New syntheses of optically active fluoroamino acids and methods for their application to the study of gamma-glutamyl hydrolase and folylpoly-gamma-glutamate synthetase.

Folylpoly-gamma-glutamate synthetase (FPGS) and gamma-glutamyl hydrolase (GH) catalyze the formation and hydrolysis of folate and antifolate poly-gamma-glutamate derivatives. Previous results from this laboratory have established the use of fluoroglutamates and fluoroglutamate-containing folates and antifolates as structural and mechanistic probes of GH and FPGS. In order to continue this work, new stereoselective synthetic routes to the fluoroglutamates and methods for their incorporation into the designed mechanistic probes are desired. Electrophilic fluorination of the lactam enolates generated from enantiomerically pure protected 5-hydroxymethyl-2-pyrrolidinones was investigated as a method for the synthesis of 4-fluorinated glutamic acids. A structure-dependent selective mono- and difluorination of the prepared 2-pyrrolidinone substrates was observed. In cases where only monofluorination was observed, the reactions proceeded with a significant degree of diastereoselectivity. In one case, a completely diastereoselective monofluorination producing the product (3R, 5S)-1-(tert-butyloxycarbonyl)-3-fluoro-5-(trityloxymethyl)-2-pyrrolidinone was observed. The reactivity differences, which were analyzed and attributed mainly to steric features of the substrates, were exploited for the synthesis of (2S)-4,4-difluoroglutamic acid and (2S, 4R)-4-fluoroglutamic acid. (4R)-2,2-Dimethyl-4-formyl-oxazolidine-3-carboxylic acid benzyl ester (Cbz-Garner aldehyde) was examined as a precursor to chiral fluoroamino acids. A general 4,4-difluoroamino acid building block was synthesized by a fluoro-Reformatsky addition to the aldehyde carbonyl followed by radical deoxygenation. The utility of this building block was demonstrated with the synthesis of N-Cbz-(2S)-4,4-difluoroglutamine and a (2 S)-4,4-difluoroglutamic acid-containing fluorescent FRET substrate to be used in a recently-designed continuous assay for GH activity. A strategy for the synthesis of (2R)-3,3-difluoroglutamic acid was also examined, but it was not successful for obtaining this compound.Ph.D.Organic chemistryPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/129800/2/3042101.pd

Synthesis of L-4,4-Difluoroglutamic Acid Via Electrophilic Difluorination of a Lactam

(matrix presented) An enantiomerically pure bicyclic lactam proved to be an excellent substrate for electrophilic difluorination using N-fluorobenzenesulfonimide. The resulting difluorinated lactam can be easily converted into L-4,4-difluoroglutamic acid. To the best of our knowledge, this is the first example of a synthetically useful electrophilic difluorination of an unactivated lactam

Electrophilic Fluorination of Pyroglutamic Acid Derivatives: Application of Substrate-Dependent Reactivity and Diastereoselectivity to the Synthesis of Optically Active 4-Fluoroglutamic Acids

Electrophilic fluorination of enantiomerically pure 2-pyrrolidinones (4) derived from (L)-glutamic acid has been investigated as a method for the synthesis of single stereoisomers of 4-fluorinated glutamic acids. Reaction of the lactam enolate derived from 9 with NFSi results in a completely diastereoselective monofluorination reaction to yield the monocyclic trans-substituted α-fluoro lactam product 21. Unfortunately, a decreased kinetic acidity in 21 and other structurally related monofluorinated products renders them resistant to a second fluorination. In contrast, the bicyclic lactam 12 is readily difluorinated under the standard conditions described to yield the α,α-difluoro lactam 24. The difference in reactivity between the two types of related lactams is attributed mainly to the presence or lack of a steric interaction between the base used for deprotonation and the protecting group present in the pyrrolidinone substrates. This conclusion was reached based on analysis of the X-ray crystal structure of 21, molecular modeling, and experimental evidence. The key intermediates 21 and 24 are converted to (2S,4R)-4-fluoroglutamic acid and (2S)-4,4-difluoroglutamic acid, respectively

Synthesis of (L)-4-Fluorotryptophan

A new synthesis of stereochemically pure (L)-4-fluorotryptophan [i.e., (2S)-4-fluorotryptophan] in seven steps from 4-fluoroindole is described. A key reaction in the synthetic strategy is a diastereoselective alkylation of the Schollkopf chiral auxiliary with a fluorinated electrophile. All reaction steps are efficient and proceed in at least 80% yield

Prediction of Interactions between Mycobacterium tuberculosis Indole Glycerol Phosphate Synthase (IGPS) and Substrate Analogs

