RESUMO
The ß-subunit of human thyroid stimulating hormone (hTSH) has been synthesized as a single glycoform bearing a chitobiose disaccharide at the native glycosylation site. Key to the successful completion of this synthesis was the introduction of an arginine-tagged acetamidomethyl group, which served to greatly facilitate handling of a glycopeptide fragment with poor aqueous solubility. This general solution to the challenge of working with intractable peptides is expected to find wide use in protein synthesis.
RESUMO
The Ras proteins are essential GTPases involved in the regulation of cell proliferation and survival. Mutated oncogenic forms of Ras alter effector binding and innate GTPase activity, leading to deregulation of downstream signal transduction. Mutated forms of Ras are involved in approximately 30% of human cancers. Despite decades of effort to develop direct Ras inhibitors, Ras has long been considered "undruggable" due to its high affinity for GTP and its lack of hydrophobic binding pockets. Herein, we report a total chemical synthesis of all-l- and all-d-amino acid biotinylated variants of oncogenic mutant KRas(G12V). The protein is synthesized using Fmoc-based solid-phase peptide synthesis and assembled using combined native chemical ligation and isonitrile-mediated activation strategies. We demonstrate that both KRas(G12V) enantiomers can successfully fold and bind nucleotide substrates and binding partners with observable enantiodiscrimination. By demonstrating the functional competency of a mirror-image form of KRas bound to its corresponding enantiomeric nucleotide triphosphate, this study sets the stage for further biochemical studies with this material. In particular, this protein will enable mirror-image yeast surface display experiments to identify all-d peptide ligands for oncogenic KRas, providing a useful tool in the search for new therapeutics against this challenging disease target.
Assuntos
Proteínas Proto-Oncogênicas p21(ras)/síntese química , Sequência de Aminoácidos , Variação Genética , Humanos , Dobramento de Proteína , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
Highly concise and stereospecific routes to cis and trans fusion, carrying various functionality at one of the bridgehead carbons, have been accomplished.
Assuntos
Reação de Cicloadição/métodos , Ciclobutanos/química , Estrogênios/síntese química , Noresteroides/síntese química , Estereoisomerismo , Alquilação , Indústria Farmacêutica/métodos , Metilação , Modelos MolecularesRESUMO
Due to the emerging importance of the bromodomain binding region in the study of epigenetic effectors and the vast implications for a wide variety of human disease, the bromodomain region of human ATPase family AAA+ (ATPases associated with diverse cellular activities) domain-containing protein 2 (ATAD2) was targeted for chemical synthesis. The ATAD2 bromodomain (130 aa) was divided into five strategic fragments to be assembled using native chemical ligation with a focus on maximal convergency and efficiency. The fragments were assembled with one cysteine and three thioleucine ligations, unveiling the native alanine and leucine amino acids at the ligation points following metal-free dethiylation. Synthetic highlights of the study are a photolabile dimethoxynitrobenzyl-protected glutamic acid side chain used to impede hydrolysis of the C-terminal Glu-thioester, a thiazolidine-protected thioleucine, and an efficient assembly of three fragments in a single reaction vessel with dual-mode kinetic-standard chemical ligation. With a focus on material throughput and convergency, the five peptide fragments were assembled into the native ATAD2 bromodomain region with a total of three HPLC events in 8% overall yield from the fragments.
