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.

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.

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.