exposed to air or immersed in water35. Unlike degradation, H2O insertion is widely encountered in P2-NaxTmO2 electrodes and can be easily distinguished by X-ray diffraction (XRD), because the intercalation of H2O expands the interlayer distance of the Na+ layers from ~5 to ~7 Å and even up to ~9 Å38. Kubota et al. proposed the Na+/H+ exchange mechanisms in O3-type NaMeO2 oxides39 and Rojo’s group further identified the presence of H+ in the Na+ layers using a neutron powder diffraction technique30. In addition, in the presence of H2O, CO2 was believed to get inserted into the transition metal layers of P2-Na2/3Fe0.5Mn0.5O228. These works provide valuable insights into the structural evolutions of NaxTmO2 upon air-exposure. However, rational connections between these intertwined reactions, especially as to why and when the water molecules insert into the Na+ layers, and which factor determines the extent of the structural transformation have not been thoroughly studied yet.Recently, Manthiram’s group reported that O3-NaNixTmO2 as electrodes in both organic and aqueous SIBs, but also provides guidance to the fabrication and application of layered transition metal oxides in other battery systems, such as the favorable Ni-rich43 cathodes for lithium ion batteries. For layered sodium-based oxides, it is widely recognized that the air-instability in P2-type oxides is much more severe than that in O3-type NaxMnO25. Herein, based on the P2-Na0.67MnO2 and P2-Na0.67Ni0.33Mn0.67O2 oxides, we unearth the underlying science that triggers the hydration of P2-NaxTmO2, apply unique solid-state nuclear magnetic resonance techniques (ss-NMR) to provide solid evidence for H+ insertion, analyze critical factors that influence the hydration, and outline the structural and chemical evolution mechanisms of P2-NaxTmO2 oxides upon air exposure. Importantly, the surface-sensitive time-of-flight secondary ion mass spectroscopy (TOF-SIMS) results reveal that the CO2 cannot insert into the layered structure. Our results indicate that Na+/H+ exchange, rather than O2 oxidation, dominates the compensation of extracted Na+ and the hydration is closely related to the contents of remaining Na+ ions in the structure. The critical sodium content nc is therefore proposed to evaluate whether H2O is able to intercalate into the sodium layers. In addition, the study of a series of P2-NaxTmO2 oxides indicates that the air-stability is closely associated with the voltage features of the charge process in the first cycle, and a practical principle related to the redox couple in the 1st cycle is thus proposed to evaluate the air-stability of NaxTmO2. These new insights into the degradation mechanisms upon air-exposure will facilitate the development of practical layered Li/Na/K transition metal oxides.Fundamental understanding of structural/chemical evolutions in moisture is not only critical for the employment of Na0.67MnO2 sample attained by solid-state reaction exhibits a typical P2 structure (space group: P63/mmc) with a layer spacing of ~5.5 Å 0.45}MnO2), according to the Rietveld refinement results44 2.1}MnO2), the Na+ ions are sandwiched by the inserted H2O molecules47, as illustrated schematically in Fig. xTmO2, the hydration phase discussed below is referred to birnessite phase unless otherwise specified. It should be further pointed out that the Na+ content in hydrated Na0.67TmO2 sample is <0.67 due to the Na+ loss upon air-exposure, as shown in the following sections. XRD, NPD and 23Na magic-angle-spinning nuclear magnetic resonance spectroscopy  and 23Na{1H} rotational-echo double-resonance (REDOR)48 can be used to detect the existence of water molecules and protons in the NaxTmO2 compounds, respectively  18% (without CO2), RH 15% + CO2 (with presence of CO2), and RH 93% + CO2 for 3 days (“Methods” part). As shown in the XRD patterns  emerge and their intensities increase with the presence of CO2. With further increase in the relative humidity (RH 93% + CO2), all of the XRD peaks can be indexed to the hydration phase and NaHCO3 (labeled with ∇), indicating that the Na0.67MnO2 is totally hydrated and partial Na+ diffuses out from the sodium layers. For Na0.67Ni0.33Mn0.67O2, no obvious hydration peaks can be identified in all of the exposed samples  images in Fig. 0.67MnO2 and Na0.67Ni0.33Mn0.67O2 particles are clean and smooth. After exposure, severe delamination damages and a massive of intragranular cracks can be observed in hydrated Na0.67MnO2 sample  elemental mapping results  and Mn ions (MnO− and MnO2−) appear at both surface and bulk of the powder . Considering that the water content in hydrated Na0.67MnO2 is ~0.45 mol per chemical formula unit  is much lower than OH− and Mn-based ions and the remaining carbonate trace is mainly distributed on the surface rather than the bulk of the particles. This fact coincides well with the depth profile results in Fig. 2 is to increase the acidity at the particle’s surface and accelerate the Na+ loss rather than insert into the bulk of the NaxTmO2 phases.The presence of COure Fig. . To charies Fig. , we adopder Fig. , in goodder Fig. . The sig0.67Ni0.33Mn0.67O2 