Synthesis of stable-isotope-labeled N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide and N-(3-dimethylaminopropyl)-N'-ethylurea.

N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) is a carbodiimide coupling reagent commonly used for the preparation of amides from carboxylic acids and amines. Because of initial concerns regarding the genotoxicity of EDC and its use in GMP syntheses at Bristol Myers Squibb, the quantitation of residual EDC and its by-product N-(3-dimethylaminopropyl)-N'-ethylurea (EDU) by liquid chromatography-mass spectrometry (LCMS) impurity analysis was required. These analyses required the use of stable-isotope-labeled EDC and EDU to serve as internal standards. To meet this need, stable-isotope-labeled EDC 9 and EDU 10 were prepared from [1,2-13 C2 ] ethylene glycol and [13 C,15 N] potassium cyanide in overall yields of 6% and 8%, respectively.

Adnectin-drug conjugates for Glypican-3-specific delivery of a cytotoxic payload to tumors.

Tumor-specific delivery of cytotoxic agents remains a challenge in cancer therapy. Antibody-drug conjugates (ADC) deliver their payloads to tumor cells that overexpress specific tumor-associated antigens-but the multi-day half-life of ADC leads to high exposure even of normal, antigen-free, tissues and thus contributes to dose-limiting toxicity. Here, we present Adnectin-drug conjugates, an alternative platform for tumor-specific delivery of cytotoxic payloads. Due to their small size (10 kDa), renal filtration eliminates Adnectins from the bloodstream within minutes to hours, ensuring low exposure to normal tissues. We used an engineered cysteine to conjugate an Adnectin that binds Glypican-3, a membrane protein overexpressed in hepatocellular carcinoma, to a cytotoxic derivative of tubulysin, with the drug-to-Adnectin ratio of 1. We demonstrate specific, nanomolar binding of this Adnectin-drug conjugate to human and murine Glypican-3; its high thermostability; its localization to target-expressing tumor cells in vitro and in vivo, its fast clearance from normal tissues and its efficacy against Glypican-3-positive mouse xenograft models.

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.

Synthesis of unlabelled and stable-isotope-labelled glucuronide metabolites of dapagliflozin and synthesis of stable-isotope-labelled dapagliflozin.

Two regioisomeric glucuronide metabolites of dapagliflozin (BMS-512148) were synthesized and used to elucidate the structures of dapagliflozin metabolites observed in human urine samples. The structures of the synthetic metabolites were assigned by heteronuclear multiple-bond correlation, ROESY, and total correlation spectroscopy experiments. Analogues of these metabolites containing carbon-13 as a stable label were also prepared for use as internal standards for the analysis of urine samples obtained from patients participating in clinical studies.

Characterization and quantification of histidine degradation in therapeutic protein formulations by size exclusion-hydrophilic interaction two dimensional-liquid chromatography with stable-isotope labeling mass spectrometry.

Two dimensional liquid chromatography (2D-LC) coupling size exclusion (SEC) and hydrophilic interaction chromatography (HILIC) is demonstrated as a useful tool to study polar excipients, such as histidine and its degradant, in protein formulation samples. The SEC-HILIC setup successfully removed interferences from complex sample matrices and enabled accurate mass measurement of the histidine degradation product, which was then determined to be trans-urocanic acid. Because the SEC effluent is a strong solvent for the second dimension HILIC, experimental parameters needed to be carefully chosen, i.e., small transferring loop, fast gradient at high flow rates for the second dimension gradient, in order to mitigate the solvent mismatch and to ensure good peak shapes for HILIC separations. In addition, the generation of trans-urocanic acid was quantified by single heart-cutting SEC-HILIC 2D-LC combined with stable-isotope labeling mass spectrometry. Compared with existing 2D quantification methods, the proposed approach is fast, insensitive to solvent mismatch between dimensions, and tolerant of small retention time shifts in the first dimension. Finally, the first dimension diode array detector was found to be a potential degradation source for photolabile analytes such as trans-urocanic acid.

Synthesis of 2-(14)C-iminothiolane and 2-(13)C,(15)N-iminothiolane (Traut's reagent).

2-Iminothiolane has found utility in the growing area of antibody-drug conjugates by serving as a lysine-thiolating agent and the junction between the antibody and the cytotoxic payload during random conjugation of a monoclonal antibody. 2-(14)C-Iminothiolane was prepared from commercially available [(14)C]KCN using a four-step sequence in an overall 10% radiochemical yield. Stable-labeled 2-(13)C,(15)N-iminothiolane was also prepared from [(13)C(15)N]KCN in a similar manner. The ˙ labeled Traut's reagent produced by this sequence showed comparable reactivity as the commercially available unlabeled reagent with a representative monoclonal antibody and could serve as highly informative analytical tools to investigate antibody-drug conjugate formation via the random conjugation process.

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.

Progress toward the total synthesis of N-methylwelwitindolinone B isothiocyanate.

Progress toward the welwitindolinone alkaloid N-methylwelwitindolinone B isothiocyanate is reported. A key reaction to synthesize the [4.3.1] bicycle embedded in the core of the molecule is a furan type 2 intramolecular Diels-Alder reaction with a tetrasubstituted dienophile, which sets the two vicinal quaternary centers present in the natural product. The sterically encumbered cycloaddition precursor was synthesized using a Horner-Wadsworth-Emmons reaction followed by a Suzuki cross-coupling reaction. Finally, introduction of the secondary alkyl chloride was achieved by a regio- and diastereoselective opening of a [2.2.1] oxobicycloheptane functionality.

Probing the stability of nonglycosylated wild-type erythropoietin protein via reiterative alanine ligations.

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.

Advances in proline ligation.

Application of native chemical ligation logic to the case of an N-terminal proline is described. Two approaches were studied. One involved incorporation of a 3R-substituted thiyl-proline derivative. Improved results were obtained from a 3R-substituted selenol function, incorporated in the context of an oxidized dimer.

Synthesis of the bicyclic welwitindolinone core via an alkylation/cyclization cascade reaction.

Synthesis of an advanced welwitindolinone intermediate via an alkylation/cyclization reaction is reported. The key step involves a one pot Lewis acid-mediated alkylation of a silylketene aminal with a furan alcohol followed by an intramolecular cyclization. The reaction is stereoselective and takes place at low temperature. The cycloadduct was highly functionalized and contains the welwitindolinone core structure.

Enantioselective synthesis of bridged bicyclic ring systems.

The type 2 intramolecular Diels-Alder (IMDA) reaction is a valuable method for synthesis of both carbocyclic and heterocyclic bridged bicyclo[5.3.1]undecane and bicyclo[4.3.1]decane ring systems. These structures are common to a number of biologically important natural products. Asymmetric variants of the type 2 IMDA reaction incorporating oxazolidinone chiral auxiliaries have been evaluated. This study has resulted in systems that deliver bridged bicyclic [5.3.1] and [4.3.1] ring systems in high diastereomeric (97-99% de) and enantiomeric purity.