Chemical biology of glycoproteins: From chemical synthesis to biological impact.

Recent advances have demonstrated the feasibility and robustness of chemical synthesis for the production of homogeneously glycosylated protein forms (glycoforms). By taking advantage of the unmatchable flexibility and precision provided by chemical synthesis, the quantitative effects of glycosylation were obtained using chemical glycobiology approaches. These findings greatly advanced our fundamental knowledge of glycosylation. More importantly, analysis of these findings has led to the development of glycoengineering guidelines for rationally improving the properties of peptides and proteins. In this chapter, we present the key experimental steps for chemical biology studies of protein glycosylation, with the aim of facilitating and promoting research in this important but significantly underexplored area of biology.

Total Chemical Synthesis of Human Thyroid-Stimulating Hormone (hTSH) ß-Subunit: Application of Arginine-tagged Acetamidomethyl (AcmR) Protecting Groups.

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.

Antibodies Against Specific MUC16 Glycosylation Sites Inhibit Ovarian Cancer Growth.

Expression of the retained C-terminal extracellular portion of the ovarian cancer glycoprotein MUC16 induces transformation and tumor growth. However, the mechanisms of MUC16 oncogenesis related to glycosylation are not clearly defined. We establish that MUC16 oncogenic effects are mediated through MGAT5-dependent N-glycosylation of two specific asparagine sites within its 58 amino acid ectodomain. Oncogenic signaling from the C-terminal portion of MUC16 requires the presence of Galectin-3 and growth factor receptors colocalized on lipid rafts. These effects are blocked upon loss of either Galectin-3 expression or activity MGAT5. Using synthetic MUC16 glycopeptides, we developed novel N-glycosylation site directed monoclonal antibodies that block Galectin-3-mediated MUC16 interactions with cell surface signaling molecules. These antibodies inhibit invasion of ovarian cancer cells, directly blocking the in vivo growth of MUC16-bearing ovarian cancer xenografts, elucidating new therapeutic modalities.

Total Chemical Synthesis and Folding of All-l and All-d Variants of Oncogenic KRas(G12V).

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.

Vaccine Elicitation of High Mannose-Dependent Neutralizing Antibodies against the V3-Glycan Broadly Neutralizing Epitope in Nonhuman Primates.

Induction of broadly neutralizing antibodies (bnAbs) that target HIV-1 envelope (Env) is a goal of HIV-1 vaccine development. A bnAb target is the Env third variable loop (V3)-glycan site. To determine whether immunization could induce antibodies to the V3-glycan bnAb binding site, we repetitively immunized macaques over a 4-year period with an Env expressing V3-high mannose glycans. Env immunizations elicited plasma antibodies that neutralized HIV-1 expressing only high-mannose glycans-a characteristic shared by early bnAb B cell lineage members. A rhesus recombinant monoclonal antibody from a vaccinated macaque bound to the V3-glycan site at the same amino acids as broadly neutralizing antibodies. A structure of the antibody bound to glycan revealed that the three variable heavy-chain complementarity-determining regions formed a cavity into which glycan could insert and neutralized multiple HIV-1 isolates with high-mannose glycans. Thus, HIV-1 Env vaccination induced mannose-dependent antibodies with characteristics of V3-glycan bnAb precursors.

Staged induction of HIV-1 glycan-dependent broadly neutralizing antibodies.

A preventive HIV-1 vaccine should induce HIV-1-specific broadly neutralizing antibodies (bnAbs). However, bnAbs generally require high levels of somatic hypermutation (SHM) to acquire breadth, and current vaccine strategies have not been successful in inducing bnAbs. Because bnAbs directed against a glycosylated site adjacent to the third variable loop (V3) of the HIV-1 envelope protein require limited SHM, the V3-glycan epitope is an attractive vaccine target. By studying the cooperation among multiple V3-glycan B cell lineages and their coevolution with autologous virus throughout 5 years of infection, we identify key events in the ontogeny of a V3-glycan bnAb. Two autologous neutralizing antibody lineages selected for virus escape mutations and consequently allowed initiation and affinity maturation of a V3-glycan bnAb lineage. The nucleotide substitution required to initiate the bnAb lineage occurred at a low-probability site for activation-induced cytidine deaminase activity. Cooperation of B cell lineages and an improbable mutation critical for bnAb activity defined the necessary events leading to breadth in this V3-glycan bnAb lineage. These findings may, in part, explain why initiation of V3-glycan bnAbs is rare, and suggest an immunization strategy for inducing similar V3-glycan bnAbs.

A Phase I Study of Unimolecular Pentavalent (Globo-H-GM2-sTn-TF-Tn) Immunization of Patients with Epithelial Ovarian, Fallopian Tube, or Peritoneal Cancer in First Remission.

