Design and Synthesis of Esters as Protein Kinase C(PKC)-CI Domain Regulators

In addition, we investigated in vitro protein binding properties with C1 domain of PKC isoforms using the Trp fluorescence quenching method. Further, we investigated in vitro protein binding properties with C1 domain of PKC isoforms using FRET.

Protein kinase C and its Ester Based Activators

Introduction

However, there are only a few reported esterification methods in the literature suitable for the synthesis of such biologically active esters in high yield. This forced the researchers to develop new methods suitable for the esterification of the biologically complex acids or alcohols.

Review of the Literature

Protein kinases
Protein kinase C

Structure, Activation and Regulation
The C1 Domain of Protein Kinase C

The C1-Domain as a Target for Natural and Synthetic Compounds

In the inactive conformation, the pseudosubstrate is bound to the catalytic site of the kinase domain. Here, some of the natural compounds targeted at the C1 domain and simplified synthetic derivatives are presented.

Prostratin and its Synthetic Analogues

As mentioned earlier, the C1 domain is unique to PKC and few other proteins, providing a selectivity advantage among the protein kinases and making the C1 domain an attractive drug target. Among these ligands, most of them interact with the C1 domain through similar interaction mode as of DAG (4) / phorbol ester (5).

Ingenol and its Synthetic Analogues

The C3 carbonyl in the five-membered ring of phorbols is replaced by an acyl or hydroxy group. These structural changes in ingenols make them more hydrophilic than phorbol esters.67 Ingenols (19 and 20) bind to the C1 domain of PKC in the same way as phorbol esters (5).

Bryostatin and its Synthetic Analogues

Interestingly, the dibenzoate derivative (23, Ki = 240 nM for PKCα) was found to induce apoptosis in Jurkat cells through caspase-3 activation, but not through PKC-mediated activation. 65, 68 Because compound 23 may not form the same interaction with the PKC C1 domain binding site groove as 21. However, the addition of a hydroxyl group to C7 (compounds 30 and 31) further reduced the binding affinity. 78 These differences were hypothesized to be in binding affinities due to the C8 gem-dimethyl group found in natural bryostatins protecting the polar C7 group.

Indolactam, Benzolactam and its Synthetic Analogues

After that, various research groups synthesized several benzolactam derivatives (36-41) and indolactams (42 and 43) (Fig. PKC with the same interactions as indolactams.

Iridals and its Synthetic Analogues

DAG-lactones

General Methods for the Synthesis of Hydroxyl Group Bearing Esters

Recently, Rafael Robles and co-workers investigated the mild and selective esterification of carboxylic acids based on Garegg–Samuelsson reaction conditions in the presence of triphenylphosphine, iodine, and imidazole, as shown in Scheme 2.4.3.114. In addition, Mashima and co-workers demonstrated that enzyme-like tetranuclear zinc cluster [Zn4(OCOCF3)6O] and alkoxy-bridged dinuclear cobalt complex [Co8(OCOtBu)12O2] promote chemoselective transesterification methods in the presence of alkyl amines (Scheme 2.4. 7 ).116e, f.

Concluding Remarks

Design, Synthesis and Protein Binding Properties of Diacyltetrol Lipids

Background and Focus of the Present Work

Design of Tetrol-Based C1 Domain Ligands - The structures of C1 domain binding natural product ligands revealed the presence of more than one hydroxyl group. Molecular docking analysis ─The analyzed molecular models of ligand-bound protein revealed the binding orientation of the DATs within the DAG/phorbol ester binding site of the PKCδ-C1b subdomain.