has been reported to be one of the most air-stable P2-NaxTmO2 oxides, the chemical change is observed in the EDS mapping as demonstrated in Fig. 3 diffraction peaks increases while the (002) diffraction peak shifts to lower 2-theta  was carried out. According to the previous results49, during the first charge process of Na0.67Ni0.33Mn0.67O2 electrode within 2.0–4.4 V (vs. Na+/Na), Mn4+ ions remain stable and the oxidation/reduction of Ni ions compensates the electrochemical extraction/insertion of Na+. As shown in Supplementary Fig. 0.67Ni0.33Mn0.67O2 is nearly the same as in the pristine sample. The valence state of Mn ions in pristine and hydrated Na0.67MnO2 samples has been also investigated by XAS. The pre-edge peak and main edge of Mn K-edge XAS correspond to 1s → 3d and 1s → 4p transitions, respectively. However, the structural changes during hydration , P2-NaxTmO2 materials are vulnerable for hydration and vice versa. The correlation between hydration degree and sodium contents x is also observed in NaxMnO2 compounds. As representatively shown in Fig. 3 absorption peaks are positively associated with the hydration degrees of Na0.67MnO2 sample.To gain deeper insight into the structural/chemical transformation mechanisms of P2-Nanc. As shown in Fig. 15. The energy difference between P2-NaxTmO2 with and without water molecules is defined as hydration energy. DFT calculation results in Fig. x > 0.52, the hydration energy of NaxMnO2 is higher than 0 eV, suggesting that water molecules cannot get inserted into the material. When the sodium content is lower than 0.52, the hydration energy is lower than 0 eV, thus the hydrated structure is more stable than the un-hydrated structure. The foregoing energy analysis provides a chemical basis for understanding the sodium-content-dependent property in the hydration of NaxMnO2 samples. Due to the low formation energy, the layered Na0.67TmO2 oxides will lose Na+ spontaneously once in contact with moist air. When the sodium contents are lower than nc, there is a strong repulsion between adjacent oxide layers (TmO2) of P2-NaxTmO2 oxides, and the insertion of H2O could lower the energy of Na0.67-x-zHxTmO2  due to the shielding effect of the water molecule and the expansion of sodium layer spacings. The above results also proved and interpreted the fact that hydration takes place after the extraction of Na+ during air-exposure.The density function theory (DFT) calculation was carried out to further understand the critical sodium content 0.67TmO2 to different ambient atmospheres are summarized in Eqs. , and Na0.67Ni0.33Mn0.67O2 immersed in water for 20 days (denoted as immersed-Na0.67Ni0.33Mn0.67O2) are presented in Fig. 0.67Ni0.33Mn0.67O2, exposed-Na0.67Ni0.33Mn0.67O2, and immersed-Na0.67Ni0.33Mn0.67O2 samples have very similar initial charge-discharge curves, suggesting that their structure and redox center are nearly identical8. However, the initial charge capacities of the exposed-Na0.67Ni0.33Mn0.67O2 (70 mAh g−1) and immersed-Na0.67Ni0.33Mn0.67O2 (77 mAh g−1) are lower than that of the pristine Na0.67Ni0.33Mn0.67O2 (85 mAh g−1), further confirming the loss of Na+ ions during the exposure and immersing processes. Fig. 0.67Ni0.33Mn0.67O2 exhibits much lower coulombic efficiency than the pristine Na0.67Ni0.33Mn0.67O2, due to the influence of the sodium bicarbonate on the surface of exposed-Na0.67Ni0.33Mn0.67O2 particles. At the voltage range of 2.0–4.0 V, the pristine Na0.67Ni0.33Mn0.67O2 material shows no evidence of capacity degradation after 100 cycles. In contrast, the immersed-Na0.67Ni0.33Mn0.67O2 and exposed-Na0.67Ni0.33Mn0.67O2 samples decay fast in capacity, and retain low discharge capacity of 56 and 53 mAh g−1, corresponding to the capacity retention of 74% and 70%, respectively. To get more insight into the degradation mechanisms, the electrochemical impedance spectra (EIS) have been employed to study the resistance changes. The interface and charge-transfer resistances were estimated based on the equivalent circuit in Supplementary Fig. SEI) and charge transfer resistance  for the exposed-Na0.67Ni0.33Mn0.67O2 and immersed-Na0.67Ni0.33Mn0.67O2  and H2O insertion are two continuous structural transition processes during the exposure of P2-NaxTmO2 samples, which usually impair their intrinsic electrochemical performances. The following questions need to be clarified: (i) whether or not these exposed samples can be recovered to their original structure and how, and (ii) what is the stoichiometry of hydrated phases (birnessite)? Therefore, in this section, the structural transitions of hydrated samples at the temperature range of 25–570 °C were investigated by in situ variable-temperature XRD technique. As shown in Fig. 0.67MnO2. The mass loss at stage a (25–130 °C) is ~15.1%, which includes decomposition of NaHCO351 and extraction of water. At this stage, the hydration structure is well maintained. From stage a to stage b, the hydrated phase is most probably transformed into a protonated phase  is much lower than stage b, which might be resulted from the loss of protons. In addition, the sodium carbonate begins