Synthesis and in vivo antitumor evaluation of an orally active potent phosphonamidate derivative targeting IDO1/IDO2/TDO.

Targeting Trp-Kyn pathways has been identified as an attractive approach for the cancer immunotherapies. In this study, a novel phosphonamidate containing compound was designed, synthesized and evaluated for its inhibitory activity against key dioxygenases in Trp-Kyn pathway, including IDO1, IDO2 and TDO. This compound showed potent IDO1 inhibitory activity with an IC50 value of 94 nM in an enzymatic assay and 12.6 nM in HeLa cells. In addition, this compound showed promising IDO2 inhibition and TDO inhibition with IC50 values of 310 nM and 2.6 µM, respectively, in enzyme assay. Based on the promising enzyme inhibitory activity toward IDO/TDO, compound F04 was evaluated of its antitumor effects in two tumor models. Further evaluation of mechanism demonstrated compound F04 with the remarkable capacity of reducing kynurenine level in plasma/TME and restoring anti-tumor immune response. F04 could be further developed as a potential immunotherapeutic agent combined with immune checkpoint inhibitors or chemotherapeutic drugs for cancer treatment.

Syntheses of 5'-Nucleoside Monophosphate Derivatives with Unique Aminal, Hemiaminal, and Hemithioaminal Functionalities: A New Class of 5'-Peptidyl Nucleotides.

A number of synthetically useful transformations have been developed to generate novel 5'-peptidyl nucleoside monophosphate analogues that incorporate sensitive phosphoaminal, -hemiaminal or -hemithioaminal functionalities. The strategies adopted entailed the coupling between dipeptides, which enclose a reactive Cα-functionalized glycine residue and phosphate or phosphorothioate moieties. These developments led to potentially powerful and general methodologies for the preparation of α-phosphorylated pseudopeptides as well as nucleoside monophosphate mimics. The resulting conjugates are of interest for a variety of important applications, which range from drug development to synthetic biology, as pronucleotides or artificial building blocks for the enzymatic synthesis of xenobiotic information systems. The potential of all dipeptide-TMP conjugates as pyrophosphate mimics in the DNA polymerization reaction was tested, and the influence of the nature of the linker was evaluated by in vitro chain elongation assay in the presence of wild-type microbial DNA polymerases.

Development of caged non-hydrolyzable phosphoamino acids and application to photo-control of binding affinity of phosphopeptide mimetic to phosphopeptide-recognizing protein.

The design and synthesis of caged non-hydrolyzable phospho-serine, -threonine, and -tyrosine derivatives that generate parent non-hydrolyzable phosphoamino acids, containing a difluoromethylene unit instead of the oxygen of a phosphoester, after UV-irradiation are described. The caged non-hydrolyzable amino acids were incorporated into peptides by standard Fmoc solid-phase peptide synthesis, and the obtained peptides were successfully converted to the parent non-hydrolyzable phosphopeptides by UV-irradiation. Application of the caged non-hydrolyzable phosphoserine-containing peptide to photo-control the binding affinity of the peptide to 14-3-3ß protein is also reported.

Effects on polo-like kinase 1 polo-box domain binding affinities of peptides incurred by structural variation at the phosphoamino acid position.

Protein-protein interactions (PPIs) mediated by the polo-box domain (PBD) of polo-like kinase 1 (Plk1) serve important roles in cell proliferation. Critical elements in the high affinity recognition of peptides and proteins by PBD are derived from pThr/pSer-residues in the binding ligands. However, there has been little examination of pThr/pSer mimetics within a PBD context. Our current paper compares the abilities of a variety of amino acid residues and derivatives to serve as pThr/pSer replacements by exploring the role of methyl functionality at the pThr ß-position and by replacing the phosphoryl group by phosphonic acid, sulfonic acid and carboxylic acids. This work sheds new light on structure activity relationships for PBD recognition of phosphoamino acid mimetics.

A general preparation of protected phosphoamino acids.

Fmoc-O-benzyl-l-phosphoserine is an important building block in the synthesis of Forigerimod, a phosphopeptide being investigated for Systemic Lupus Erythematosus (SLE). An efficient one-pot process was developed using inexpensive, readily available starting materials. This general procedure was used to prepare a variety of protected phosphoamino acids.

Sequential activation and deactivation of protein function using spectrally differentiated caged phosphoamino acids.