Conclusion

Experimental Sections

Instrumentations and Characterization
General procedure for the deprotection of benzyl groups (II) 135 : To a solution of benzyl ether containing compounds (1 equiv.) in 10 mL of ethyl acetate/ethanol (3
General procedure for the deprotection of the cyclopentylidene and dimethyl acetal groups (III) 130,132 : To a stirring solution of protected tetrols (1 equiv.)
General procedure for the preparation of cyclopentylidene ketal and acetonide of diols (IV) 130,131 : Method-A: To a stirring solution of diol (1 equiv.) in
General procedure for the reduction of esters with LiAlH 4 (V) 132
General procedure for the mono protection of diol and triol with Ag 2 O (VI) 136 : To a solution of alcohol (1 equiv.) in anhydrous dichloromethane (20 mL),
Purification of Proteins from Bacterial Cells: The PKCδ C1b and PKCθC1b subdomains fused with glutathione S-transferase (GST) were expressed in BL21
Measurement of Binding Parameters using Fluorescence Spectroscopic Analysis: Fluorescence measurements were performed on a Fluoromax-4

General procedure for the deprotection of benzyl groups (II)135: To a solution of compounds containing benzyl ether (1 eq.) in 10 mL ethyl acetate/ethanol (3 solutions of compounds containing benzyl ether (1 eq.) in 10 mL ethyl acetate /ethanol (ratio 3:1) was added with 10% Pd–C (0.1 equiv) and stirring was continued under H2 (80 psi) at room temperature for 8–9 h.General Procedure for Deprotection of Cyclopentylidene and Dimethyl Acetal Groups (III)130,132: To a mixed solution of protected tetrols (1 eq.) of dimethyl acetal groups (III)130,132: To a mixed solution of protected tetrols (1 eq.) in dichloromethane (10 mL), 3–5 mL of 80% acetic acid was added and continued stirring at 40 °C for 4–5 h.

Spectroscopic Characterization of the Synthesized Compounds

Then benzyl bromide (1.4 mL, 11.76 mmol) was added dropwise to the reaction mixture and stirred overnight at room temperature.132 The reaction was quenched with a saturated solution of ammonium chloride and the residue was extracted with diethyl ether (3 x 20 mL).

NMR Spectra of the Synthesized Compounds

Development of Diacyltetrol Based Anionic Hybrid Lipids as Protein Kinase C (PKC)-C1 Domain

Regulators

Background and Focus of the Present Work

Therefore, these hybrid lipids are structural mimics of DAG and PS/PA/PG lipids. Therefore, the binding affinities of hybrid lipids highlight the importance of ligand hydrophobicity and binding orientation. However, these binding parameters do not indicate an authentic C1 domain binding site for the hybrid lipids.

Experimental Section

General Procedure for the Deprotection of Isopropylidene Group (I) 151
General Procedure for the Deprotection of Benzyl Groups (III): The removal of benzyl ether group using 0.1 equivalents of 10% Pd-C, under 80 psi of H 2 gas for 3 h
General Procedure for the Deprotection of TBDPS Group (IV) 156 : To a stirring solution of TBDPS protected compound (1.0 equiv) in THF (5.0 mL) was added
General Procedure for the One-pot Phosphorylation of Protected Alcohols to Prepare DAT-PS and DAT-PG Derivatives (V): To an ice-cooled

After completion of the reaction, the catalyst (Pd-C) was filtered through a pad of Celite and washed with 30 mL of MeOH. After completion of the reaction, the solvent was removed under reduced pressure to give a residue. The degree (r) of anisotropy in protein tryptophan fluorescence was calculated using eq 2, at.

Characterization of the Synthesized Compounds

Extent of Membrane Localization: The extent of localization of the ligands at the liposome interface was investigated by the NBD fluorescence quenching method using PC/Cholesterol/Ligand16/NBD-PE liposomes in 50 mM Tris buffer, pH 8.2, containing 150 mM NaCl, according to the reported procedure.151,159.

