Samuel J. Danishefsky

The B‐Alkyl Suzuki–Miyaura Cross‐Coupling Reaction: Development, Mechanistic Study, and Applications in Natural Product Synthesis

The development of new reactions that facilitate the creative and efficient synthesis of molecular structures with desirable properties continues to fascinate chemists. The test of a significant contribution is its acceptance over time by the scientific community. The B-alkyl Suzuki-Miyaura cross-coupling reaction appears to be one such reaction. Since its disclosure by Suzuki and Miyaura in 1986, this reaction has been an attractive solution to challenging synthetic problems.

Native Chemical Ligation at Valine: A Contribution to Peptide and Glycopeptide Synthesis†

Our laboratory has been pursuing the total synthesis of naturally occurring glycoproteins bearing multiple oligosaccharide domains. Specifically, efforts are well underway to accomplish a de novo total synthesis of erythropoietin alpha (EPO), in homogeneous form.[1] Although a variety of peptide ligation strategies have been developed to facilitate the merger of large, complex peptide and glycopeptide fragments,[2–9] the need for highly efficient methodology continues to motivate the chemical community to develop more powerful strategies. Our pursuit of the total synthesis of homogeneous erythropoietin (EPO) as well as other biologically active glycopeptides has inspired new glycopeptide ligations.[10] To achieve our most complex goals, we must learn how to overcome the serious obstacles in joining glycopeptides in an iterative fashion.

Immunization of metastatic breast cancer patients with a fully synthetic globo H conjugate: A phase I trial

The carbohydrate antigen globo H commonly found on breast cancer cells is a potential target for vaccine therapy. The objectives of this trial were to determine the toxicity and immunogenicity of three synthetic globo H-keyhole limpet hemocyanin conjugates plus the immunologic adjuvant QS-21. Twenty-seven metastatic breast cancer patients received five vaccinations each. The vaccine was well tolerated, and no definite differences were observed among the three formulations. Serologic analyses demonstrated the generation of IgM antibody titers in most patients, with minimal IgG antibody stimulation. There was significant binding of IgM antibodies to MCF-7 tumor cells in 16 patients, whereas IgG antibody reactivity was observed in a few patients. There was evidence of complement-dependent cytotoxicity in several patients. Affinity column purification supported the specificity of IgM antibodies for globo H. On the basis of these data, globo H will constitute one component of a polyvalent vaccine for evaluation in high-risk breast cancer patients.

DieB-Alkyl-Suzuki-Miyaura-Kreuzkupplung: Entwicklung, Untersuchungen zum Mechanismus und Anwendungen in der Naturstoffsynthese

Die Entwicklung neuer Reaktionen zur Erleichterung effizienter und kreativer Synthesen von Zielmolekulen jedweder Art ubt eine anhaltende Faszination auf Chemiker aus. Ob eine neue Reaktion einen bedeutenden Beitrag leistet, wird mit der Zeit anhand der Akzeptanz unter moglichen Anwendern entschieden. Die B-Alkyl-Suzuki-Miyaura-Kreuzkupplung scheint eine solche bedeutende Reaktion zu sein. Seit ihrer Veroffentlichung durch Suzuki und Miyaura im Jahre 1986 hat sie sich wiederholt als attraktive Losung fur herausfordernde Syntheseprobleme erwiesen.

Fully Synthetic Carbohydrate-Based Vaccines in Biochemically Relapsed Prostate Cancer: Clinical Trial Results With α-N-Acetylgalactosamine-O-Serine/Threonine Conjugate Vaccine

PURPOSE We report the synthesis of a mucin-related O-linked glycopeptide, alpha-N-acetylgalactosamine-O-serine/threonine (Tn), which is highly simplistic in its structure and can induce a relevant humoral response when given in a trimer or clustered (c) formation. We tested for an antitumor effect, in the form of a change in the posttreatment versus pretreatment prostate-specific antigen (PSA) slopes, that might serve as a surrogate for effectiveness of vaccines in delaying the time to radiographic progression. METHODS We compared the antibody response to immunization with two conjugates, Tn(c)-keyhole limpet hemocyanin (KLH) and Tn(c)-palmitic acid (PAM) with the saponin immunologic adjuvant QS21, in a phase I clinical trial in patients with biochemically relapsed prostate cancer. Patients received Tn(c)-KLH vaccine containing either 3, 7, or 15 microg of Tn(c) per vaccination. Ten patients received 100 microg of Tn(c)-PAM. QS21 was included in all vaccines. Five vaccinations were administered subcutaneously during 26 weeks with an additional booster vaccine at week 50. RESULTS Tn(c), when given with the carrier molecule KLH and QS21, stimulated the production of high-titer immunoglobulin M (IgM) and IgG antibodies. Inferior antibody responses were seen with T(c)-PAM. There was no evidence of enhanced immunogenicity with increasing doses of vaccine. An antitumor effect in the form of a decline in posttreatment versus pretreatment PSA slopes was also observed. CONCLUSION A safe synthetic conjugate vaccine in a trimer formation was developed that can break immunologic tolerance by inducing specific humoral responses. It seemed to affect the biochemical progression of the disease as determined by a change in PSA log slope.