Assuntos
Adenosina Trifosfatases/síntese química , Proteínas de Ligação a DNA/síntese química , Fragmentos de Peptídeos/química , Estrutura Terciária de Proteína , Técnicas de Síntese em Fase Sólida/métodos , ATPases Associadas a Diversas Atividades Celulares , Cromatografia Líquida de Alta Pressão , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de MassasRESUMO
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a medicinally important glycoprotein, used as an immunostimulant following bone-marrow transplant. On the basis of reports of its potential utility as an anticancer vaccine adjuvant, we undertook to develop a synthetic route toward single-glycoform GM-CSF. We describe herein a convergent total synthesis of GM-CSF aglycone and two homogeneous glycoforms. Analytical and biological studies confirm the structure and activity of these synthetic congeners.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/síntese química , Modelos Moleculares , Conformação Proteica , Alanina/química , Sequência de Aminoácidos , Cisteína/química , Escherichia coli , Glicosilação , Dados de Sequência Molecular , Estrutura MolecularRESUMO
The development of anticancer vaccines requires the identification of unique epitope markers, preferably expressed exclusively on the surface of cancer cells. This Account describes the path of development of a carbohydrate-based vaccine for metastatic breast cancer, including the selection and synthesis of Globo-H as the target, the development of the vaccine conjugate and adjuvant design, the study of the immune response and consideration of class switch, and the analysis of Globo-H distribution on the surface of various cancer cells, cancer stem cells, and normal cells. The first synthesis of Globo-H was accomplished through the use of glycal chemistry; this approach delivered sufficient material for evaluation in phase I human trials. The development of a programmable one-pot synthesis method rendered the synthesis more practical and enabled the midstage proof-of-concept phase II trial and late-stage phase III trial. Finally, enzymatic synthesis of Globo-H coupled with cofactor regeneration was used for the late-stage multicenter trials and manufacture of the product. Along this path of development, it was discovered that the vaccine induced antibodies to target not only Globo-H, but also SSEA3 and SSEA4. Moreover, these three glycolipids were found to be uniquely expressed not only on the cell surface of breast cancer but on 15 additional cancer types, suggesting the broad application of this vaccine in cancer treatment and perhaps cancer prevention. In addition, a new glycolipid adjuvant was designed to target the CD1d receptor on dendritic cells and B cells for presentation to and activation of T cells to modulate the immune response and induce a class switch from IgM to IgG, thereby overcoming the common problem of carbohydrate-based vaccines that often induce mainly IgM antibodies. As demonstrated in this vaccine development, the chemical approach to the synthesis and conjugation of carbohydrate-based immunogens provides the flexibility for access to various structures and linkers to identify optimal compositions for development. The enzymatic method was then introduced to enable the practical synthesis of the vaccine candidate for clinical development and commercialization. Overall, this Account illustrates the path of development of a cancer vaccine, from selection of a unique glycan marker on breast cancer cells and the cancer stem cells as target to the use of chemistry in combination with immunology and cancer biology to enable the design and development of the Globo-H vaccine to target three specific glycan markers exclusively expressed on the cell surface of a number of different types of cancer.
Assuntos
Antígenos Glicosídicos Associados a Tumores/imunologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/imunologia , Vacinas Anticâncer/imunologia , Antígenos Glicosídicos Associados a Tumores/metabolismo , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/imunologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Vacinas Anticâncer/química , Feminino , Humanos , Células-Tronco Neoplásicas/imunologiaRESUMO
The synthesis of polypeptides on solid phase via mediation by isonitriles is described. The acyl donor is a thioacid, which presumably reacts with the isonitrile to generate a thio-formimidate carboxylate mixed anhydride intermediate. Applications of this chemistry to reiterative solid-phase peptide synthesis as well as solid-phase fragment coupling are described.
Assuntos
Amidas/química , Nitrilas/química , Peptídeos/síntese química , Técnicas de Síntese em Fase Sólida/métodos , Estrutura Molecular , Vasopressinas/síntese química , Vasopressinas/químicaRESUMO
The identification of synthesizable substructural domains within more complex structural targets is of significant value in designing a workable plan of synthesis. We term this process "pattern recognition analysis" (PRA). In this paper we continued to build on the theme of PRA as a potential resource in retrosynthetic blueprints to reach highly challenging targets. The paper operates at two levels. First, there is provided a clear sense of definitions of categories by which patterns are related to hypothetical reaction types. Although the required reaction type may for the moment not exist, we believe that this method of analysis is likely to promote innovation that identifies unmet needs and opportunities to advance the cause of complex target synthesis. In addition, we describe reductions to practice in expanding the menu of achievable patterns. It is likely that the future value of PRA will be associated with its utility in leading the way to new and exploitable chemical innovation.
