Synthesis of 2-methyl- and 2-methylenecyclobutane amino acids

An efficient and easy formal [2+2] cycloaddition (Michael-Dieckmann-type reaction) on methyl 2-acetamidoacrylate with ketene diethyl acetal gave the cyclobutane core. Two kinds of 2-substituted cyclobutane amino acids have been obtained from this compound by means of stereocontrolled interconversion of functional groups: 1-amino-2-methylcyclobutane-1-carboxylic acids (2,4-methanovalines) and 1-amino-2-methylenecyclobutane-1-carboxylic acid. The latter amino acid can be regarded as a restricted -methyl-- vinylglycine. © 2005 Elsevier Ltd. All rights reserved

Conformationally-Locked C-Glycosides: Tuning Aglycone Interactions for Optimal Cheperone Behaviour in Gaucher Fibroblasts

A series of conformationally locked C-glycosides based on the 3-aminopyrano[3,2-b]pyrrol-2(1H)-one (APP) scaffold has been synthesized. The key step involved a totally stereocontrolled C-Michael addition of a serine-equivalent C-nucleophile to tri-O-benzyl-2-nitro-D-galactal, previously published by the authors. Stereoselective transformations of the Michael adduct allowed us the synthesis of compounds with mono- or diantennated aglycone moieties and different topologies. In vitro screening showed highly selective inhibition of bovine liver β-glucosidase/β-galactosidase and specific inhibition of human β-glucocerebrosidase among lysosomal glycosidases for compounds bearing palmitoyl chains in the aglycone, with a marked dependence of the inhibition potency upon their number and location. Molecular dynamics simulations highlighted the paramount importance of an optimal orientation of the hydrophobic substituent to warrant efficient non-glycone interactions, which are critical for the binding affinity. The results provide a rationale for the strong decrease of the inhibition potency of APP compounds on going from neutral to acidic pH. The best candidate was found to behave as pharmacological chaperone in Gaucher fibroblasts with homozygous N370S and F213I mutations, with enzyme activity enhancements similar to those encountered for the reference compound AmbroxolMinisterio de Economía y Competitividad CTQ2012-36365, SAF2013-44021-RJunta de Andalucía FQM-1467European Union Seventh Framework Programme FP7-People-2012-CI

Methyl-to-Ethyl Replacement Makes the Difference: Structure-Guided Design of a New Cancer Vaccine Based on a Tn An-gen Surrogate

Mucins are large extracellular glycoproteins that exhibit different glycosylation patterns and post-translational modifications between healthy and cancer cells[1]. Mucin 1 (MUC1) is a common glycoprotein in cancer cells that plays a multifaceted role in cancer development, cell proliferation, and migration[2,3]. These properties make MUC1 an excellent antigen for cancer vaccine candidates. Several works[3,4] have used MUC1-derived GalNAc glycopeptides, especially the sequence APDT(-O-GalNAc-Thr)RP, for cancer vaccine development but with limited success due to the low immunogenicity and stability of the glycopeptide. We have developed a novel Tn antigen following a structure-guided design in which the threonine of the above sequence has been replaced by the unnatural amino acid L-4-hydroxynorvaline (Hnv) to increase the antigen/antibody affinity. We have confirmed by X-crystallography analysis of the complex that the ethyl group at the C of the unnatural residue favors the CH/ interactions between the Tn antigen and the SM3 antibody, resulting in a slight increase in affinity due to enthalpy-entropy balance. The chemical modification (HnvThr) allows the synthetic glycopeptide to exhibit similar properties to the naturally occurring derivative, similar serum stability, and a similar conformational landscape in solution. A vaccination campaign in mice is currently underway in which the synthetic antigen has been conjugated to evaluate the biological impact of this chemical modification