to decompose and results in the mass loss in stage c. With further increase in temperature, the crystallinity of Na0.67-x-zMnO2 increases, as shown in stage d. It should be pointed out that the decomposition temperature of Na2CO3 is lower than 800 °C due to the small sizes of the Na2CO3 particles  and (103) peaks  of the P2 phase emerge gradually, indicating the P2-Na0.67MnO2 phase gradually recovers at stage e. However, the mass loss of 0.45% during 130–217 °C corresponds to x = 0.61 in Na0.67-x-zHxMnO2, exceeding the amount of lost Na+ ions . In conclusion, with the increase of temperature, the hydrated Na0.67MnO2 sample undergoes dehydration and NaHCO3 decomposition (70–130 °C), deprotonation (130–217 °C), Na2CO3 decomposition (>130 °C) and the recrystallization of P2 phase (>483 °C) process. In our recent work23, we demonstrate that Na0.67Zn0.1Mn0.67O2 is a promising Na-ion battery cathode with outstanding cycling stability and better air-stability than Na0.67MnO2. Therefore, we also performed the in situ variable-temperature XRD of totally hydrated Na0.67Zn0.1Mn0.9O2 powder. Obviously, the structural transformation of the hydrated Na0.67Zn0.1Mn0.9O2 in Supplementary Fig. 0.67MnO2. From the hydration phase (stage a) to the protonated phase (stage b), the combined mechanism of two-phase and solid-solution reactions can be recognized. Moreover, the temperature range of deprotonation of the hydrated Na0.67Zn0.1Mn0.9O2 sample is elusive and the temperature of the recrystallization of P2-type Na0.67Zn0.1Mn0.9O2 phase (376 °C) is lower than that of Na0.67MnO2 (483 °C). The above results indicate that if the Na0.67TmO2 samples undergo sodium-loss and further hydration processes upon air-exposure, high-temperature annealing can be used to recover their original structures , it is more difficult to extract Na+ from the lattice chemically. Therefore, the potential of redox couples could be considered as one of the key principles to evaluate the air-stability of Na0.67TmO2 electrodes. Based on the above conclusion, we summarize the main redox couples in P2-NaxTmO2 electrodes  starts to transform into the layered Na0.67MnO2. The XRD peaks of β-MnO2 disappear at ~540 °C and all of the Na2CO3 is decomposed at ~ 640 °C. We compared the air-stability of the Na0.67MnO2 samples at 640 °C, 900 °C, and the sample calcinated at 900 °C for 900 min, which exhibit increased crystallinity , we proposed a general and in-depth picture on the chemical/structural evolutions of P2-NaxTmO2 during moisture-exposure. In addition, by extending the study to a variety of layered sodium-based oxides  we have also demonstrated that the redox potential properties in the first charge process could be used as an empirical rule for evaluating the air-stability of NaxTmO2 electrodes. Our results provide significant new clues for the design, synthesis, storage, and application of layered sodium cathodes, as well as other alkali-metal transition metal oxides.In summary, a comprehensive investigation of the structural and chemical transformations of P2-NaxTmO2 cathodes have been synthesized by high-temperature solid-state reactions23. Stoichiometric amounts of raw materials, e.g. MnO2 , ZnO , NiO , Al2O3 , TiO2 , CuO , Li2CO3 , and Na2CO3  were ball-milled with acetone solvent for 3.5 h at 500 rpm. Then dried at 120 °C overnight, pressed into pellets and heated at 900 °C for 15 h in air. After slowly cooled to 150 °C in the furnace, the pellets were transferred to an Ar-filled glove box immediately, ground and kept the final products in the Ar-filled glove box.The pristine Na65. To study the effect of different ambient atmospheres on the structural and chemical stability of NaxTmO2, the prepared Na0.67TmO2 oxides are put in a centrifuge tube and then aged on an airtight container under four different storage conditions at the constant temperature of 40 °C. They are dry CO2, controlled humidity of RH 18% (without CO2), RH 15% with CO2, and RH 93% with CO2. To maintain the ambience conditions of dry CO2, RH 15% with CO2 and RH 93% with CO2, the dry silica gel, saturated aqueous LiCl and NaHCO3 solutions are placed below the samples, respectively. After that, the containers are filled with CO2. The RH 18% (without CO2) atmosphere was obtained by placing the saturated NaOH solution at the bottom of the container and then filled with dry O2. The accurate RH is determined by a hygrometer, whose accuracy is ±1%. The elemental compositions of pristine and exposed samples were confirmed by inductively coupled plasma-atomic emission spectrometry (ICP-AES) analysis . Before ICP-AES and Time-of-flight secondary ion mass spectroscopy (TOF-SIMS) testing, we applied a scavenging process to remove the Na2CO3/NaHCO3 byproducts in the exposed samples. Specifically, the exposed samples were immersed and stirred in distilled water for 3 min, then centrifuged and dried at 80 °C overnight.The saturated salt solutions could produce stable relative humidity in a closed system at a certain temperatureλ = 1.5406 Å). The patterns were refined by the General Structure Analysis System (GSAS) software44. The morphologies of samples