Lee F. Kuyper

Site-specific Conjugation on Serine → Cysteine Variant Monoclonal Antibodies

We have engineered a cysteine residue at position 442 (EU/OU numbering) in the third constant domain (CH3) of the heavy chain of several IgGs with different specificities, isoforms, and variants with the intent to introduce a site for chemical conjugation. The variants were expressed in NS0 mouse myeloma cells, where monomeric IgG is the major form and formation of aggregate was minimal. Monomeric IgG contained no free thiol; however, it was discovered that the engineered thiols were reversibly blocked and could be reduced under controlled conditions. Following reduction, reactive thiol was conjugated with a cysteine-specific bifunctional chelator, bromoacetyl-TMT to a humanized 323/A3 IgG4 variant. Conjugation had no significant effect on antibody affinity. To prove that the conjugation was site-specific, an antibody-TMT conjugate was labeled with lutetium-177 and subjected to peptide mapping followed by sequence analysis. Glu-C digestion demonstrated that 91% of the label was recovered in the COOH-terminal peptide fragment containing the engineered cysteine.

Pyrrolo[2,3-d]pyrimidines and pyrido[2,3-d]pyrimidines as conformationally restricted analogues of the antibacterial agent trimethoprim

Conformationally restricted analogues of the antibacterial agent trimethoprim (TMP) were designed to mimic the conformation of drug observed in its complex with bacterial dihydrofolate reductase (DHFR). This conformation of TMP was achieved by linking the 4-amino function to the methylene group by one- and two-carbon bridges. A pyrrolo[2,3-d]pyrimidine, a dihydro analogue, and a tetrahydropyrido[2,3-d]pyrimidine were synthesized and tested as inhibitors of DHFR. One analogue showed activity equivalent to that of TMP against DHFR from three species of bacteria. An X-ray crystal structure of this inhibitor bound to Escherichia coli DHFR was determined to evaluate the structural consequences of the conformational restriction.

X-Ray Crystallographic Studies of Candida Albicans Dihydrofolate Reductase. High Resolution Structures of the Holoenzyme and an Inhibited Ternary Complex.

The recent rise in systemic fungal infections has created a need for the development of new antifungal agents. As part of an effort to provide therapeutically effective inhibitors of fungal dihydrofolate reductase (DHFR), we have cloned, expressed, purified, crystallized, and determined the three-dimensional structure ofCandida albicans DHFR. The 192-residue enzyme, which was expressed in Escherichia coli and purified by methotrexate affinity and cation exchange chromatography, was 27% identical to human DHFR. Crystals of C. albicans DHFR were grown as the holoenzyme complex and as a ternary complex containing a pyrroloquinazoline inhibitor. Both complexes crystallized with two molecules in the asymmetric unit in space group P21. The final structures had R-factors of 0.199 at 1.85-Å resolution and 0.155 at 1.60-Å resolution, respectively. The enzyme fold was similar to that of bacterial and vertebrate DHFR, and the binding of a nonselective diaminopyrroloquinazoline inhibitor and the interactions of NADPH with protein were typical of ligand binding to other DHFRs. However, the width of the active site cleft of C. albicans DHFR was significantly larger than that of the human enzyme, providing a basis for the design of potentially selective inhibitors.

Resolving crystal polymorphisms by finding “stationary points” from quantitative analysis of crystal growth response surfaces

Quantitative analysis of statistically sampled experimental designs have shown that a functionally interesting crystal growth polymorphism for E. coli cytidine deaminase can be resolved into pure states by finding where each state has a crystal growth optimum.

Regioselectivity in the Lithiation of O-(4-Methoxyphenyl)-N,N-diethylthiocarbamate: Relative Directed Metalation Abilities of Methoxy and N,N-diethylthiocarbamate Functionalities

Abstract Directed ortho metalation is a useful approach to the regioselective synthesis of polysubstituted aromatics.1 The regioselectivity of the metalation can depend, in some instances, on the relative positions and directing abilities of two or more functional groups, and studies to establish the relative directing abilities of ortho metalation groups have been reported.1 The O-aryl carbamates are particularly powerful ortho-metalation directors and have been employed effectively in the construction of a variety of polysubstituted aromatic compounds.1,2