Assuntos
Técnicas de Química Sintética/métodos , Reação de Cicloadição/métodos , Estrutura Molecular , EstereoisomerismoRESUMO
Current HIV-1 vaccines elicit strain-specific neutralizing antibodies. Broadly neutralizing antibodies (BnAbs) are not induced by current vaccines, but are found in plasma in â¼20% of HIV-1-infected individuals after several years of infection. One strategy for induction of unfavored antibody responses is to produce homogeneous immunogens that selectively express BnAb epitopes but minimally express dominant strain-specific epitopes. Here we report that synthetic, homogeneously glycosylated peptides that bind avidly to variable loop 1/2 (V1V2) BnAbs PG9 and CH01 bind minimally to strain-specific neutralizing V2 antibodies that are targeted to the same envelope polypeptide site. Both oligomannose derivatization and conformational stabilization by disulfide-linked dimer formation of synthetic V1V2 peptides were required for strong binding of V1V2 BnAbs. An HIV-1 vaccine should target BnAb unmutated common ancestor (UCA) B-cell receptors of naïve B cells, but to date no HIV-1 envelope constructs have been found that bind to the UCA of V1V2 BnAb PG9. We demonstrate herein that V1V2 glycopeptide dimers bearing Man5GlcNAc2 glycan units bind with apparent nanomolar affinities to UCAs of V1V2 BnAbs PG9 and CH01 and with micromolar affinity to the UCA of a V2 strain-specific antibody. The higher-affinity binding of these V1V2 glycopeptides to BnAbs and their UCAs renders these glycopeptide constructs particularly attractive immunogens for targeting subdominant HIV-1 envelope V1V2-neutralizing antibody-producing B cells.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/imunologia , Glicopeptídeos/imunologia , HIV-1/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Dimerização , Eletroforese em Gel de Poliacrilamida , Glicopeptídeos/síntese química , Glicopeptídeos/metabolismo , Ligação Proteica , Receptores de Antígenos de Linfócitos B/genética , Ressonância de Plasmônio de SuperfícieRESUMO
Human granulocyte colony-stimulating factor (G-CSF) is an endogenous glycoprotein involved in hematopoiesis. Natively glycosylated and nonglycosylated recombinant forms, lenograstim and filgrastim, respectively, are used clinically to manage neutropenia in patients undergoing chemotherapeutic treatment. Despite their comparable therapeutic potential, the purpose of O-linked glycosylation at Thr133 remains a subject of controversy. In light of this, we have developed a synthetic platform to prepare G-CSF aglycone with the goal of enabling access to native and designed glycoforms with site-selectivity and glycan homogeneity. To address the synthesis of a relatively large, aggregation-prone sequence, we advanced an isonitrile-mediated ligation method. The chemoselective activation and coupling of C-terminal peptidyl Gly thioacids with the N-terminus of an unprotected peptide provide ligated peptides directly in a manner complementary to that with conventional native chemical ligation-desulfurization strategies. Herein, we describe the details and application of this method as it enabled the convergent total synthesis of G-CSF aglycone.
Assuntos
Fator Estimulador de Colônias de Granulócitos/química , Peptídeos/química , Sequência de Aminoácidos , Eletroforese em Gel de Poliacrilamida , Dados de Sequência MolecularRESUMO
Glycoproteins are an important class of naturally occurring biomolecules which play a pivotal role in many biological processes. They are biosynthesized as complex mixtures of glycoforms through post-translational protein glycosylation. This fact, together with the challenges associated with producing them in homogeneous form, has hampered detailed structure-function studies of glycoproteins as well as their full exploitation as potential therapeutic agents. By contrast, chemical synthesis offers the unique opportunity to gain access to homogeneous glycoprotein samples for rigorous biological evaluation. Herein, we review recent methods for the assembly of complex glycopeptides and glycoproteins and present several examples from our laboratory towards the total chemical synthesis of clinically relevant glycosylated proteins that have enabled synthetic access to full-length homogeneous glycoproteins.
Assuntos
Glicoproteínas/síntese química , Glicosilação , Estrutura Molecular , Engenharia de ProteínasRESUMO
Nonglycosylated erythropoietin bearing acetamidomethyl protecting groups at the cysteine residues has been synthesized via chemical methods. Alanine ligation was used to assemble four peptide fragments, themselves prepared by solid phase peptide synthesis. This work outlines a route for the synthesis of homogeneous glycosylated erythropoietin.
