Modular and Photoreversible Polymer-Nanoparticle Hydrogels via Host-Guest Interactions.

Polymer-nanoparticle (PNP) hydrogels are a class of nanocomposite materials showing potential as injectable platforms for biomedical applications. Their design is limited by incomplete knowledge of how the binding motif impacts the viscoelastic properties of the material and is generally constrained to non-responsive supramolecular interactions. Expanding the scope of available interactions and advancing the understanding of how defined interactions influence network formation would accelerate PNP hydrogel design. To address this gap in the design of PNP hydrogels, the study designs and investigates a tunable platform based on beta-cyclodextrin (ßCD) host-guest cross-links between functionalized polymers and nanoparticles. A host-functionalized polymer (ßCD hyaluronic acid) and guest harboring block co-polymer (poly(ethylene glycol)-b-poly(lactic acid)) NPs are synthesized. The presence and accessibility for binding of the host and guest moieties are characterized via isothermal titration calorimetry. PNP hydrogels with varying concentrations of functionalized polymer and NPs reveal a limited window of concentrations for gelation. It is hypothesized that network formation is governed by the capacity of polymer chains to effectively bridge NPs, which is related to the host-guest ratios present in the system. Further, photo-responsive guests are incorporated to engineer photoreversible gelation of PNP hydrogels via exposure to specific wavelengths of light.

Peptide-Directed Attachment of Hydroxylamines to Specific Lysines of IgG Antibodies for Bioconjugations with Acylboronates.

The role of monoclonal antibodies as vehicles to deliver payloads has evolved as a powerful tool in cancer therapy in recent years. The clinical development of therapeutic antibody conjugates with precise payloads holds great promise for targeted therapeutic interventions. The use of affinity-peptide mediated functionalization of native off-the-shelf antibodies offers an effective approach to selectively modify IgG antibodies with a drug-antibody ratio (DAR) of 2. Here, we report the traceless, peptide-directed attachment of two hydroxylamines to native IgGs followed by chemoselective potassium acyltrifluoroborate (KAT) ligation with quinolinium acyltrifluoroborates (QATs), which provide enhanced ligation rates with hydroxylamines under physiological conditions. By applying KAT ligation to the modified antibodies, conjugation of small molecules, proteins, and oligonucleotides to off-the-shelf IgGs proceeds efficiently, in good yields, and with simultaneous cleavage of the affinity peptide-directing moiety.

Identification of two α-tocodienol isomers in palm oil after countercurrent chromatographic enrichment.

Countercurrent chromatography (CCC) was used for the enrichment of α-tocodienol (α-T2), a rare vitamin E-related minor compound previously tentatively detected in palm oil. Hitherto, only one isomer has been mentioned to occur at traces in palm oil. However, CCC fractionation followed by GC/MS measurements of all fractions resulted in the detection of two α-T2 isomers in five different palm oil vitamin E dietary supplement capsules. Five repetitive CCC separations of ~ 1 g sample and additional purification steps by column chromatography provided ~ 2 mg of two equally abundant α-T2 isomers with a purity of ~ 85%. The positions of the double bonds in the alkyl side chain could be assigned by means of two characteristic chemical shifts in the 1H NMR spectrum. Accordingly, the structures of the α-T2 isomers were 2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridec-3,11-dienyl)chroman-6-ol (double bonds in 3',11'-position) and 2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridec-7,11-dienyl)chroman-6-ol (double bonds in 7',11'-position). Natural occurrence of both isomers was proven by GC/MS screening of crude palm oil after saponification and CCC separation. Moreover, GC/MS analysis allowed the tentative assignment of γ-tocomonoenol (γ-T1) and ß-tocomonoenol (ß-T1) as trace compounds in palm oil.

α-Tocomonoenol Is Bioavailable in Mice and May Partly Be Regulated by the Function of the Hepatic α­Tocopherol Transfer Protein.