We conducted a phase I study in ovarian cancer patients to evaluate the safety and immunogenicity of a synthetic unimolecular pentavalent carbohydrate vaccine (Globo-H, GM2, sTn, TF, and Tn) supported on a peptide backbone, conjugated to keyhole limpet haemocyanin (KLH), and mixed with immunological adjuvant QS-21. Twenty-four advanced-stage, poor-risk, first-remission ovarian cancer patients were enrolled from January 2011-Septermber 2013. Three dose levels were planned (25, 50, 100 mcg) with three cohorts of six patients each, with an additional 6-patient expansion cohort at the MTD. ELISA serologic IgM and IgG responses for each antigen was defined as positive response if antibody titers were ≥1:80 over the respective patient's pre-vaccination serum. The study would be considered positive if at least four of 12 patients treated at the MTD showed immune responses for at least three of the five antigens. Twenty-four patients (median age, 54 years [range, 36-68]) were included in the safety analysis. Histology was high-grade serous in 22 patients (92%); 18 had stage III and six stage IV disease. The vaccine was well-tolerated at all doses, with no DLTs. At the highest treated dose, IgG and/or IgM responses were recorded against ≥3 antigens in 9/12 patients (75%), ≥4 in 7/12 (58%), and 5 in 3/12 (25%). With a median follow-up of 19 months (range, 2-39), 20 patients (83%) recurred and six (25%) died. The unimolecular pentavalent vaccine construct was shown to be safe and immunogenic. Such a construct greatly simplifies regulatory requirements and manufacturing, facilitates scalability, and provides adaptability.

Human Cancer Antigen Globo H Is a Cell-Surface Ligand for Human Ribonuclease 1.

Pancreatic-type ribonucleases are secretory enzymes that catalyze the cleavage of RNA. Recent efforts have endowed the homologues from cow (RNase A) and human (RNase 1) with toxicity for cancer cells, leading to a clinical trial. The basis for the selective toxicity of ribonuclease variants for cancerous versus noncancerous cells has, however, been unclear. A screen for RNase A ligands in an array of mammalian cell-surface glycans revealed strong affinity for a hexasaccharide, Globo H, that is a tumor-associated antigen and the basis for a vaccine in clinical trials. The affinity of RNase A and RNase 1 for immobilized Globo H is in the low micromolar-high nanomolar range. Moreover, reducing the display of Globo H on the surface of human breast adenocarcinoma cells with a small-molecule inhibitor of biosynthesis or a monoclonal antibody antagonist decreases the toxicity of an RNase 1 variant. Finally, heteronuclear single quantum coherence (HSQC) NMR spectroscopy showed that RNase 1 interacts with Globo H by using residues that are distal from the enzymic active site. The discovery that a systemic human ribonuclease binds to a moiety displayed on human cancer cells links two clinical paradigms and suggests a mechanism for innate resistance to cancer.

Fully Synthetic Granulocyte Colony-Stimulating Factor Enabled by Isonitrile-Mediated Coupling of Large, Side-Chain-Unprotected Peptides.

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.

Designing synthetic vaccines for HIV.

Despite three decades of intensive research efforts, the development of an effective prophylactic vaccine against HIV remains an unrealized goal in the global campaign to contain the HIV/AIDS pandemic. Recent characterization of novel epitopes for inducing broadly neutralizing antibodies has fueled research in the design and synthesis of new, well-defined antigenic constructs for the development of HIV envelope-directed vaccines. The present review will cover previous and recent efforts toward the design of synthetic vaccines based on the HIV viral envelope glycoproteins, with special emphasis on examples from our own laboratories. The biological evaluation of some of the most representative vaccine candidates, in terms of their antigenicity and immunogenicity, will also be discussed to illustrate the current state-of-the-art toward the development of fully synthetic HIV vaccines.

Total synthesis of glycosylated proteins.

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.

Development of Globo-H cancer vaccine.

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.

Studies toward the total synthesis of pluraflavin A.

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 α C-aryl glycoside by Stille cross-coupling and subsequent hydrogenation of the aryl glycal. Chemo- and stereoselective O-glycosylations of α oliose and ß 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.

Chemical synthesis of the ß-subunit of human luteinizing (hLH) and chorionic gonadotropin (hCG) glycoprotein hormones.

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.

Stereospecific cis- and trans-ring fusions arising from common intermediates.

Highly concise and stereospecific routes to cis and trans fusion, carrying various functionality at one of the bridgehead carbons, have been accomplished.

Synthesis of granulocyte-macrophage colony-stimulating factor as homogeneous glycoforms and early comparisons with yeast cell-derived material.

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.

Chemical synthesis of the ATAD2 bromodomain.

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.

Intramolecular Diels-Alder reactions of cycloalkenones: stereoselectivity, Lewis acid acceleration, and halogen substituent effects.

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.

Erythropoietin derived by chemical synthesis.

Erythropoietin is a signaling glycoprotein that controls the fundamental process of erythropoiesis, orchestrating the production and maintenance of red blood cells. As administrated clinically, erythropoietin has a polypeptide backbone with complex dishomogeneity in its carbohydrate domains. Here we describe the total synthesis of homogeneous erythropoietin with consensus carbohydrate domains incorporated at all of the native glycosylation sites. The oligosaccharide sectors were built by total synthesis and attached stereospecifically to peptidyl fragments of the wild-type primary sequence, themselves obtained by solid-phase peptide synthesis. The glycopeptidyl constructs were joined by chemical ligation, followed by metal-free dethiylation, and subsequently folded. This homogeneous erythropoietin glycosylated at the three wild-type aspartates with N-linked high-mannose sialic acid-containing oligosaccharides and O-linked glycophorin exhibits Procrit-level in vivo activity in mice.