were characterized by scanning electron microscopy . The EDS elemental mapping experiments were conducted using FEI Talos-F200s TEM instrument. The 23Na ss-NMR experiments were acquired on a Bruker AVANCE III 400 MHz spectrometer using a double resonance 1.3 mm MAS probe spinning at frequencies of up to 55 kHz with a Hahn-echo pulse sequence . The 90o pulse length of 1.2 μs and a recycle delay of 2 ms were used. The 23Na shifts were referenced to 1 M NaCl aqueous solution (0 ppm). For 1H, the 2.5 mm MAS probe was used spinning at 25 kHz to record the 1H spectra with the rotor-synchronized Hahn Echo sequence and a cycle delay of 2 s and a 90o pulse length of 4 μs. No window functions were added and the chemical shift of 1H was referenced against adamantane (1.87 ppm). By using 2.5 mm MAS probe with a MAS rate of 25 kHz, the rotational echo double resonance (REDOR) NMR sequence  is applied to confirm the presence of H+ in the exposed materials. Geometry-independent information about the 23Na-1H dipole couplings can be conveniently obtained from the plot of signal attenuation  versus the dipolar evolution time (2tau). In all MAS NMR measurements, the variable temperature (VT) gas temperature was set to 315 K. The in situ XRD experiments were performed in air and on a BrukerD8 Discover diffractometer equipped with a Cu Kα radiation. Infrared spectra were recorded on a Nicolet is50 FT-IR  spectrometer. X-ray absorption spectroscopy (XAS) data were acquired in the transmission mode at the BL14W1 beamline of the Shanghai synchrotron radiation facility (SSRF) at room temperature, and the incident beam was monochromatized by a Si (111) double-crystal monochromator. TOF-SIMS were performed on a TOF-SIMS5 spectrometer (ION TOF GmbH). The depth profiling and high-resolution mapping were conducted at the high current mode and burst alignment mode, respectively, with a pulsed Bi1+ ion beam (30 keV). The sputtering was conducted by a 500 eV Cs+ ion beam with the area of 100 μm × 100 μm, and the analyzed areas for depth profiling and high-resolution mapping are typically 100 μm × 100 μm and 50 μm × 50 μm with the pixel of 128 × 128.The X-ray diffraction (XRD) patterns were obtained on a Rigaku Ultima IV diffractometer by a Persee instrument with Cu Kα radiation  and Fluoroethylene carbonate  as the electrolyte, Whatman glass fiber filter as the separator, and sodium metal as the counter electrode. The galvanostatic charge-discharge processes were conducted on multichannel battery tester . The electrochemical impedance spectroscopy (EIS) was conducted by a four-channel multifunctional electrochemical workstation (Versa STAT MC. America), at the cell voltage of 4.0 V and with the frequency range of 100 kHz to 0.01 Hz.The electrodes are composed of 80 wt% of active materials, 10 wt% of polyvinylidene fluoride (PVDF) and 10 wt% acetylene carbon black. The mass loading of active materials is 2.5–3 mg cm66 and the exchange-correlation interactions of electron were described with spin-polarized generalized gradient approximation (GGA) and parameterized by PBE formula67. The projector-augmented wave approach and GGA + U method were used to evaluate the electron-ion interactions and the localization of the d electrons of the TM ions, respectively69. The U value of Mn set to 3.9 eV and the wave functions were expended by plane wave with a kinetic energy cut-off of 520 eV. The Monkhorst–Pack scheme70 was used for the integration in the irreducible Brillouin zone with a k-point mesh resolution of 2π × 0.025 Å−1. The lattice parameters and atomic coordinates were fully relaxed, and the final forces on all atoms were <0.01 eV Å−1. For the DFT calculations, a 3 × 3 × 1 supercell, which contains 12 Na, 36 O, and 18 Mn has been adopted. Sodium contents of x = 0.667, 0.556, 0.500, 0.444 and 0.389 in NaxMnO2 correspond to 12, 10, 9, 8 and 7 Na ions in the supercell, and the number of different structures for each Na contents is x = 0.667, 0.556, 0.500, 0.444 and 0.389 in NaxMnO2 are 1, 2, 2, 2, 2, respectively, as shown in Fig. The DFT calculations were performed on VASP (Vienna ab initio Simulation Package)Supplementary Information


The progress of the conversion and ee was also monitored in a selected case. This is the first report of a PIKAT using an immobilized ωTA in a non-aqueous environment.Comprising approximately 40% of the commercially available optically active drugs, α-chiral amines are pivotal for pharmaceutical manufacture. In this context, the enzymatic asymmetric amination of ketones represents a more sustainable alternative than traditional chemical procedures for chiral amine synthesis. Notable advantages are higher atom-economy and selectivity, shorter synthesis routes, milder reaction conditions and the elimination of toxic catalysts. A parallel interconnected kinetic asymmetric transformation (PIKAT) is a cascade in which one or two enzymes use the same cofactor to convert two reagents into more useful products. Herein, we describe a PIKAT catalyzed by an immobilized ω-transaminase (ωTA) in neat toluene, which concurrently combines an asymmetric transamination of a ketone with