Assuntos
Alanina/química , Cisteína/química , Eritropoetina/síntese química , Fragmentos de Peptídeos/química , Sequência de Aminoácidos , Sequência de Carboidratos , Eritropoetina/química , Eritropoetina/metabolismo , Glicosilação , Modelos Químicos , Dados de Sequência Molecular , Estrutura Molecular , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Fragmentos de Peptídeos/metabolismo , Estabilidade Proteica , Estrutura Secundária de ProteínaRESUMO
Human luteinizing hormone (hLH) and human chorionic gonadotropin (hCG) are human glycoprotein hormones each consisting of two subunits, an identical α-subunit and a unique ß-subunit, that form noncovalent heterodimers. Structurally, ß-hCG shares a high degree of sequence similarity with ß-hLH, including a common N-glycosylation site at the N-terminus but differs mainly in the presence of an extended C-terminal portion incorporating four closely spaced O-linked glycans. These glycoproteins play important roles in reproduction and are used clinically in the treatment of infertility. In addition, the role of hCG as a tumor marker in a variety of cancers has also attracted significant interest for the development of cancer vaccines. In clinical applications, these hormones are administered as mixtures of glycoforms due to limitations of biological methods in producing homogeneous samples of these glycoproteins. Using the powerful tools of chemical synthesis, the work presented herein focuses on the highly convergent syntheses of homogeneous ß-hLH and ß-hCG bearing model glycans at all native glycosylation sites. Key steps in these syntheses include a successful double Lansbury glycosylation en route to the N-terminal fragment of ß-hCG and the sequential installation of four O-linked glycosyl-amino acid cassettes into closely spaced O-glycosylation sites in a single, high-yielding solid-supported synthesis to access the C-terminal portion of the molecule. The final assembly of the individual glycopeptide fragments involved a stepwise native chemical ligation strategy to provide the longest and most complex human glycoprotein hormone (ß-hCG) as well as its closely related homologue (ß-hLH) as discrete glycoforms.
Assuntos
Gonadotropina Coriônica Humana Subunidade beta/síntese química , Glicopeptídeos/química , Hormônio Luteinizante Subunidade beta/síntese química , Fragmentos de Peptídeos/química , Sequência de Aminoácidos , Gonadotropina Coriônica Humana Subunidade beta/química , Humanos , Hormônio Luteinizante Subunidade beta/química , Dados de Sequência Molecular , Conformação ProteicaRESUMO
The intramolecular Diels-Alder reactions of cycloalkenones and terminal dienes occur with high endo stereoselectivity, both thermally and under Lewis-acidic conditions. Through computations, we show that steric repulsion and tether conformation govern the selectivity of the reaction, and incorporation of either BF3 or α-halogenation increases the rate of cycloaddition. With a longer tether, isomerization from a terminal diene to the more stable internal diene results in a more facile cycloaddition.
Assuntos
Cicloparafinas/química , Halogênios/química , Cetonas/química , Ácidos de Lewis/química , Catálise , Modelos Moleculares , TermodinâmicaRESUMO
A synthetic strategy towards the potent cytostatic agent pluraflavin A has been developed. Formation of the enantioenriched anthrapyran core bearing a halogen atom enabled the introduction of the αâ C-aryl glycoside by Stille cross-coupling and subsequent hydrogenation of the aryl glycal. Chemo- and stereoselective O-glycosylations of αâ oliose and ßâ 3-epi vancosamine residues afforded a fully glycosylated aromatic core. Attempts to install the dimethylamino group of the C-disaccharide suggest that introduction of an azide group by displacement and subsequent reduction may pave the way to the total synthesis of pluraflavin A.
Assuntos
Antraquinonas/síntese química , Produtos Biológicos/síntese química , Antraquinonas/química , Produtos Biológicos/química , Glicosilação , EstereoisomerismoRESUMO
The power of chemical synthesis of large cysteine-free polypeptides has been significantly enhanced through the use of nonproteogenic constructs which bear strategically placed thiol groups, enabling native chemical ligation. Central to these much expanded capabilities is the specific, radical-induced, metal-free dethiolation, which can be accomplished in aqueous medium.
Assuntos
Cisteína/química , Hormônio Paratireóideo/síntese química , Sequência de Aminoácidos , Substituição de Aminoácidos , Dicroísmo Circular , Humanos , Dados de Sequência Molecular , Hormônio Paratireóideo/químicaRESUMO
The conversion of peptide and proteins from their soluble state into well-organized aggregates, together with the accompanied oxidation of methionine residue, presents a significant challenge to human health, to the manufacture of protein therapeutics, and to the synthesis of proteins and glycoproteins. Despite their fundamental importance, little is known about the molecular basis of these two side reactions and their control. Here, using chemical peptide synthesis, we further confirmed the importance of the balance between hydrophobic interactions and electrostatic repulsive forces in inducing and inhibiting aggregation and methionine oxidation. Most importantly, through extending the established principle, we are able to effectively stabilize the problematic peptide fragment through the attachment of cleavable arginine tags. Future applications of our approach are expected to facilitate the synthesis and study of difficult peptides, proteins, and glycoproteins and will provide more opportunities for the optimization of protein biopharmaceuticals and for the development of cell-permeable biomolecules.