Tocomonoenols are vitamin E derivatives present in foods with a single double bond at carbon 11' in the sidechain. The α-tocopherol transfer protein (TTP) is required for the maintenance of normal α-tocopherol (αT) concentrations. Its role in the tissue distribution of α-11'-tocomonoenol (αT1) is unknown. We investigated the tissue distribution of αT1 and αT in wild-type (TTP+/+) and TTP knockout (TTP-/-) mice fed diets with either αT or αT1 for two weeks. αT1 was only found in blood, not tissues. αT concentrations in TTP+/+ mice were in the order of adipose tissue > brain > heart > spleen > lungs > kidneys > small intestine > liver. Loss of TTP function depleted αT in all tissues. αT1, contrary to αT, was still present in the blood of TTP-/- mice (16% of αT1 in TTP+/+). Autoclaving and storage at room temperature reduced αT and αT1 in experimental diets. In conclusion, αT1 is bioavailable, reaches the blood in mice, and may not entirely depend on TTP function for secretion into the systemic circulation. However, due to instability of the test compounds in the experimental diets, further in vivo experiments are required to clarify the role of TTP in αT1 secretion. Future research should consider compound stability during autoclaving of rodent feed.

Prospective feasibility analysis of a novel off-line approach for MR-guided radiotherapy.

BACKGROUND: The present work aimed to analyze the feasibility of a shuttle-based MRI-guided radiation therapy (MRgRT) in the treatment of pelvic malignancies. PATIENTS AND METHODS: 20 patients with pelvic malignancies were included in this prospective feasibility analysis. Patients underwent daily MRI in treatment position prior to radiotherapy at the German Cancer Research Center. Positional inaccuracies, time and patient compliance were assessed for the application of off-line MRgRT. RESULTS: In 78% of applied radiation fractions, MR imaging for position verification could be performed without problems. Additionally, treatment-related side effects and reduced patient compliance were only responsible for omission of MRI in 9% of radiation fractions. The study workflow took a median time of 61 min (range 47-99 min); duration for radiotherapy alone was 13 min (range 7-26 min). Patient positioning, MR imaging and CT imaging including patient repositioning and the shuttle transfer required median times of 10 min (range 7-14 min), 26 min (range 15-60 min), 5 min (range 3-8 min) and 8 min (range 2-36 min), respectively. To assess feasibility of shuttle-based MRgRT, the reference point coordinates for the x, y and z axis were determined for the MR images and CT obtained prior to the first treatment fraction and correlated with the coordinates of the planning CT. In our dataset, the median positional difference between MR imaging and CT-based imaging based on fiducial matching between MR and CT imaging was equal to or less than 2 mm in all spatial directions. The limited space in the MR scanner influenced patient selection, as the bore of the scanner had to accommodate the immobilization device and the constructed stereotactic frame. Therefore, obese, extremely muscular or very tall patients could not be included in this trial in addition to patients for whom exposure to MRI was generally judged inappropriate. CONCLUSION: This trial demonstrated for the first time the feasibility and patient compliance of a shuttle-based off-line approach to MRgRT of pelvic malignancies.

Accelerated separation of GC-amenable lipid classes in plant oils by countercurrent chromatography in the co-current mode.

Triacylglycerols represent the major part (>90%) in most plant oils and have to be eliminated, when the minor compounds such as phytosterols or tocopherols should be analyzed. Here, we used an all liquid-liquid chromatographic technique, countercurrent chromatography (CCC), to fractionate the minor lipids before gas chromatography (GC) analysis. To cover the wide range of polarity of the minor compounds, we used the co-current mode, in which both mobile and stationary phase are pumped through the system. This allowed to elute substances which partitioned almost exclusively in the stationary phase within 90 min. After testing with standard compounds, the method was applied to the separation of sesame oil and sunflower oil samples. The abundant triacylglycerols could be effectively separated from tocopherols, phytosterols, diacylglycerols, and free fatty acids in the samples, and these compounds could be analyzed (after trimethylsilylation) by GC coupled with mass spectrometry. After the enrichment caused by the CCC fractionation, we were also able to identify the tocopherol derivative α-tocomonoenol, which had not been described in sunflower oil before. Also, separation of sesame oil yielded a mixture of the polar compounds sesamin and sesamolin without further impurities.

Discovery of Glutamate Carboxypeptidase III Ligands to Compete the Uptake of [177Lu]Lu-PSMA-617 in Healthy Organs.

Prostate-specific membrane antigen (PSMA)-targeted radio ligand therapeutics (RLTs), such as [177Lu]Lu-PSMA-617 (Pluvicto), have been shown to accumulate in salivary glands and kidneys, potentially leading to undesired side effects. As unwanted accumulation in normal organs may derive from the cross-reactivity of PSMA ligands to glutamate carboxypeptidase III (GCPIII), it may be convenient to block this interaction with GCPIII-selective ligands. Parallel screening of a DNA-encoded chemical library (DEL) against GCPIII and PSMA allowed the identification of GCPIII binders. Structure-activity relationship (SAR) studies resulted in the identification of nanomolar GCPIII ligands with up to 1000-fold selectivity over PSMA. We studied the ability of GCPIII ligands to counteract the binding of [177Lu]Lu-PSMA-617 to human salivary glands by autoradiography and could demonstrate a partial radioprotection.