an anti-parallel kinetic resolution of an amine racemate. The applicability of the PIKAT was tested on a set of prochiral ketones and racemic α-chiral amines in a 1:2 molar ratio, which yielded elevated conversions (up to >99%) and enantiomeric excess ( This w) and using inexpensive 2-propylamine as the amine donor [3-ωTA biocatalyst also led to excellent activities for the asymmetric synthesis of α-chiral amines from prochiral ketones in neat organic solvents when the system’s water activity (aw) was controlled with hydrate salt pairs [R)-phenoxy-2-propylamine from phenoxyacetone by using only one or two equivalents of 2-propylamine as the amine donor, respectively. Thus, we could increase the substrate concentration from 50 mM to 400 mM without any apparent loss in catalytic performance. The use of the immobilized EziG3-ωTA in an organic solvent as a reaction medium did not negatively affect—and possibly even increased—biocatalyst stability, as the reaction could be conducted in a continuous flow reactor for several days.As mentioned above, recurrent challenges in ωTA-catalyzed reactions using the “classical” alanine (or another α-amino acid) as the sacrificial amine donor in an aqueous environment are unfavorable reaction equilibrium and frequent enzyme inhibition by the amine product and pyruvate co-product ,30,31,32ne donor ,34,35. Bne donor . In thisne donor ,39,40,41ne donor ,48,49,50ne donor . Notably+/NAD(P)H) [Nature benefits from a large number of chemically interconnected processes that simultaneously occur in the same living systems. Concurrent artificial catalytic processes inspired by such systems have been developed for the synthesis of enantioenriched compounds ,54,55,56NAD(P)H) ,58. An eNAD(P)H) ,60; in tNAD(P)H) .3-immobilized and (R)-selective ωTA from Arthrobacter sp. (EziG3-AsR-ωTA) in a neat organic solvent. The immobilization of AsR-ωTA on EziG controlled porosity glass beads was previously investigated and exhibited high retention of enzyme activity and excellent stability in both aqueous and organic media [3-AsR-ωTA, toluene was found to be the best solvent when the system’s water activity (aw) was fixed and controlled at 0.7 using salt hydrate pairs [rac-4–6b) undergoes resolution to yield the ketone by-product with a theoretical maximum conversion of 50% and, in principle, leaving the (S)-configured amine enantiomer untouched; in the same pot and anti-parallel direction, the prochiral ketone substrate (1–3a) gets converted to an enantiopure (R)-configured amine product.In this work, we describe a novel PIKAT using an EziGic media ,51. Herete pairs . Therefo3 (Fe Amber) beads were used as immobilization carrier material. These beads are made of high porosity glass with pores of well-defined size and geometry . The beads’ glass surface  is coated with a semi-hydrophobic polymer , which is further derivatized to chelate Fe(III) cations. These Fe(III) centers enable the chemical coordination binding between the enzyme and the carrier [w w−1 was reported with this type of carrier material, due to the high surface area of the porous glass per unit of volume [EziG carrier . Enzyme f volume . The advR)-selective ωTA from Arthrobacter sp. on EziG3 (forming EziG3-AsR-ωTA), through the Fe(III)-ion affinity binding between the enzyme and the carrier material. In practice, a genetically fused and N-terminal poly-histidine (His-6) tag of the ωTA interacts with high affinity with the Fe(III) ions onto the polymeric surface of the EziG beads. We previously employed EziG3-AsR-ωTA with an enzyme loading of 10% w w−1 (ratio of enzyme to bead), and quantitative enzyme immobilization was achieved within 2 h incubation time [3 (Fe Amber) beads (20 mg) in KPi buffer  supplemented with PLP (0.1 mM). Under the optimized aqueous reaction conditions , the specific catalytic activity was determined for the transamination of rac-methylbenzylamine 6b (100 mM) with sodium pyruvate (50 mM) at 30 °C [−1enzyme, whereas immobilized EziG3-AsR-ωTA exhibited an apparent activity of approximately 380 U µmol−1enzyme. The lower apparent activity of the immobilized biocatalyst must be, at least in part, attributed to additional mass transfer limitations in heterogeneous catalysis ; this is particularly valid for the kinetic resolution of rac-methylbenzylamine that has an elevated reaction rate in a homogeneous aqueous system.We previously reported the immobilization of an  was controlled using a hydrate salt pair , as reported in our previous study [R)-selective enzyme originated from Arthrobacter sp.