Interaction of antigenic peptides with MHC class I molecules on living cells studied by photoaffinity labeling.

Using a direct binding assay based on photoaffinity labeling, we have studied the interaction of antigenic peptides with murine MHC class I molecules on living cells. Photoreactive derivatives were prepared by N-terminal amidation with iodo, 4-azido salicylic acid of the Kd restricted Plasmodium berghei circumsporozoite (P.b. CS) peptide 253-260 (YIPSAEKI) and the Db-restricted Adenovirus 5 early region 1A (Ad5 E1A) peptide 234-243 (SGPSNTPPEI). As assessed in functional competition experiments, both peptide derivatives retained the specific binding activity of the parental peptides for Kd or Dd, respectively. The P.b. CS photoprobe specifically labeled Kd molecules on P815 (H-2d) cells, but failed to label RMA (H-2b) cells. Conversely, the Ad5 E1A photoprobe specifically labeled Db molecules on RMA cells, but failed to label P815 cells. When the two photoprobes were tested on a panel of Con A-activated spleen cells expressing 10 different H-2 haplotypes, significant photoaffinity labeling was observed only on H-2d cells with the P.b. CS photoprobe and on H-2b cells with the Ad5 E1A photoprobe. Labeling of cell-associated Kd or Db molecules with the photoprobes was specifically inhibited by antigenic peptides known to be presented by the same class I molecule. Photoaffinity labeling of Kd with the P.b. CS photoprobe was used to study the dynamics of peptide binding on living P815 cells. Binding increased steadily with the incubation period (up to 8 h) at 37 degrees C and at ambient temperature, but was greatly reduced (greater than 95%) at 0 to 4 degrees C or in the presence of ATP synthesis inhibitors. The magnitude of the labeling was twofold higher at room temperature than at 37 degrees C. In contrast, binding to isolated Kd molecules in solution rapidly reached maximal binding, particularly at 37 degrees C. Dissociation of the photoprobe from either cell-associated or soluble Kd molecules was similar, with a half time of approximately 1 h at 37 degrees C, whereas the complexes were long-lived at 4 degrees C in both instances.

Differential T cell receptor photoaffinity labeling among H-2Kd restricted cytotoxic T lymphocyte clones specific for a photoreactive peptide derivative. Labeling of the alpha-chain correlates with J alpha segment usage.

Using a direct binding assay based on photoaffinity labeling, we studied the interaction of T cell receptor (TCR) with a Kd-bound photoreactive peptide derivative on living cells. The Kd-restricted Plasmodium berghei circumsporozoite (PbCS) peptide 253-260 (YIPSAEKI) was reacted NH2-terminally with biotin and at the TCR contact residue Lys259 with photoreactive iodo, 4-azido salicylic acid (IASA) to make biotin-YIPSAEK(IASA)I. Cytotoxic T lymphocyte (CTL) clones derived from mice immunized with this derivative recognized this conjugate, but not a related one lacking the IASA group nor the parental PbCS peptide. The clones were Kd restricted. Recognition experiments with variant conjugates, lacking substituents from IASA, revealed a diverse fine specificity pattern and indicated that this group interacted directly with the TCR. The TCR of four clones could be photoaffinity labeled by biotin-YIPSAEK(125IASA)I. This labeling was dependent on the conjugates binding to the Kd molecule and was selective for the TCR alpha (2 clones) or beta chain (1 clone), or was common for both chains (1 clone). TCR sequence analysis showed a preferential usage of J alpha TA28 containing alpha chains that were paired with V beta 1 expressing beta chains. The TCR that were photoaffinity labeled at the alpha chain expressed these J alpha and V beta segments. The tryptophan encoded by the J alpha TA28 segment is rarely found in other J alpha segments. Moreover, we show that the IASA group interacts preferentially with tryptophan in aqueous solution. We thus propose that for these CTL clones, labeling of the alpha chain occurs via the J alpha-encoded tryptophan residue.

Reverse transcriptase-dependent and -independent phases of infection with mouse mammary tumor virus: implications for superantigen function.