DNA-encoded chemical libraries enable the discovery of potent PSMA-ligands with substantially reduced affinity towards the GCPIII anti-target.

Prostate-specific membrane antigen (PSMA) is a tumor-associated protein that has been successfully targeted with small organic ligands and monoclonal antibodies. Pluvicto™ is a PSMA-targeted radioligand therapeutic (RLT) recently approved by the FDA for the treatment of metastatic castration-resistant prostate cancer (2022 FDA marketing authorization). Although a large Phase III clinical trial (VISION trial) demonstrated clinical benefits in patients treated with Pluvicto™, the therapeutic window of the drug is narrowed by its undesired accumulation in healthy organs. Glutamate carboxypeptidase III (GCPIII), an enzyme sharing 70% identity with PSMA, may be responsible for the off-target accumulation of PSMA-RLTs in salivary glands and kidneys. In this work, we designed and synthesized affinity and selectivity maturation DNA-encoded chemical libraries (ASM-DELs) comprising 18'284'658 compounds that were screened in parallel against PSMA and GCPIII with the aim to identify potent and selective PSMA ligands for tumor-targeting applications. Compound A70-B104 was isolated as the most potent and selective ligand (KD of 900 pM for PSMA, KD of 40 nM for GCPIII). 177Lu-A70-B104-DOTA, a radiolabeled derivative of compound A70-B104, presented selective accumulation in PSMA-positive cancer lesions (i.e., 7.4% ID g-1, 2 hour time point) after systemic administration in tumor-bearing mice. The results of autoradiography experiments showed that 177Lu-A70-B104-DOTA selectively binds to PSMA-positive cancer tissues, while negligible binding on human salivary glands was observed.

Some chemistry of tris(pyrazolyl)methylthiolate derivatives.

An efficient synthetic method for the preparation of TAS tris(pyrazolyl)methylthiolate (3) is reported. Nucleophilic exchange reactions with 3 gave (pyr)C(=S)SC(pyr)3 (4) and MeSC(pyr)3 (5). 5 acts as scorpionate ligand in [MeSC(pyr)3Cr(CO)3] (6), from the decomposition of TDAE2+ [SC(pyr)3Mo(CO)3-]2 by SO2FCl TDAE2+[O=MoF4-F-Mo(=O)Cl4]2- (8) was isolated. The X-ray structures of 3-6 and 8 are discussed.

Targeted quantitation of furan fatty acids in edible oils by gas chromatography/triple quadrupole tandem mass spectrometry (GC-TQ/MS).

Furan fatty acids (FuFAs) have been recognized as beneficial food ingredients to human health. Herein, a targeted quantitation approach by gas chromatography coupled to triple quadrupole tandem mass spectrometry (GC-TQ/MS) was developed for the identification of FuFAs in common marine and other edible oils in multiple reaction monitoring (MRM) mode without any isolation and enrichment. The limit-of-quantitation (LOQ, 0.6 pg) was determined under the optimized parameters in MRM mode. Identification of FuFAs in common edible oils demonstrated that marine fish oils were concentrated sources of 9-(3-methyl-5-pentylfuran-2-yl)nonanoic acid (9M5), 11-(3,4-dimethyl-5-propylfuran-2-yl)undecanoic acid (11D3) and 11-(3,4-dimethyl-5-pentylfuran-2-yl)undecanoic acid (11D5). However, FuFAs were not identified in common plant oils. Additionally, 11D5 was identified in the lipids of Schizochytrium limacinum at a comparable level with that in marine fish oil. We believe that this protocol could facilitate the qualitative and quantitative analysis of FuFAs in food and biological samples.

The indole motif is essential for the antitrypanosomal activity of N5-substituted paullones.