—was immobilized on EziG3 (Fe Amber) controlled porosity glass beads and prepared for reactions in organic solvent, as previously reported [For screening of the PIKAT with the immobilized AsR-ωTA, we selected three prochiral ketones and three racemic α-chiral primary amines comprising aliphatic and aromatic substrates . Thus, pus study . AsR-ωTAreported ,51. The ee) of the final products proved difficult. Therefore, we decided to modify the established work-up procedure for enzymatic transamination reactions such that ketones and amines could be separately extracted from the mixture and analyzed in two discrete steps. Although this work-up procedure would require improvements for a preparative or industrial scale application, possibly in a continuous process, separation of the products allowed us to quantify conversions and ee by adding an internal standard (IS). Conversions were determined according to the gas chromatography (GC) peak area ratios between the products and IS in the different extraction phases. ee values were obtained after derivatization to acetoamido and analyzed using a chiral GC column.Initial experiments indicated the formation of imine side products in the final reaction mixture; these imines arose from the reaction between ketones and amines that were present as substrates and products in the reaction mixture. In fact, the (reversible) imine formation between ketones and amines is more favored in an organic solvent than in an aqueous buffer. Due to the complexity of the obtained reaction mixture, quantification of conversion and enantiomeric excess  depending on the amine donor (R)-1b was always obtained with >99% ee. The conversion of 1a into (R)-1b and the outcome of the anti-parallel kinetic resolution of rac-4–6b were obviously mutually influenced; in particular, kinetic resolution theoretically could not exceed the 50% value, as the prochiral ketone and the racemic amine were always applied in a 1:2 molar ratio. In contrast, the kinetic resolution of rac-6b afforded an apparent conversion of 51 ± 1%, although the ketone 1a was converted into (R)-1b with 90 ± 7% conversion. This apparent discrepancy must be attributed to the probably higher volatility of rac-6b, thereby resulting in an apparent slightly higher formation of acetophenone 6a. Furthermore, the unreacted amine donor 6b resulted in 80% ee for the (S)-configured enantiomer, thus indicating that both enantiomers can be accepted by the AsR-ωTA during the kinetic resolution. The imperfect enantioselectivity of the kinetic resolution step was a general trend, as amines 4b and 5b were obtained in average values of 40% and 2% ee, respectively.The PIKAT of ketone ne donor a; howeve2a yielded higher conversion for the asymmetric formal reductive amination step such that (R)-2b was obtained with conversion between 77% and 91% -configured enantiomers at the end of the reaction (between 64 ± 1% and 80 ± 1% ee).The PIKAT of ketone  and 91% b. Notabl3a; the asymmetric formal reductive amination step afforded quantitative conversion for all of the three investigated cases -3b in optically pure form (ee >99%). As observed in the previous cases, AsR-ωTA accepted both enantiomers of the racemic amine donors with a preference for the (R)-configured ones, thereby leading to enantioenriched mixture of (S)-4–6b at the end of the process (between 52 ± 2% and 59 ± 9% ee).The best performing PIKAT was observed with ketone ed cases c. As pre1a and racemic amine donor 6b. The progress of the conversion  until 50 ± 1% conversion.In order to examine the progress of the PIKAT reaction, we performed a time study with ketone nversion a and enasion ee, b were moee), whereas the ee of the amine donor mixture gradually increased from 0% to ca. 80% (S) at the end of the reaction. It is evident that the ee of (S)-6b increased more rapidly at the beginning of the PIKAT, as (R)-6b is virtually the only accepted enantiomer by the AsR-ωTA at this stage; however, the ee curve flattens after 20 h reaction time, thus indicating that a considerable portion (S)-6b eventually starts to be accepted by AsR-ωTA. This behavior results in the imperfect selectivity of the anti-parallel kinetic resolution step.ee of the desired amine product. Ketone 1a and amine donor rac-6b were chosen as model substrates, and the results are reported in S)-6b was always obtained with an ee around 70% when 1a was added from 50 mM to 80 mM concentration. Moreover, increasing the concentration of 1a up to 90 mM and 100 mM was detrimental for the selectivity of the kinetic resolution, as the ee of (S)-6b decreased to average values of 48% and 28%, respectively. In contrast, as observed in all this study’s experiments, the asymmetric formal reductive amination step proceeded with excellent enantioselectivity such that (R)-1b was always obtained in >99% ee.Since the PIKAT is based on a reversible amine transfer, we decided to study the influence of the molar ratio between prochiral ketone and a racemic amine donor on the overall thermodynamic equilibrium of the process. Therefore, we decided to increase the prochiral ketone substrate concentration from 50 mM up to 100 mM while keeping the racemic amine concentration constant at 100 mM. Thus, we could check whether an excess of ketone  could push the anti-parallel kinetic resolution of the racemic amine to completion at a given ketone substrate concentration. Crucially, high ee values could also be accessible for the kinetic resolution of the racemic amine donor, while the formal reductive amination reaction would always provide >99% In another set of reactions, we either doubled the enzyme concentration in the reaction or added another portion of fresh enzyme after 24 h reaction