Tuberculosis (TB), which is the second most common cause of mortality worldwide, is caused by Mycobacterium tuberculosis (Mt). MtIGPS catalyzes the fourth step (transformation of 1-(o-carboxyphenylamino)-1-deoxyribulose 5’-phosphate (CdRP) into IGP) in the tryptophan biosynthetic pathway which is essential for M. tuberculosis growth. It over expresses the tryptophan pathway genes during an immune response and inhibition of MtIGPS allows CD4 T-cells to fight against M. tuberculosis more effectively therefore, MtIGPS could be a new potential drug target. Our goal is to computationally predict the binding of different compounds resembling CdRP to MtIGPS using molecular docking tools. Molecular docking is the prediction of interaction between protein and ligand using software. Consider a lock and key model in which the protein active site is the lock and various inhibitors are the keys to open this lock. For this purpose, the Maestro 13.4 version was used. It predicts types of interactions and binding energy (kcal/mol) for a ligand-MtIGPS pair. Using this program, protein (PDB ID: 3T44) and ligands were optimized using option “Protein preparation” and “Ligprep” respectively. A grid box was made around the active site to ensure docking will happen in that region only and Maestro displayed results in the form of binding energy and the poses of how ligands bind to MtIGPS. Our results show that Gly242 is an important interaction and removal of it can destabilize the protein-ligand complex and encourage further confirmation in the laboratory

The interconnected roles of residues N189, E168, and S220 in M. tuberculosis Indole-3 glycerol phosphate synthase catalysis

Tuberculosis (TB) is an infectious disease caused by the bacteria Mycobacterium tuberculosis that primarily attacks the lungs. Drug resistance for M. tuberculosis is emerging and therefore new drug targets need to be identified. One way to target TB is through the inhibition of the tryptophan biosynthetic pathway of M. tuberculosis since production of tryptophan is essential for the replication of M. tuberculosis. The enzyme Indole-3-glycerol phosphate synthase (MtIGPS) was chosen as a potential drug target due to its role in tryptophan biosynthesis. Residue N189 interacts with active site residues E168 and S220. It is inferred from previous studies done with IGPS from Sulfolobus solfataricus that E168 is important for catalysis and S220 for substrate binding. In this study, the residue N189 in MtIGPS was mutated to a variety of other amino acids. The effects were studied through steady-state kinetics and rate-pH profiles to elucidate the role of N189 in the interactions of active site residues E168 and S220. It is possible that N189 plays a role in catalysis through its proximity to E168, which is suspected to play a role in the MtIGPS mechanism. It was found that mutation of N189 to Q, D or K has a drastic impact on MtIGPS catalytic activity, indicating that N189 is catalytically important. Once the role of N189 and the other residues is better understood, then inhibitors for IGPS can be designed

The Synthesis of (2S)-4,4-Difluoroglutamyl Γ-Peptides Based On Garner\u27s Aldehyde and Fluoro-Reformatsky Chemistry

The development of optically active fluorinated synthetic building blocks of general utility is a current goal of organo-fluorine chemists. The serine-derived Garner aldehyde was converted to a general 4,4-difluoroamino acid building block via fluoro-Reformatsky reaction with ethyl bromodifluoroacetate. The utility of this building block was demonstrated by the synthesis of derivatives of (2S)-4,4-difluoroglutamine, (2S)-4,4-difluoroglutamic acid, and its incorporation into a fluorophore-containing isopeptide 2 designed as a mechanistic probe of γ-glutamyl hydrolase. Compound 2 proved to be a substrate for γ-glutamyl hydrolase and was hydrolyzed at a rate significantly slower than the corresponding non-fluorinated analog

The Impact of double mutant residues E57D/E219Q and E57Q/E219Q on M.tuberculosis indole-3-glycerol phosphate synthase catalysis

Tuberculosis (TB) is a progressive infectious disease mostly caused by Mycobacterium tuberculosis and that primarily affects the lungs. Tuberculosis remains the leading cause of death from an infectious disease among adults worldwide, with more than 10 million people becoming newly sick from tuberculosis each year. There is a high increase in drug resistance for tuberculosis, paving a way for new drug targets to be developed. The potential drug target chosen is the enzyme Indole-3-glycerol phosphate synthase (MtIGPS), which plays an important role in tryptophan biosynthesis and catalyzes the conversion of 1-(o-carboxyphenylamino)-1-deoxyribulose 5’-phosphate (CdRP) into indole-glycerol-phosphate (IGP) in the bacterial tryptophan biosynthetic pathway. The introduction of mutations to residues that play an important role in catalysis or binding between the ligands and MtIGPS can provide information about the roles of these residues. The two mutants E57D/E219Q and E57Q/E219Q were expressed, purified and their catalytic activities were measured. A kcat value of 4.89× 10-5 1/s for E57D/E219Q, 2.13× 10-5 1/s for E57Q/E219Q and 7.4× 10-2 1/s for the wild type were obtained. These data Indicate that residues E57D and E219Q on MtIGPS are catalytically important due to the amount of CdRP converted per unit time shown from the kcat values

Preparation of IGPS Product Analogs through Reductive Amination and Wittig Reaction

Indole-3-glycerol phosphate synthase (IGPS) catalyzes the indole-forming reaction in the multi-step bacterial tryptophan biosynthetic pathway. This pathway appears to be necessary for bacterial growth and it could represent a target for potential new anti-infective agents in the future. In order to realize this possibility, we are currently investigating two reaction strategies for the synthesis of IGPS product analogs. These two reaction types are the Wittig reaction and reductive amination. This presentation will describe our progress towards obtaining these two different IGPS product analogs