Severe infections with potentially fatal outcomes are caused by parasites from the genera Trypanosoma and Leishmania (class Kinetoplastea). The diseases affect people of remote areas in the tropics and subtropics with limited access to adequate health care. Besides insufficient diagnostics, treatment options are limited, with tenuous developments in recent years. Therefore, new antitrypanosomal antiinfectives are required to fight these maladies. In the presented approach, new compounds were developed and tested on the target trypanothione synthetase (TryS). This enzyme is crucial to the kinetoplastids' unique trypanothione-based thiol redox metabolism and thus for pathogen survival. Preceding studies have shown that N5-substituted paullones display antitrypanosomal activity as well as TryS inhibition. Herein, this compound class was further examined regarding the structure-activity relationships (SAR). Diverse benzazepinone derivatives were designed and tested in cell-based assays on bloodstream Trypanosoma brucei brucei (T. b. brucei) and intracellular amastigotes of Leishmania infantum (L. infantum) as well as in enzyme-based assays on L. infantum TryS (LiTryS) and T. b. brucei TryS (TbTryS). While an exchange of just the substituent in the 9-position of paullones led to potent inhibitors on LiTryS and T. b. brucei parasites, new compounds lacking the indole moiety showed a total loss of activity in both assays. Conclusively, the indole as part of the paullone structure is pivotal for keeping the TryS inhibitory and antitrypanosomal activity of this substance class.

Discovery of Antitrypanosomal Indolylacetamides by a Deconstruction-Optimization Strategy Applied to Paullones.

The parasitic kinetoplastid diseases Leishmaniasis, Chagas disease and Human African Trypanosomiasis constitute serious threats for populations throughout the (sub-)tropics. Most available drugs to treat these diseases possess inadequate properties and candidates to fill the drug pipeline are urgently needed. Paullone-N5 -acetamides inhibit trypanothione synthetase (TryS), an essential kinetoplastid enzyme, and exhibit antiparasitic activity in the low micromolar range, but lack the desired selectivity against mammalian cells (selectivity index (SI):<10). With the aim to identify the paullones' moieties responsible for TryS inhibition and bioactivity, we applied molecular simplification and ring disconnection approaches. The new indolylacetamides lost activity against the expected molecular target (TryS) compared to the reference paullone MOL2008 (Leishmania infantum TryS IC50 : 150 nM; Trypanosoma brucei bloodstream form EC50 : 4.3 µM and SI: 2.4). However, several of them retained potency (T. b. brucei EC50 : 2.4-12.0 µM) and improved selectivity (SI: 5 to >25).

Coaxial waveguide MRI.

As ultrahigh-field MR imaging systems suffer from the standing wave problems of conventional coil designs, the use of antenna systems that generate travelling waves was suggested. As a modification to the original approach, we propose the use of a coaxial waveguide configuration with interrupted inner conductor. This concept can focus the radiofrequency energy to the desired imaging region in the human body and can operate at different Larmor frequencies without hardware modifications, as it is not limited by a lower cut-off frequency. We assessed the potential of the method with a hardware prototype setup that was loaded with a tissue equivalent phantom and operated with imaging areas of different size. Signal and flip angle distributions within the phantom were analyzed, and imaging at different Larmor frequencies was performed. Results were compared to a finite difference time domain simulation of the setup that additionally provides information on the spatial distribution of the specific absorption rate load. Furthermore, simulation results with a human model (virtual family) are presented. It was found that the proposed method can be used for MRI at multiple frequencies, achieving transmission efficiencies similar to other travelling wave approaches but still suffers from several limitations due to the used mode of wave propagation.

3D-QSAR based on quantum-chemical molecular fields: toward an improved description of halogen interactions.

Current 3D-QSAR methods such as CoMFA or CoMSIA make use of classical force-field approaches for calculating molecular fields. Thus, they can not adequately account for noncovalent interactions involving halogen atoms like halogen bonds or halogen-π interactions. These deficiencies in the underlying force fields result from the lack of treatment of the anisotropy of the electron density distribution of those atoms, known as the "σ-hole", although recent developments have begun to take specific interactions such as halogen bonding into account. We have now replaced classical force field derived molecular fields by local properties such as the local ionization energy, local electron affinity, or local polarizability, calculated using quantum-mechanical (QM) techniques that do not suffer from the above limitation for 3D-QSAR. We first investigate the characteristics of QM-based local property fields to show that they are suitable for statistical analyses after suitable pretreatment. We then analyze these property fields with partial least-squares (PLS) regression to predict biological affinities of two data sets comprising factor Xa and GABA-A/benzodiazepine receptor ligands. While the resulting models perform equally well or even slightly better in terms of consistency and predictivity than the classical CoMFA fields, the most important aspect of these augmented field-types is that the chemical interpretation of resulting QM-based property field models reveals unique SAR trends driven by electrostatic and polarizability effects, which cannot be extracted directly from CoMFA electrostatic maps. Within the factor Xa set, the interaction of chlorine and bromine atoms with a tyrosine side chain in the protease S1 pocket are correctly predicted. Within the GABA-A/benzodiazepine ligand data set, PLS models of high predictivity resulted for our QM-based property fields, providing novel insights into key features of the SAR for two receptor subtypes and cross-receptor selectivity of the ligands. The detailed interpretation of regression models derived using improved QM-derived property fields thus provides a significant advantage by revealing chemically meaningful correlations with biological activity and helps in understanding novel structure-activity relationship features. This will allow such knowledge to be used to design novel molecules on the basis of interactions additional to steric and hydrogen-bonding features.