Photolabile caging groups, including the 1-(2-nitrophenyl)ethyl (NPE) group, have been applied to probe many biological processes, including protein phosphorylation. Although studies with NPE-caged phosphoamino acids have provided valuable information, these investigations have been limited to the use of only one caged species in a single experiment. To expand the scope of these tools, we have developed an approach for sequentially uncaging two different phosphopeptides in one system, enabling interrogation of multiple phosphorylation events. We present the synthesis of [7-(diethylamino)coumarin-4-yl]methyl (DEACM)-caged phosphorylated serine, threonine, and tyrosine building blocks for Fmoc-based solid-phase peptide synthesis to allow convenient incorporation of these residues into peptides and proteins. Exposure of DEACM- and NPE-caged phosphopeptides to 420 nm light selectively releases the DEACM group without affecting the NPE-caged peptide. This then enables a subsequent irradiation event at 365 nm to remove the NPE group and liberate a second phosphopeptide. We demonstrate the versatility of this general sequential uncaging approach by applying it to control Wip1 phosphatase with two wavelengths of light.

[Prebiotic phosphate: a problem insoluble in water ? ]. / Fosforo prebiotico: un problema insolubile in acqua?

It is well-known that in water phosphate readily reacts with calcium, precipitating as insoluble apatite. How phosphorus could have been available for prebiotic reactions is still an open problem. We suggest that phosphorus-containing compounds might have accumulated in a hydrophobic medium, since the absence of calcium ions would have prevented them from precipitating as apatite. Hydrophobic compounds may have been synthesized on the early Earth through the polymerization of methane or through Fischer-Tropsch-type reactions. Moreover, hydrophobic compounds would have been delivered to the early Earth by extraterrestrial infall. In previous articles (Morchio and Traverso [1999], Morchio et al. [2001]) we suggested that such hydrophobic material would have formed a hydrophobic layer on the surface of the sea, which would have provided an environment thermodynamically more suitable than water for the concentration and polymerization of organic molecules fundamental to life, particularly amino acids and (pyrimidine) bases. It may be hypothesized that elemental phosphorus or phosphorus-containing compounds (such as phosphite) deriving from volcanic eruptions would have ended up raining down into the hydrophobic layer, accumulating due to the absence of calcium ions, in an environment protected against hydrolysis. Phosphorus-containing compounds might have interacted with hydrophobic molecules in the layer giving rise to polymers. In particular, phosphite might have reacted with the hydrophobic amino acids, giving rise to phosphoamino acids, which, in turn, might have interacted with pyrimidine bases (relatively abundant in the layer) giving rise to peptides and oligonucleotide-like polymers. Indeed, it has been experimentally shown (Zhou et al. [1996]) that, in an anhydrous organic medium (pyridine), dialkilphosphite reacts with amino acids to form phosphoamino acids, which interact with pyrimidine nucleosides to give nucleotides, short oligonucleotides and phosphoryl peptides.

Synthesis of fluorine-containing bioisosteres corresponding to phosphoamino acids and dipeptide units.

It has been shown that fluorinated analogues of naturally occurring biological active compounds including amino acids often exhibit unique physiological activity. Among wide varieties of fluorine-containing amino acids, nonhydrolyzable phosphoamino acids possessing a substituent of the difluoromethylene (CF(2)) unit for the phosphoryl ester oxygen are of value in the medicinal and biological fields. We have engaged in the synthesis of these classes of nonhydrolyzable phosphoamino acids corresponding to pTyr 3, pSer 4, and pThr 5 with their incorporation into peptides using newly developed deprotecting procedures. In this article, stereoselective synthesis of the CF(2)-substituted pThr mimetics and development of a two-step deprotecting methodology for the nonhydrolyzable analogues are reviewed. In the course of the above synthetic study, we found that gamma,gamma-difluoro-alpha,beta-enoates were reduced to gamma-fluoro-beta,gamma-enoates by organocopper reagents and then applied to the synthesis of (Z)-fluoroalkene dipeptide isosteres, which have served as potential dipeptide mimetics having structural as well as electrostatic similarity to the parent peptide bonds. Furthermore, mechanistic investigation of the organocopper-mediated reduction led us to development of a SmI(2)-mediated approach toward the synthesis of the fluoroalkene isosteres.