APPLICATION OF AUGMENTED REALITY IN CHEMISTRY TEACHING

Augmented reality (AR) is the combination of real environment and virtual elements that have the taskof enriching reality with information or other elements. In this sense, chemistry, and organic chemistryin particular, are disciplines for which students require an adequate spatial vision to understand theconcepts and we propose that augmented reality can support and promote spatial skills.Although different studies have been carried out in recent years on the use of augmented reality inthis context, it is necessary to deepen into its application from a multidisciplinary point of view, being inclose contact with the developers of the applications and considering the needs and criteria of professorsof different levels and students.To this end, this project has counted on the collaboration with the company CreativiTIC, throughits MetAClass Studio platform. From our side, we needed to create markers and the linked chemicalstructures, which students can then visualise the molecular structures in 3D directly from their mobiledevices. The development has been carried out in a multidisciplinary way and covering differenteducational levels, from Secondary Education to University.Finally, we conducted an evaluation of this tool to qualitatively assess whether students achieved abetter understanding and improved their visualisation of molecular structures.This project demonstrates how the appropriate use of augmented reality allows for a betterunderstanding of chemical structures and their reactions, as well as increasing student motivation, butalso highlights the need for proper teacher training and the appropriate implementation of the technolog

Deciphering the Non-Equivalence of Serine and Threonine O-Glycosylation Points: Implications for Molecular Recognition of the Tn Antigen by an anti-MUC1 Antibody

© 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. The structural features of MUC1-like glycopeptides bearing the Tn antigen (α-O-GalNAc-Ser/Thr) in complex with an anti MUC-1 antibody are reported at atomic resolution. For the α-O-GalNAc-Ser derivative, the glycosidic linkage adopts a high-energy conformation, barely populated in the free state. This unusual structure (also observed in an α-S-GalNAc-Cys mimic) is stabilized by hydrogen bonds between the peptidic fragment and the sugar. The selection of a particular peptide structure by the antibody is thus propagated to the carbohydrate through carbohydrate/peptide contacts, which force a change in the orientation of the sugar moiety. This seems to be unfeasible in the α-O-GalNAc-Thr glycopeptide owing to the more limited flexibility of the side chain imposed by the methyl group. Our data demonstrate the non-equivalence of Ser and Thr O-glycosylation points in molecular recognition processes. These features provide insight into the occurrence in nature of the APDTRP epitope for anti-MUC1 antibodies.Peer Reviewe

The use of fluoroproline in MUC1 antigen enables efficient detection of antibodies in patients with prostate cancer

A structure-based design of a new gene22ration tumor-associated glycopeptides with improved affinity against two anti-MUC1 antibodies is described. These unique antigens feature a fluorinated proline residue, such as a (4S)-4-fluoro-L-proline or 4,4-difluoroproline, at the most immunogenic domain. Binding assays using bio-layer interferometry reveal 3-fold to 10-fold affinity improvement with respect to the natural (glyco)peptides. According to X-ray crystallography and MD simulations, the fluorinated residues stabilize the antigen-antibody complex by enhancing key CH/ interactions. Interestingly, a notable improvement in detection of cancer-associated anti-MUC1 antibodies from serum of patients with prostate cancer is achieved with the non-natural antigens, which proves that these derivatives can be considered better diagnostic tools than the natural antigen for this type of cancer.We thank the Ministerio de Economía y Competitividad (projects CTQ2015-67727-R, UNLR13-4E-1931, CTQ2013-44367-C2-2-P, CTQ2015-64597-C2-1P, and BFU2016-75633-P). I. A. B. thanks the Asociación Española Contra el Cancer en La Rioja for a grant. I. S. A. and G. J. L. B. thank FCT Portugal (PhD studentship and FCT Investigator, respectively) and the EPSRC for funding. G. J. L. B. holds a Royal Society URF and an ERC StG (TagIt). F.C. and G. J. L. B thank the EU (Marie-Sklodowska Curie ITN, Protein Conjugates). R.H-G. thanks Agencia Aragonesa para la Investigación y Desarrollo (ARAID) and the Diputación General de Aragón (DGA, B89) for financial support. The research leading to these results has also received funding from the FP7 (2007-2013) under BioStruct-X (grant agreement N°283570 and BIOSTRUCTX_5186). We thank synchrotron radiation source DIAMOND (Oxford) and beamline I04 (number of experiment mx10121-19). Hokkaido University group acknowledges to JSPS KAKENHI Grant Number 25220206 and JSPS Wakate B KAKENHI Grant Number 24710242. We also thank CESGA (Santiago de Compostela) for computer support