Toward optimized carbohydrate-based anticancer vaccines: Epitope clustering, carrier structure, and adjuvant all influence antibody responses to Lewisy conjugates in mice

The feasibility of using carbohydrate-based vaccines for the immunotherapy of cancer is being actively explored at the present time. Although a number of clinical trials have already been conducted with glycoconjugate vaccines, the optimal design and composition of the vaccines has yet to be determined. Among the candidate antigens being examined is Lewisy (Ley), a blood group-related antigen that is overexpressed on the majority of human carcinomas. Using Ley as a model for specificity, we have examined the role of epitope clustering, carrier structure, and adjuvant on the immunogenicity of Ley conjugates in mice. A glycolipopeptide containing a cluster of three contiguous Ley-serine epitopes and the Pam3Cys immunostimulating moiety was found to be superior to a similar construct containing only one Ley-serine epitope in eliciting antitumor cell antibodies. Because only IgM antibodies were produced by this vaccine, the effect on immunogenicity of coupling the glycopeptide to keyhole limpet hemocyanin was examined; although both IgM and IgG antibodies were formed, the antibodies reacted only with the immunizing structure. Reexamination of the clustered Ley-serine Pam3Cys conjugate with the adjuvant QS-21 resulted in the identification of both IgG and IgM antibodies reacting with tumor cells, thus demonstrating the feasibility of an entirely synthetic carbohydrate-based anticancer vaccine in an animal model.

Preparation and evaluation of unimolecular pentavalent and hexavalent antigenic constructs targeting prostate and breast cancer: a synthetic route to anticancer vaccine candidates.

Several novel, fully synthetic, carbohydrate-based antitumor vaccines have been assembled. Each construct consists of multiple cancer-related antigens displayed on a single polypeptide backbone. Recent advances in synthetic methodology have allowed for the incorporation of a complex oligosaccharide terminating in a sialic acid residue (i.e., GM2) as one of the carbohydrate antigens. Details of the vaccine synthesis as well as the results of preliminary immunological investigations are described herein.

Erythropoietin Derived by Chemical Synthesis

EPO via Total Synthesis Erythropoietin (EPO) is a hormone involved in the production of red blood cells. Synthetic EPO produced via genetically engineered cell cultures is used to treat anemia and—more controversially—to boost athletic performance. EPO is a glycoprotein, and though its protein component is well-defined, both natural and synthetic EPO exhibit a wide range of attached oligosaccharides. Wang et al. (p. 1357; see the Perspective by Hsieh-Wilson and Griffin) prepared an EPO sample by a chemical synthesis that maintains a uniform pattern of attached sugars throughout, which may prove helpful in the analysis of how variation in the sugar components of EPO impact function. Chemical synthesis of a glycoprotein hormone provides a sample uniformly substituted with specific sugar chains. [Also see Perspective by Hsieh-Wilson and Griffin] 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.

New Chemistry with Old Functional Groups: On the Reaction of Isonitriles with Carboxylic Acids—A Route to Various Amide Types

Thermolysis of isonitriles with carboxylic acids provides, in one step, N-formyl imides (see, for example, 8 + 19 --> 21). The resultant N-formyl group can be converted to N-H, NCH2OH, or NCH3. This chemistry allows for a new route for synthesizing beta-N (asparagine)-linked glycosyl amino acids.

The synthesis, discovery, and development of a highly promising class of microtubule stabilization agents: Curative effects of desoxyepothilones B and F against human tumor xenografts in nude mice

We have evaluated two synthetic epothilone analogues lacking the 12,13-epoxide functionality, 12,13-desoxyepothilone B (dEpoB), and 12,13-desoxyepothilone F (dEpoF). The concentrations required for 50% growth inhibition (IC50) for a variety of anticancer agents were measured in CCRF-CEM/VBL1000 cells (2,048-fold resistance to vinblastine). By using dEpoB, dEpoF, aza-EpoB, and paclitaxel, the IC50 values were 0.029, 0.092, 2.99, and 5.17 μM, respectively. These values represent 4-, 33.5-, 1,423- and 3,133-fold resistance, respectively, when compared with the corresponding IC50 in the parent [nonmultiple drug-resistant (MDR)] CCRF-CEM cells. We then produced MDR human lung carcinoma A549 cells by continuous exposure of the tumor cells to sublethal concentrations of dEpoB (1.8 yr), vinblastine (1.2 yr), and paclitaxel (1.8 yr). This continued exposure led to the development of 2.1-, 4,848-, and 2,553-fold resistance to each drug, respectively. The therapeutic effect of dEpoB and paclitaxel was also compared in vivo in a mouse model by using various tumor xenografts. dEpoB is much more effective in reducing tumor sizes in all MDR tumors tested. Analysis of dEpoF, an analog possessing greater aqueous solubility than dEpoB, showed curative effects similar to dEpoB against K562, CCRF-CEM, and MX-1 xenografts. These results indicate that dEpoB and dEpoF are efficacious antitumor agents with both a broad chemotherapeutic spectrum and wide safety margins.