Mouse mammary tumor virus (MMTV) encodes a superantigen (SAg) that promotes stable infection and virus transmission. Upon subcutaneous MMTV injection, infected B cells present SAg to SAg-reactive T cells leading to a strong local immune response in the draining lymph node (LN) that peaks after 6 d. We have used the reverse transcriptase inhibitor 3'-azido-3'-deoxythymidine (AZT) to dissect in more detail the mechanism of SAg-dependent enhancement of MMTV infection in this system. Our data show that no detectable B or T cell response to SAg occurs in AZT pretreated mice. However, if AZT treatment is delayed 1-2 d after MMTV injection, a normal SAg-dependent local immune response is observed on day 6. Quantitation of viral DNA in draining LN of these infected mice indicates that a 4,000-fold increase in the absolute numbers of infected cells occurs between days 2 and 6 despite the presence of AZT. Furthermore MMTV DNA was found preferentially in surface IgG+ B cells of infected mice and was not detectable in SAg-reactive T cells. Collectively our data suggest that MMTV infection occurs preferentially in B cells without SAg involvement and is completed 1-2 d after virus challenge. Subsequent amplification of MMTV infection between days 2 and 6 requires SAg expression and occurs in the absence of any further requirement for reverse transcription. We therefore conclude that clonal expansion of infected B cells via cognate interaction with SAg-reactive T cells is the predominant mechanism for increasing the level of MMTV infection. Since infected B cells display a memory (surface IgG+) phenotype, both clonal expansion and possibly longevity of the virus carrier cells may contribute to stable MMTV infection.

Synthesis and in vitro evaluation of a novel radioligand for αvβ3 integrin receptor imaging: [(18)F]FPPA-c(RGDfK).

The development of RGD-based antagonist of αvβ3 integrin receptor has enhanced the interest in PET probes to image this receptor for the early detection of cancer, to monitor the disease progression and the response to therapy. In this work, a novel prosthetic group (N-(4-fluorophenyl)pent-4-ynamide or FPPA) for the (18)F-labeling of an αvβ3 selective RGD-peptide was successfully prepared. [(18)F]FPPA was obtained in three steps with a radiochemical yield of 44% (decay corrected). Conjugation to c(RGDfK(N3)) by the Cu(II) catalyzed Huisgen azido alkyne cycloaddition provided the [(18)F]FPPA-c(RGDfK) with a radiochemical yield of 29% (decay corrected), in an overall synthesis time of 140min.

Modeling of the TCR-MHC-peptide complex.

The methodology for generating a homology model of the T1 TCR-PbCS-K(d) class I major histocompatibility complex (MHC) class I complex is presented. The resulting model provides a qualitative explanation of the effect of over 50 different mutations in the region of the complementarity determining region (CDR) loops of the T cell receptor (TCR), the peptide and the MHC's alpha(1)/alpha(2) helices. The peptide is modified by an azido benzoic acid photoreactive group, which is part of the epitope recognized by the TCR. The construction of the model makes use of closely related homologs (the A6 TCR-Tax-HLA A2 complex, the 2C TCR, the 14.3.d TCR Vbeta chain, the 1934.4 TCR Valpha chain, and the H-2 K(b)-ovalbumine peptide), ab initio sampling of CDR loops conformations and experimental data to select from the set of possibilities. The model shows a complex arrangement of the CDR3alpha, CDR1beta, CDR2beta and CDR3beta loops that leads to the highly specific recognition of the photoreactive group. The protocol can be applied systematically to a series of related sequences, permitting the analysis at the structural level of the large TCR repertoire specific for a given peptide-MHC complex.

Synthesis of a non-peptidic PET tracer designed for α5β1 integrin receptor.