The use of fluoroproline in MUC1 antigen enables efficient detection of antibodies in patients with prostate cancer

A structure-based design of a new generation of tumor-associated glycopeptides with improved affinity against two anti-MUC1 antibodies is described. These unique antigens feature a fluorinated proline residue, such as a (4S)-4-fluoro-l-proline or 4,4-difluoro-l-proline, at the most immunogenic domain. Binding assays using biolayer interferometry reveal 3-fold to 10-fold affinity improvement with respect to the natural (glyco)peptides. According to X-ray crystallography and MD simulations, the fluorinated residues stabilize the antigen-antibody complex by enhancing key CH/π interactions. Interestingly, a notable improvement in detection of cancer-associated anti-MUC1 antibodies from serum of patients with prostate cancer is achieved with the non-natural antigens, which proves that these derivatives can be considered better diagnostic tools than the natural antigen for prostate cancer.We thank the Ministerio de Economía y Competitividad (projects CTQ2015-67727-R, UNLR13-4E-1931, CTQ2013-44367-C2-2-P, CTQ2015-64597-C2-1P, and BFU2016-75633-P). I.A.B. thanks the Asociación Española Contra el Cancer en La Rioja for a grant. I.S.A. and G.J.L.B. thank FCT Portugal (Ph.D. studentship and FCT Investigator, respectively) and EPSRC. G.J.L.B. holds a Royal Society URF and an ERC StG (TagIt). F.C. and G.J.L.B thank the EU (Marie-Sklodowska Curie ITN, Protein Conjugates). R.H-G. thanks Agencia Aragonesa para la Investigación y Desarrollo (ARAID) and the Diputación General de Aragón (DGA, B89) for financial support. The research leading to these results has also received funding from the FP7 (2007-2013) under BioStruct-X (grant agreement no. 283570 and BIOSTRUCTX_5186). We thank synchrotron radiation source DIAMOND (Oxford) and beamline I04 (number of experiment mx10121-19). The Hokkaido University group acknowledges JSPS KAKENHI grant no. 25220206 and JSPS Wakate B KAKENHI grant no. 24710242. We also thank CESGA (Santiago de Compostela) for computer support.Peer reviewedPeer Reviewe

Structure-Guided Approach for the Development of MUC1-Glycopeptide-Based Cancer Vaccines with Predictable Responses