Cytotoxicity, cellular uptake, and metabolism to short-chain metabolites of 11'-α-tocomonoenol is similar to RRR-α-tocopherol in HepG2 cells.

Contrary to the major vitamin E congener α-tocopherol, which carries a saturated sidechain, and α-tocotrienol, with a threefold unsaturated sidechain, little is known about the intracellular fate of α-tocomonoenol, a minor vitamin E derivative with a single double bond in C11'-position of the sidechain. We hypothesized that, due to structural similarities, the uptake and metabolism of α-tocomonoenol will resemble that of α-tocopherol. Cytotoxicity, cellular uptake of α-tocomonoenol, α-tocopherol and α-tocotrienol and conversion into the short-chain metabolites αCEHC and αCMBHC were studied in HepG2 cells. α-Tocomonoenol did not show significant effects on cell viability and its uptake was similar to that observed for α-tocopherol and significantly lower than for α-tocotrienol. α-Tocomonoenol was mainly metabolized to αCMBHC in liver cells, but to a lower extent than α-tocotrienol, while α-tocopherol was not metabolized in quantifiable amounts at all. In summary, the similarities in the cytotoxicity, uptake and metabolism of α-tocomonoenol and α-tocopherol suggest that this minor vitamin E congener deserves more attention in future research with regard to its potential vitamin E activity.

Asymmetric, visible light-mediated radical sulfinyl-Smiles rearrangement to access all-carbon quaternary stereocentres.

The asymmetric construction of all-carbon quaternary centres within acyclic settings represents a long-standing challenge for synthetic chemists. Alongside polar and radical methods, rearrangement reactions represent an attractive platform, but still broadly applicable methods are in high demand. Here we report an asymmetric, radical sulfinyl-Smiles rearrangement to access acyclic amides that bear an α-all-carbon quaternary centre. Our strategy uses enantioenriched N-arylsulfinyl acrylamides as acceptors for a variety of radicals produced in situ under mild photoredox conditions. The sulfinamido group not only directs the 1,4-migration of the aryl moiety onto the α-carbon of the amide, which thus governs its absolute configuration, but also functions as a traceless chiral auxiliary. The amides obtained in this multicomponent process are prevalent in pharmaceuticals, agrochemicals and bioactive natural products, and can be transformed into valuable chiral α,α-disubstituted acids, oxindoles as well as into ß,ß-disubstituted amines, highlighting the synthetic potential of this transformation.

Fast isolation of the environmentally relevant halogenated natural product MHC-1 by means of countercurrent chromatography.

Environmentally relevant halogenated natural products (HNPs) are frequently similarly high concentrated in marine biota as major anthropogenic persistent organic pollutants (POPs). The lack of widely available reference standards, however, hampers the in-depth research of several HNPs. For instance, (1R,2S,4R,5R,1'E)-2-bromo-1-bromomethyl-1,4-dichloro-5-(2'-chloroethenyl)-5-methylcyclohexane (MHC-1), which is produced by species referred to the red seaweed Plocamium cartilagineum has not yet been synthesized due to its complex structure and stereochemistry. For this reason, we aimed to establish a method for fast isolation of mg-amounts of MHC-1 from its natural producer based on countercurrent chromatography (CCC). Five biphasic solvent systems were tested and finally, the solvent system acetonitrile/n-hexane/toluene (9:9:2, v/v/v) was selected for the separations due to its suitable partition coefficient of MHC-1 (KU/L = 0.52). n-Hexane extracts of dried P. cartilagineum were directly injected into the CCC system. Four subsequent CCC runs from three samples of Plocamium cartilagineum (two from Heligoland, Germany and one from Brittany, France) could be performed with high reproducibility. Together, the main fraction provided ~16 mg MHC-1 in a purity of >97% according to GC/FID, GC/ECNI-MS and NMR analysis. This amount could be used to prepare ~1600 quantitative standard solutions of MHC-1. The high MHC-1 content in the seaweed sample collected at Brittany indicated that this area was another hotspot of MHC-1.