Selected Spectra of DAT-Anionic Lipids

Hydroxylmethyl)phenyl Ester Analogues as Protein Kinase C-C1 Domain Regulators

Background and Focus of the Present Work

An ether group was also introduced to understand the importance of the ester group at the ortho position in C1 domain binding. The hydrophobic amino acids surrounding the binding site of the C1 domain also interact with the hydrophobic side chains of the ligands. In the next chapter, we described the synthesis, spectral analysis, and binding properties of the C1 domain of alkyl cinnamates.

Experimental Section

General Procedure for the Synthesis of Formyl-phenyl Monoester Analogues: Hexadecanic acid/octonic acid (1.1 equiv), dicyclohexylcarbodiimide (1.1
General Procedure for the Synthesis of Formylphenyl Diester Analogues 167 : Palmitic acid/octonic acid (2.2 equiv), dicyclohexylcarbodiimide (2.2
General procedure for the Reduction of Aldehydes: To a stirring solution of formylphenyl ester analogues (1.0 equiv) in THF, NaBH 4 (2.0 equiv) was added portion-
Fluorescence Measurements

General procedure for the synthesis of formylphenyl diestra analogues 167: palmitic acid/octonic acid (2.2 equiv), dicyclohexylcarbodiimide (2.2 Analogs 167: palmitic acid/octonic acid (2.2 equiv), dicyclohexylcarbodiimide (2.2 equiv) and N ,N-dimethylaminopyridine (0.1 eq. ) was added to a stirred solution of dihydroxybenzaldehyde (1.0 eq.) in anhydrous dichloromethane (8 mL) under N2 General procedure for the reduction of aldehydes: To a stirred solution of formylphenyl ester analogs (1, 0 equiv) in THF, NaBH4 (2.0 equiv) was added portionwise to the formylphenyl ester analogs (1.0 equiv) in THF, NaBH4 (2.0 equiv) was added portionwise at 0 °C over 5 min and stirring was continued for another 15 minutes at room temperature. General procedure for Synthesis of 2-tert-butoxy and 2-(benzyloxy)-4-formylphenyl hexadecanoate/octanoate: To a solution of 4-(benzyloxy)-4-formylphenyl hexadecanoate/octanoate: To a solution of 4-formyl-2-hydroxyphenyl hexadecanoate/octanoate (1.0 equiv.) in anhydrous dichloromethane (10 mL), silver oxide (1.5 equiv.) and benzyl bromide/tert-butyl iodide (1.2 equiv.) were added under N2 atmosphere.

Characterization of the Synthesized Compounds

IITG-Chemistry Chapter 4 sepharose column, and the GST tag was removed by thrombin treatment using methods similar to those described previously. Extent of Membrane Localization: The extent of localization of the ligands at the liposome interface was investigated by the fluorescence quenching method using PC/cholesterol/ligand16/NBD-PE liposomes in 50 mM Tris buffer, pH 8.2, containing 150 mM NaCl, according to the reported procedure.125,160. Molecular modeling: Molecular docking modeling was performed using the crystal structure of PKCδ C1b (Protein Data Bank code 1PTR).127 Generation of energy-minimized three-dimensional structures of ligands and ligand-protein docking was performed using methods similar to those, that is described. earlier.151.160.

Selected Spectra of (Hydroxymethyl)phenyl Ester Analogues

Synthesis of Protein Kinase C (PKC)-C1 Domain Targeted Alkyl Cinnamates

Background and Focus of the Present Work

Design and Synthesis of Alkyl Cinnamates Chapter 5 Design and Synthesis─ Curcumin is one of the most thoroughly studied natural compounds. Design and Synthesis of Alkyl Cinnamates Chapter 5 The compounds also contain hydrophobic alkyl chains for their potential interaction with either the hydrophobic residues of the C1 domain or with membrane lipids. Design and Synthesis of Alkyl Cinemas Chapter 5 Alkyl chain length did not show any significant effect on absorption and emission maxima.