Assuntos
Bioquímica/métodos , Proteínas/química , Sequência de Aminoácidos , Cromatografia Líquida , Eritropoetina/química , Humanos , Espectrometria de Massas , Metionina/metabolismo , Dados de Sequência Molecular , Oxirredução , Peptídeos/química , Estrutura Quaternária de ProteínaRESUMO
We describe herein the discovery of a series of panaxytriol (PXT)-derived polyacetylene small molecules with promising cytoprotective activity. In mouse xenograft models, we have demonstrated the capacity of our synthetic analogs to mitigate a range of cancer therapeutic agent-induced toxicities, including body weight loss, lethality, neurotoxicity, and hematotoxicity. Our PXT analogs have also been found to reduce radiation-induced body weight loss and lethality in mouse models. Moreover, several PXT analogs appear to exhibit moderate in vivo antiinflammatory activity as well as in vitro immunoenhancing capabilities. These compounds appear to derive their activity through induction of cancer preventive phase 2 enzymes. The studies described herein suggest that coadministration of a PXT-derived agent with cancer chemotherapeutics or radiation therapy may serve to mitigate a range of therapy-associated toxicities.
Assuntos
Produtos Biológicos/farmacologia , Citoproteção/efeitos dos fármacos , Enedi-Inos/farmacologia , Álcoois Graxos/farmacologia , Panax/química , Poli-Inos/farmacologia , Animais , Produtos Biológicos/química , Morte Celular/efeitos dos fármacos , Morte Celular/efeitos da radiação , Citoproteção/efeitos da radiação , Enedi-Inos/química , Álcoois Graxos/química , Feminino , Fluoruracila/farmacologia , Humanos , Isomerismo , Longevidade/efeitos dos fármacos , Longevidade/efeitos da radiação , Camundongos , Camundongos Nus , Paclitaxel/farmacologia , Poli-Inos/química , Redução de Peso/efeitos dos fármacos , Redução de Peso/efeitos da radiação , Raios X , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Migrastatin is a biologically active natural product isolated from Streptomyces that has been shown to inhibit tumor cell migration. Upon completion of the first total synthesis of migrastatin, a number of structurally simplified analogs were prepared. Following extensive in vitro screening, a new generation of analogs was identified that demonstrates substantially higher levels of in vitro inhibitory activity, stability and synthetic accessibility when compared to the parent natural product. Herein, we describe two promising ether-derivative analogs, the migrastatin core ether (ME) and the carboxymethyl-ME (CME), which exhibit high efficacy in blocking tumor cell migration and metastasis in lung cancer. These compounds show an in vitro migration inhibition in the micromolar range (IC(50): ME 1.5 to 8.2 µM, CME 0.5 to 5 µM). In a human small-cell lung carcinoma (SCLC) primary xenograft model, ME and CME compounds were found to be highly potent in inhibiting overall metastasis even at the lowest dosage used (degree of inhibition: 96.2% and 99.3%, respectively). Together these very encouraging findings suggest that these analogs have promise as potent antimetastatic agents in lung cancer.
Assuntos
Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Macrolídeos/síntese química , Macrolídeos/uso terapêutico , Metástase Neoplásica/tratamento farmacológico , Piperidonas/síntese química , Piperidonas/uso terapêutico , Animais , Linhagem Celular Tumoral , Movimento Celular , Éteres/síntese química , Éteres/química , Humanos , Macrolídeos/química , Masculino , Camundongos , Metástase Neoplásica/patologia , Piperidonas/química , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cancer cells may be distinguished from normal cells by cell surface displays of aberrant levels and types of carbohydrate domains. Accordingly, these tumor-associated carbohydrate antigens (TACAs) represent promising target structures for the design of anticancer vaccines. Over the past 20 years, our laboratory has sought to use the tools of chemical synthesis to develop TACA-based anticancer vaccine candidates. We provide herein a personal accounting of our laboratory's progress toward the long-standing goal of developing clinically viable fully synthetic carbohydrate-based anticancer vaccines.