Promoting Photocatalytic Hydrogen Evolution Activity of Graphitic Carbon Nitride with Hole-Transfer Agents.

Visible light-driven photocatalytic reduction of protons to H2 is considered a promising way of solar-to-chemical energy conversion. Effective transfer of the photogenerated electrons and holes to the surface of the photocatalyst by minimizing their recombination is essential for achieving a high photocatalytic activity. In general, a sacrificial electron donor is used as a hole scavenger to remove photogenerated holes from the valence band for the continuation of the photocatalytic hydrogen (H2 ) evolution process. Here, for the first time, the hole-transfer dynamics from Pt-loaded sol-gel-prepared graphitic carbon nitride (Pt-sg-CN) photocatalyst were investigated using different adsorbed hole acceptors along with a sacrificial agent (ascorbic acid). A significant increment (4.84 times) in H2 production was achieved by employing phenothiazine (PTZ) as the hole acceptor with continuous H2 production for 3 days. A detailed charge-transfer dynamic of the photocatalytic process in the presence of the hole acceptors was examined by time-resolved photoluminescence and in situ electron paramagnetic resonance studies.

Conversion of Cannabidiol (CBD) into Psychotropic Cannabinoids Including Tetrahydrocannabinol (THC): A Controversy in the Scientific Literature.

Cannabidiol (CBD) is a naturally occurring, non-psychotropic cannabinoid of the hemp plant Cannabis sativa L. and has been known to induce several physiological and pharmacological effects. While CBD is approved as a medicinal product subject to prescription, it is also widely sold over the counter (OTC) in the form of food supplements, cosmetics and electronic cigarette liquids. However, regulatory difficulties arise from its origin being a narcotic plant or its status as an unapproved novel food ingredient. Regarding the consumer safety of these OTC products, the question whether or not CBD might be degraded into psychotropic cannabinoids, most prominently tetrahydrocannabinol (THC), under in vivo conditions initiated an ongoing scientific debate. This feature review aims to summarize the current knowledge of CBD degradation processes, specifically the results of in vitro and in vivo studies. Additionally, the literature on psychotropic effects of cannabinoids was carefully studied with a focus on the degradants and metabolites of CBD, but data were found to be sparse. While the literature is contradictory, most studies suggest that CBD is not converted to psychotropic THC under in vivo conditions. Nevertheless, it is certain that CBD degrades to psychotropic products in acidic environments. Hence, the storage stability of commercial formulations requires more attention in the future.

Improving the resolution of overlapping peaks by heartcut two-dimensional countercurrent chromatography with the same solvent system in both dimensions.

The countercurrent chromatographic (CCC) isolation of structurally related compounds with high chemical purity can be challenging, especially in the field of lipids, were the range of biphasic solvent systems is limited. In the past, special elution modes like recycling CCC or re-injection of pooled fractions into a subsequent CCC (off-line re-injection CCC) were applied successfully to improve the separation of interfering compounds. However, isolation of minor compounds is often hampered by overlapping major compounds in natural samples. In this study we used heartcut two-dimensional CCC (2D CCC) to transfer the analyte completely to the next dimension while the interfering compound was partly removed. This procedure not only reduced the amount of the interfering major compound but also its peak width by up to 20% in the second dimension despite the longer elution time. Since the same solvent system and coil dimensions were used in the second dimension, this positive effect was attributed to the "transfer volume" which can be compared to an injection volume into the second dimension. As a consequence, improved peak resolution was generally observed for peaks which were partly transferred to the next dimension. This unexpected beneficial effect of 2D CCC on peak resolution was demonstrated by means of two examples in comparison with CCC in recycling and off-line re-injection mode. Applying the same CCC conditions, heartcut 2D CCC yielded >14 mg of the pentacyclic triterpenol lupeol, isolated from the unsaponifiable matter of shea butter, while recycling CCC and one-dimensional CCC only yielded ∼5 and ∼1 mg, respectively. Moreover, the resolution of three different ergosterol derivates (ergosta-5,7-dienol, ergosta-7,22-dienol and ergost-7-enol) which were separated from the very abundant ergosterol in the unsaponifiable matter of freeze-dried button mushrooms could be improved with heartcut 2D CCC compared with off-line re-injection CCC.