Funding Information: This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 956544. F.S.L., A.E.B., T.K.R., and S.T. are recipients of Sklodowska Curie ITN, DIRNANO, grant agreement No. 956544. F.C. thanks the Mizutani Foundation for Glycoscience (grant 220115). I.A.B. and A.A. thank the Asociación Española Contra el Cancer (AECC), sección La Rioja, for doctoral fellowship. We thank the ALBA (Barcelona, Spain) synchrotron beamline XALOC. We thank ARAID, the Agencia Estatal de Investigación (AEI, BFU2016-75633-P and PID2019-105451GB-I00 to R.H.-G., PID2021-127622OB-I00 and PDC2022-133725-C21 to F.C., PID2022-136735OB-I00 to A.M.), Universidad de La Rioja (REGI22/47 and REGI22/16), Gobierno de Aragón (E34_R17 and LMP58_18 to R.H.-G.) with FEDER (2014-2020) funds for “Building Europe from Aragón” for financial support, and the COST Action CA18103 INNOGLY: Innovation with Glycans new frontiers from synthesis to new biological targets. F.M. acknowledges Fundação para a Ciência e Tecnologia Portugal (FCT-Portugal) for 2020.00233.CEECIND and PTDC/BIA-MIB/31028/2017. A.S.G. thanks FCT-Portugal for PhD fellowships (SFRH/BD/140394/2018 and COVID/BD/152986/2023). F.M and A.S.G. thank UCIBIO project (UIDP/04378/2020 and UIDB/04378/2020), and Associate Laboratory Institute for Health and Bioeconomy - i4HB project (LA/P/0140/2020) and the National NMR Facility supported by FCT-Portugal (ROTEIRO/0031/2013-PINFRA/22161/2016, cofinanced by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC). The authors thank Dr Vikki Cantrill for her help with the editing of this manuscript. Funding Information: This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 956544. F.S.L., A.E.B., T.K.R., and S.T. are recipients of Sklodowska Curie ITN, DIRNANO, grant agreement No. 956544. F.C. thanks the Mizutani Foundation for Glycoscience (grant 220115). I.A.B. and A.A. thank the Asociación Española Contra el Cancer (AECC), sección La Rioja, for doctoral fellowship. We thank the ALBA (Barcelona, Spain) synchrotron beamline XALOC. We thank ARAID, the Agencia Estatal de Investigación (AEI, BFU2016-75633-P and PID2019-105451GB-I00 to R.H.-G., PID2021-127622OB-I00 and PDC2022-133725-C21 to F.C., PID2022-136735OB-I00 to A.M.), Universidad de La Rioja (REGI22/47 and REGI22/16), Gobierno de Aragón (E34_R17 and LMP58_18 to R.H.-G.) with FEDER (2014–2020) funds for “Building Europe from Aragón” for financial support, and the COST Action CA18103 INNOGLY: Innovation with Glycans new frontiers from synthesis to new biological targets. F.M. acknowledges Fundação para a Ciência e Tecnologia Portugal (FCT-Portugal) for 2020.00233.CEECIND and PTDC/BIA-MIB/31028/2017. A.S.G. thanks FCT-Portugal for PhD fellowships (SFRH/BD/140394/2018 and COVID/BD/152986/2023). F.M and A.S.G. thank UCIBIO project (UIDP/04378/2020 and UIDB/04378/2020), and Associate Laboratory Institute for Health and Bioeconomy - i4HB project (LA/P/0140/2020) and the National NMR Facility supported by FCT-Portugal (ROTEIRO/0031/2013–PINFRA/22161/2016, cofinanced by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC). The authors thank Dr Vikki Cantrill for her help with the editing of this manuscript. Publisher Copyright: © 2023 The Authors. Published by American Chemical SocietyMucin-1 (MUC1) glycopeptides are exceptional candidates for potential cancer vaccines. However, their autoantigenic nature often results in a weak immune response. To overcome this drawback, we carefully engineered synthetic antigens with precise chemical modifications. To be effective and stimulate an anti-MUC1 response, artificial antigens must mimic the conformational dynamics of natural antigens in solution and have an equivalent or higher binding affinity to anti-MUC1 antibodies than their natural counterparts. As a proof of concept, we have developed a glycopeptide that contains noncanonical amino acid (2S,3R)-3-hydroxynorvaline. The unnatural antigen fulfills these two properties and effectively mimics the threonine-derived antigen. On the one hand, conformational analysis in water shows that this surrogate explores a landscape similar to that of the natural variant. On the other hand, the presence of an additional methylene group in the side chain of this analog compared to the threonine residue enhances a CH/π interaction in the antigen/antibody complex. Despite an enthalpy-entropy balance, this synthetic glycopeptide has a binding affinity slightly higher than that of its natural counterpart. When conjugated with gold nanoparticles, the vaccine candidate stimulates the formation of specific anti-MUC1 IgG antibodies in mice and shows efficacy comparable to that of the natural derivative. The antibodies also exhibit cross-reactivity to selectively target, for example, human breast cancer cells. This investigation relied on numerous analytical (e.g., NMR spectroscopy and X-ray crystallography) and biophysical techniques and molecular dynamics simulations to characterize the antigen-antibody interactions. This workflow streamlines the synthetic process, saves time, and reduces the need for extensive, animal-intensive immunization procedures. These advances underscore the promise of structure-based rational design in the advance of cancer vaccine development.publishersversionpublishe