time. No improvements were observed (data not shown).1–3a were successfully employed with racemic amine donors 4–6b in a parallel interconnected kinetic asymmetric transformation (PIKAT). The asymmetric synthesis step yielded (R)-configure amines 1–3b with average conversions from 48% to >99% and virtually perfect enantioselectivity (>99% ee). Notably, ketone 3a was quantitatively converted in the three investigated cases and amination of ketone 2a also led to elevated conversions (77% or higher), whereas ketone 1a was converted with slightly lower conversions   and nearly racemic amine was obtained (ee 2%); accordingly, the concurrent asymmetric formal reductive amination step led to moderate conversion, albeit with an excellent ee of >99% (1a with rac-6b (1a was converted to (R)-1b with 90 ± 7% conversion after 72 h of reaction time, whereas the rac-6b concentration gradually decreased to reach 50 ± 1% after 72 h. The ee percentage values displayed in R)-1b was formed. Furthermore, the originally racemic 6b became an enantioenriched mixture of the (S)-enantiomer over time, as mainly the (R)-enantiomer was converted to 6a. After 72 h, the (S)-enantiomer comprised approximately 90% of 6b.Overall, the asymmetric formal reductive amination step appeared to be only minorly affected by the type of racemic amine donor that was applied. The anti-parallel kinetic resolution step showed a consistent trend depending on the applied amine donor; for instance,  of >99% , entry 2h rac-6b a showed ee in the kinetic resolution of rac-6b by adding more of the prochiral ketone 1a to the reaction. In this manner, we would sacrifice some conversion in the asymmetric synthesis of (R)-1b while aiming to gain a higher enantioenrichment of (S)-6b. Unfortunately, the use of an excess of 1a (up to equimolar with rac-6b) did not provide any evident improvement of the reaction outcome (see 1a to (R)-1b was slightly lower than in the previous experiments (1a. With the exception of R)-1b from 1a -1b was 31 mM and 20 mM, respectively. We attribute this effect to the known ωTA enzyme’s inhibition at higher concentrations of some ketone substrates [rac-6b was not improved by increasing the concentration of 1a, as the ee values never exceeded 78 ± 2%  were purchased from Sigma-Aldrich . EziG3 (Fe Amber) enzyme carrier material was provided by EnginZyme AB . Further details for equipment and analytical determination are presented in Ketones R)-selective ωTA from Arthrobacter sp. (AsR-ωTA) [C-terminal His-tagged (AsR-ωTA) ,64 was eAsR-ωTA) .−1 protein concentration according to the supplier-provided protocol. A low-concentration assay was used in cases of lower protein concentrations (<25 µg mL−1). Samples were prepared by mixing 980 µL diluted stock solution and 20 µL protein sample (for low-concentration assay: 800 µL diluted stock solution and 200 µL protein sample) followed by incubation for 5–10 min at room temperature. Absorption at 595 nm was measured and plotted against the protein concentration. Diluted ωTA enzyme samples were then prepared and measured in the same fashion in order to determine their concentration using the albumin calibration line.Protein assay dye reagent concentrate  was diluted 5 times with MilliQ water and filtered through a paper filter. This diluted stock solution was freshly prepared before use and kept in the dark at 4 °C. The albumin calibration line was determined in the standard range of 200–1000 µg mL3 carrier material (20 ± 0.2 mg) was cooled down in an ice bath and suspended in an immobilization buffer  supplemented with PLP (0.1 mM). Purified ω-TA  was added to the suspension. The mixture was shaken on an orbital shaker (120 rpm) for 3 h at 4 °C. Small aliquots from the aqueous phase (20 μL) were taken before and after the immobilization procedure, and their concentrations were determined using the Bradford assay  and hydrate salt pair  were suspended in EtOAc  and shaken for 15 min . The EziG3-AsR-ωTA was allowed to sediment and the solvent was removed by pipetting. The EziG3-AsR-ωTA with hydrate salt pair was resuspended in another aliquot of EtOAc, and the above-described process was repeated twice. Next, the EziG3-AsR-ωTA with hydrate salt pair was washed with toluene  and then allowed to sediment, after which the solvent was removed by pipetting. Finally, toluene  was added as a reaction solvent. The ketone substrate 1–3a  and racemic amine donor 4–6b  were added, and the reaction vials were shaken in an upright position  for 72 h at 25 °C. Work-up was performed by separating ketone and amine compounds from each other by extraction. First, the EziG3-AsR-ωTA was left to sediment and the organic reaction mixture was separated from the biocatalyst by pipetting. Then, naphthalene was added as an internal standard to the organic reaction mixture from a concentrated stock solution . The amine compounds were selectively extracted from the toluene reaction phase using an aqueous 2 M HCl solution (400 µL), thereby minimizing the possible extraction of ketones. Following mixing and centrifugation , the two layers were separated and the organic layer was kept