Arginine-glycine-aspartic acid (RGD)-containing peptides have been traditionally used as PET probes to noninvasively image angiogenesis, but recently, small selective molecules for α5 β1 integrin receptor have been developed with promising results. Sixty-one antagonists were screened, and tert-butyl (S)-3-(2-((3R,5S)-1-(3-(1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)propanoyl)-5-((pyridin-2-ylamino)methyl)pyrrolidin-3-yloxy)acetamido)-2-(2,4,6-trimethylbenzamido)propanoate (FPMt) was selected for the development of a PET tracer to image the expression of α5 β1 integrin receptors. An alkynyl precursor (PMt) was initially synthesized in six steps, and its radiolabeling was performed according to the azide-alkyne copper(II)-catalyzed Huisgen's cycloaddition by using 1-azido-2-[(18)F]fluoroethane ([(18)F]12). Different reaction conditions between PMt and [(18)F]12 were investigated, but all of them afforded [(18)F]FPMt in 15 min with similar radiochemical yields (80-83%, decay corrected). Overall, the final radiopharmaceutical ([(18)F]FPMt) was obtained after a synthesis time of 60-70 min in 42-44% decay-corrected radiochemical yield.

Covalent cell surface functionalization of human fetal osteoblasts for tissue engineering.

The chemical functionalization of cell-surface proteins of human primary fetal bone cells with hydrophilic bioorthogonal intermediates was investigated. Toward this goal, chemical pathways were developed for click reaction-mediated coupling of alkyne derivatives with cellular azido-expressing proteins. The incorporation via a tetraethylene glycol linker of a dipeptide and a reporter biotin allowed the proof of concept for the introduction of cell-specific peptide ligands and to follow the reaction in living cells. Tuning the conditions of the click reaction resulted in chemical functionalization of living human fetal osteoblasts with excellent cell survival.

Synthesis of F-18-labeled cyclic-[RGDfK] peptides for PET imaging of angiogenesis

Objectives: αvβ3 integrin is of great interest for tumor targeting because of its high concentration in tumor tissue. It recognizes ligands containing an arginine-glycine-aspartate motif (RGD), and a number of RGD-containing peptides have been developed as PET imaging probes of angiogenesis. We synthesized a series of 18F-labeled cyclic-[RGDfK] peptides for in vivo imaging of αvβ3 expression. Our F-18 labeled prosthetic groups were attached to the αvβ3 ligand via click chemistry, and the reaction conditions (time, temperature, solvent and pH) were optimized by using single modified amino acids.Methods: Seven amino acids were selected considering their different biochemical properties (polarity, total charge, presence of aromatic ring and heteroatom). All the amino acids were modified by the introduction of azido moiety to allow the interaction with alkyne prosthetic groups. Once the conditions of the click chemistry were optimized, the prosthetic groups were also coupled with the cyclic-[RGDfK] exhibiting an azido function. 4- Trimethylammonium-nitrobenzene triflate was used as precursor for the radiosynthesis of the prosthetic groups. The fluorination was carried out with K2CO3/K2.2.2 in CH3CN at 95 oC, and the nitro group was reduced with NaBH4 and Pd/C in MeOH. The resulting 18F-aniline was subsequently coupled to alkynoic acids to yield the final F-18 labeled prosthetic groups. Finally, the prosthetic groups were attached to the peptides via Huisgen's cycloaddition. Figure 1. F-18 labeled αvβ3 ligand.Results: Our new prosthetic groups were successfully clicked to the modified amino acids and to the cyclic- [RGDfK], and the reactions were almost quantitative within 1 to 3.5 h. The pH of the reaction did not influence the reaction kinetic and yield. The four steps of the F-18 labeling were completely automated providing the final products in quantities and yields practical for PET imaging. IC50 values of our ligands for αvβ3 and α5β1 demonstrated a high selectivity of our compounds towards αvβ3, as well as the negligible effect of the prosthetic groups on the affinity of the ligand to its receptor, as confirmed by the prediction of the molecular modeling.Conclusions: We have successfully synthesized novel F-18 labeled prosthetic groups, as well as novel PET imaging probes of αvβ3 expression. The reaction conditions of the Huisgen's cycloaddition were optimized with selected modified amino acids, and subsequently transposed to the cyclic-[RGDfK] peptide. IC50 data demonstrate that our 18F-labeled ligands were selective for αvβ3. In vivo microPET/CT studies in tumor bearing mice are underway.