Protein Binding Properties

Experimental Section

General experimental procedure for the preparation of half esters of malonic acid (I): Meldrum's acid (1 equiv.) and cetyl alcohol or 1-octanol (1 equiv.)
General experimental procedure for the preparation of esters of trans-cinnamic acids (II):- Method A: A solution of trans-cinnamic acid
Protein purification
Fluorescence studies
Liposome binding assay
Extent of membrane localization
Molecular docking

General experimental procedure for the preparation of half-esters of malonic acid (I): Meldrum's acid (1 eq.) and cetyl alcohol or 1-octanol (1 eq.) of malonic acid (I): Meldrum's acid (1 eq.) and cetyl alcohol or 1-octanol ( 1 equiv) was refluxed in toluene (30 mL) for 4 h. General experimental procedure for the preparation of esters of trans-cinnamic acids (II):- Method A: A solution of trans-cinnamic acids-trans-cinnamic acids (II):- Method A: A solution of trans-cinnamic acid (1.0 equiv. . ), cetyl alcohol or 1-octanol (1.0 equiv), p -toluenesulfonic acid (0.1 equiv) in toluene was refluxed for 6 h and the azeotrope collected in a Dean-Stark trap. Method B: To a stirred solution of 2-((hexadecyloxy)carbonyl)acetic acid or 2-((octyloxy)carbonyl)acetic acid, corresponding benzaldehyde derivative in toluene (15 mL) was added pyridine (1.5 equiv), piperidine (0 .1 eq.) and the resulting mixture was stirred for 8 hours at 70°C.

Characterization of the Synthesized Compounds

Selected Spectra of Alkyl Cinnamates

Zn(OTf) 2 Catalyzed Chemoselective Esterification of Carboxylic Acids Usable for the PKC Activation and

Background and Focus of the Present Work

Similar results were also obtained in the esterification of aliphatic acids with secondary benzyl alcohols (Table 6.1.3, entries 17 and 18). Similarly, selective esterification of an aliphatic acid in the presence of an aromatic acid was also observed (Table 6.1.3, entry 12). Zn(OTf)2 catalyzed chemoselective esterification, Chapter 6, Table 6.1.7: 31P and 13C chemical shifts for esterification intermediates/reaction species.

Experimental Section

General Procedure for the Zn(OTf) 2 Catalysed Esterification/Amidation of Carboxylic Acids: To a stirring solution of I 2 (2.0

In conclusion, we have developed a Zn(OTf)2-catalyzed facile and highly chemoselective esterification/amidation method for aromatic and aliphatic carboxylic acids in the presence of triphenylphosphine and I2. In addition, we demonstrated the regioselective esterification of unsymmetrical alcohols as well as the regioselective esterification of aliphatic carboxylic acids via aromatic carboxylic acids, which can be very important in the synthesis of polyfunctionalized substrates. This facile and efficient method will serve as a useful alternative to existing esterification methods and will reduce the burden of protecting groups in the synthesis of more complex molecules, which represents an important goal in modern organic chemistry.

Spectral Characterization of the New Compounds

13C-NMR in CDCl3; (A) 10 min after addition of 1a to the stoichiometric mixture of Ph3P and I2; (B) 30 min after addition of Zn(OTf)2 to the mixture of 1a, Ph3P and I2; (C) 4 h after addition of 2a to mixture of Zn(OTf)2, (1a), Ph3P and I2.*→Represents C=O of phosphate ester intermediate and compound 3aa; →Represents the phenyl protons of triphenylphosphine (Ph3P) and triphenylphosphine oxide (Ph3P=O); →Represents the OCH2 protons of the aliphatic chain of cetyl alcohol.

NMR Spectra of Some of the Selected Esters

Conclusion and Future Perspective

The best compounds (13, 5, and 8, from Chapter 4) showed 5-fold stronger binding affinity than natural DAG for the C1 domain. In addition, molecular docking studies showed that these ester analogs bind to the C1 domain of PKC isoforms at the DAG/phorbol ester binding site. In addition, this methodology has been used to synthesize PKC regulators in high yield.