separate. The acidic aqueous layer was re-extracted with toluene (2 × 500 µL) to selectively remove potential traces of ketone. The organic layers were combined and dried over MgSO4 prior to analysis. Then, the acidic aqueous layer—containing the protonated amine products—was basified using an aqueous solution of either KOH (200 µL of a 10 M stock solution) or K2CO3 (for reactions with substrate 2a), and extracted with EtOAc containing 50 mM naphthalene as an internal standard (2 × 500 µL). Drying over MgSO4 was performed prior to analysis. Analysis was performed by separately injecting ketone- and amine-containing solutions on GC equipped with an achiral column (see -1) for 30 min . Samples were quenched by the addition of water (500 μL) and shaken for 30 min . Following centrifugation, the organic layer was dried over MgSO4 and analyzed by GC equipped with a chiral column , immobilized EziG2 as carrier gas and a DB-1701 column from Agilent , or a HP-5 column from Agilent . Chiral samples were measured using a CP-DEX column from Agilent  or a HydroDex-β-TBDAc column  from Macherey-Nagel :DB1701-30 m-method-A: constant pressure 6.9 psi, T injector 250 °C, split ratio 50:1, T initial 80 °C, hold 6.5 min; gradient 10 °C/min up to 160 °C, hold 5 min; gradient 20 °C/min up to 200 °C, hold 2 min; gradient 20 °C/min up to 280 °C, hold 1 min.DB1701-30 m-method-B: constant pressure 6.9 psi, T injector 250 °C, split ratio 50:1, T initial 60 °C, hold 6.5 min; gradient 20 °C/min up to 100 °C, hold 1 min; gradient 20 °C/min up to 280 °C, hold 1 min.HP-5-method-A: constant pressure 4 psi, T injector 250 °C, split ratio 30:1, T initial 60 °C; gradient 5 °C/min up to 150 °C, hold 1 min; gradient 10 °C/min up to 250 °C, hold 1 min.HP-5-method-B: constant pressure 4 psi, T injector 250 °C, split ratio 30:1, T initial 40 °C, hold 2 min; gradient 5 °C/min up to 80 °C, hold 2 min; gradient 20 °C/min up to 250 °C.CP-DEX-method-A: constant flow 1.4 mL/min, T injector 200 °C, split ratio 40:1, T initial 100 °C, hold 2 min; gradient 1 °C/min up to 130 °C, hold 5 min; gradient 10 °C/min up to 170 °C, hold 10 min.; gradient 10 °C/min up to 180 °C, hold 1 min.CP-DEX-method-B: constant flow 1.4 mL/min, T injector 200 °C, split ratio 40:1, T initial 100 °C, hold 2 min; gradient 1 °C/min up to 118 °C, hold 5 min; gradient 10 °C/min up to 170 °C, hold 10 min.; gradient 10 °C/min up to 180 °C, hold 1 min.CP-DEX-method-C: constant flow 1.5 mL/min, T injector 200 °C, split ratio 20:1, T initial 60 °C, hold 2 min; gradient 5 °C/min up to 100 °C, hold 2 min; gradient 10 °C/min up to 180 °C, hold 1 min.HydroDex-β-TBDAc-method: constant flow 1 mL/min, T injector 220 °C, split ratio 20:1, T initial 100 °C, hold 2 min; gradient 1 °C/min up to 150 °C, hold 8 min; gradient 10 °C/min up to 170 °C, hold 1 min.The biotransformation conversions were determined using a 7890A GC system  equipped with a FID detector with HR)-configured amine product in >99% ee. Conversely, the immobilized ωTA from Arthrobacter sp. displayed only moderate selectivity in the kinetic resolution step, thereby resulting in a maximum of 80 ± 1% ee value for the less converted (S)-configured amine donor. Time studies showed that the kinetic resolution step proceeded with main (or even sole) consumption of the (R)-configured amine until a conversion of ca. 35% was obtained, at which stage it appeared that the other enantiomer started to be accepted as well, ultimately leading to a 51 ± 1% conversion and 80% ee. In contrast, the anti-parallel formal reductive amination proceeded smoothly until 90 ± 7% conversion, and >99% ee of the final amine product was obtained. The enantioselectivity of the kinetic resolution step can potentially be enhanced by testing other available ωTAs and/or engineering new and more selective variants that are customized for this process. Admittedly, one limitation of the current process is the elaborated work-up procedure entailed in the selective extraction of the amine products (and ketone by-product) from the reaction mixture. Therefore, further work is required to develop more efficient procedures for the isolation of pure products, such as using fractional distillation and/or selective ion-exchange or hydrophobic resins [In this work, we report the successful development of the first example of parallel interconnected kinetic asymmetric transformation (PIKAT) using an immobilized ω-transaminase in a neat organic solvent. PIKATs have been achieved almost exclusively using oxidoreductase enzymes, and other enzyme classes such as transferases have rarely been considered. The PIKAT with ωTA entails an asymmetric formal reductive amination of a prochiral ketone combined with a concomitant and anti-parallel kinetic resolution of a racemic amine. The use of a neat organic solvent is critical for the successful application of the PIKAT concept with ωTA, as it enables the appropriate tuning of the thermodynamic equilibrium between two anti-parallel reactions, also using structurally diverse substrates. The formal reductive amination step always proceeded with excellent selectivity, thus yielding the (c resins . In this