Surface modification of biomaterials for conjugation with human fetal osteoblasts.

Surface functionalization of hydroxyapatite (HA) and beta-tricalcium phosphate (TCP) bioceramics with chemical ligands containing a pyrrogallol moiety was developed to improve the adhesion of bone cell precursors to the biomaterials. Fast and biocompatible copper-free click reaction with azido-modified human fetal osteoblasts resulted in improved cell binding to both HA and TCP bioceramics, opening the way for using this methodology in the preparation of cell-engineered bone implants.

Synthesis, characterization and thermal decomposition of [Pd2 (C²-dmba) (µ-SO4) (SO2)2]

The bridged sulphate complex [Pd2 (C²,dmba) (µ-SO4) (SO2)2] has been obtained by reacting a saturated solution of SO2 in methanol and the cyclometallated compound [Pd(C²,N-dmba)(µ-N3)] 2; (dmba = N,N-dimethylbenzylamine), at room temperature for 24 h. Reaction product was characterized by elemental analysis, NMR comprising 13C{¹H} and ¹H nuclei and I.R. spectrum's measurements. Thermal behavior has been investigated and residual products identified by X-ray powder diffraction.

Síntese de beta-N-acetilglicosaminídeos de arila modificados em C-6 como potenciais agentes antimicrobianos

We report herein the synthesis of aryl beta-N-acetylglucosaminides containing azido, amino and acetamido groups at C-6 as potential antimicrobial agents. It was expected that these compounds could interfere with the biosynthesis and/or biotransformation of N-acetylglucosamine in fungi and bacteria. None of the compounds showed antimicrobial activity against bacteria (Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa), filamentous fungus (Aspergillus niger) and yeasts (Saccharomyces cerevisae, Candida albicans and Candida tropicallis), at the concentration of 1 mg/mL in agar diffusion assay.

Síntese de 2-iodobenzamidas e 3-(iodoacetamido)benzamidas ligadas à D-galactose e suas reações de carbociclização radicalar mediadas por hidreto de tri-n-butilestanho

Starting from methyl 6-O-allyl-4-azido-2,3-di-O-benzyl-4-deoxy-α-D-galactopyranoside, four new derivatives containing 2-iodobenzamido and 3-(iodoacetamido)benzamido groups were synthesized. These four compounds were submitted to tri-n-butyltin hydride mediated radical cyclization reactions, resulting in two macrolactams from 11- and 15-endo aryl radical cyclization. The corresponding four hydrogenolysis products were also obtained. The structures of the new compounds were elucidated by ¹H and 13C NMR spectroscopy, DEPT, COSY, HMQC and HMBC experiments.

Comportamento térmico de composto ciclopaladados binucleares contendo azo-ligantes aniônicos

No presente trabalho é descrita a investigação do comportamento térmico de uma nova série de complexos ciclopaladados, obtidos a partir de reações do azido-complexo [Pd(dmba)(mi-N3)]2 (1) (dmba = N,N-dimetilbenzilamina) com os azo-ligantes aniônicos pirazolato (pz), imidazolato (imz) e 3,5-dimetil-pirazolato (3,5-dmpz). Os compostos binucleares [Pd(dmba)(mi-pz)]2 (2), [Pd(dmba)(mi-imz)]2 (3) e [Pd(dmba)(mi-3,5-dmpz)]2 (4) foram caracterizados por análise elementar, espectroscopia de absorção na região do infravermelho, espectroscopia de RMN de 13C{¹H} e ¹H e análise termogravimétrica. O produto final de decomposição térmica, paládio metálico, foi caracterizado pela difratometria de raios-X, método do pó.

(A) BODIPY as a versatile fluorophore (B) Synthesis of PNAs via Staudinger reaction

(A) In recent years, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) fluorophores have attracted considerable interest due to their unique photochemical properties. However detailed studies on the stability of BODIPY and analogues under acidic and basic conditions have been lacking. Thus the stability of a series of BODIPY analogues in acidic (di- and trichloroacetic acid) and basic (aqueous ammonium hydroxide) conditions was investigated using 11B NMR spectroscopy. Among the analogues tested, 4,4-diphenyl BODIPY was the most stable under the conditions used in the experiments. It was found that reaction of 4,4-dimethoxy BODIPY with dichloroacetic acid gave mixed anhydride 4,4-bis(dichloroacetoxy) BODIPY in good yields. Treatment of the latter mixed anhydride with alcohols such as methanol and ethanol in the presence of a base afforded corresponding borate esters, whereas treatment with 1,2-diols such as ethylene glycol and catechol in the presence of a base gave corresponding cyclic borate esters. Furthermore treatment of 4,4-difluoro-8-methyl-BODIPY with secondary amines in dihalomethane resulted in carbon–carbon bond formation at the meso-methyl position of BODIPY via Mannich-type reactions. The resulting modified BODIPY fluorophores possess high fluorescent quantum yields. Five BODIPY analogues bearing potential ion-binding moieties were synthesized via this Mannich-type reaction. Among these, the BODIPY bearing an aza-18-crown-5 tether was found to be selective towards copper (II) ion, resulting in a large blue shift in absorption and sharp fluorescent quenching, whereas aza-15-crown-4 analogue was selected towards fluoride ion, leading to effective florescent quenching and blue shift. (B) Peptide nucleic acids (PNA), as mimics of natural nucleic acids, have been widely applied in molecular biology and biotechnology. Currently, the preparation of PNA oligomers is commonly achieved by a coupling reaction between carboxyl and amino groups in the presence of an activator. In this thesis attempts were made towards the synthesis of PNA through the Staudinger ligation reactions between C-terminal diphenylphosphinomethanethiol thioesters and N-terminal α-azido PNA building blocks.

Single-Molecule Magnets and Multifunctional Molecular Magnetic Materials Based on Polynuclear Metal Complexes

Our work on single molecule magnets and multifunctional magnetic materials is presented in four projects. In the first project we show for first time that heteroatomic-type pseudohalides, such as OCN-, can be employed as structure-directing ligands and ferromagnetic couplers in higher oxidation state metal cluster chemistry. The initial use of cyanato groups in Mn cluster chemistry has afforded structurally interesting MnII/III14 (1) and MnII/III/IV16 (2) clusters in which the end-on bridging cyanates show a preference in binding through their O-atom. The Mn14 compound shows entirely visible out-of-phase alternating currect signals below 5 K and large hysteresis loops below 2 K. Furthermore, the amalgamation of azido groups with the triethanolamine tripodal ligand in manganese carboxylate cluster chemistry has led to the isolation of a new ferromagnetic, high-nuclearity and mixed-valence MnII/III15Na2 (3) cluster with a large ground-state spin value of S = 14. In the second project we demonstrate a new synthetic route to purely inorganic-bridged, transition metal-azido clusters [CoII7 (4) and NiII7 (5)] and coordination polymers [{FeII/III2}n (6)] which exhibit strong ferromagnetic, SMM and long-range magnetic ordering behaviors. We also show that access to such a unique ferromagnetic class of inorganic, N-rich and O-free materials is feasible through the use of Me3SiN3 as the azido-ligand precursor without requiring the addition of any organic chelating/bridging ligand. In the last projects we have tried to bring together molecular magnetism and optics via the synthesis of multifunctional magnetic materials based on 3d- or 4f-metal ions. We decided to approach such challenge from two different directions: firstly, in our third project, by the deliberate replacement of non-emissive carboxylato ligands in known 3d-SMMs with their fluorescent analogues, without perturbing the metal-core structure and SMM properties (complexes 7, 8, and 9). The second route (last project) involves the use of naphthalene or pyridine-based polyalcohol bridging ligands for the synthesis of new polynuclear LnIII metal clusters (Ln = lanthanide) with novel topologies, SMM behaviors and luminescent properties arising from the increased efficiency of the “antenna” organic group. This approach has led us to the isolation of two new families of LnIII8 (complexes 10-13) and LnIII4 (complexes 14-20) clusters.