Effect of Sulfotyrosine and Negatively Charged Amino Acid of Leech-Derived Peptides on Binding and Inhibitory Activity Against Thrombin.

Hirudins, natural sulfo(glyco)proteins, are clinical anticoagulants that directly inhibit thrombin, a key coagulation factor. Their potent thrombin inhibition primarily results from antagonistic interactions with both the catalytic and non-catalytic sites of thrombin. Hirudins often feature sulfate moieties on Tyr residues in their anionic C-terminus region, enabling strong interactions with thrombin exosite-I and effectively blocking its engagement with fibrinogen. Although sulfotyrosines have been identified in various hirudin variants, the precise relationship between sulfotyrosine and the number of negatively charged amino acids within the anionic-rich C-terminus peptide domain for the binding of thrombin has remained elusive. By using Fmoc-SPPS, hirudin dodecapeptides homologous to the C-terminus of hirudin variants from various leech species were successfully synthesized, and the effect of sulfotyrosine and the number of negatively charged amino acids on hirudin-thrombin interactions was investigated. Our findings did not reveal any synergistic effect between an increasing number of sulfotyrosines or negatively charged amino acids and their inhibitory activity on thrombin or fibrinolysis in the assays, despite a higher binding level toward thrombin in the sulfated dodecapeptide Hnip_Hirudin was observed in SPR analysis.

Small molecule tractable PARP inhibitors: Scaffold construction approaches, mechanistic insights and structure activity relationship.

Diverse drug design strategies viz. molecular hybridization, substituent installation, scaffold hopping, isosteric replacement, high-throughput screening, induction and separation of chirality, structure modifications of phytoconstituents and use of structural templates have been exhaustively leveraged in the last decade to load the chemical toolbox of PARP inhibitors. Resultantly, numerous promising scaffolds have been pinpointed that in turn have led to the resuscitation of the credence to PARP inhibitors as cancer therapeutics. This review briefly presents the physiological functions of PARPs, the pharmacokinetics, and pharmacodynamics, and the interaction profiles of FDA-approved PARP inhibitors. Comprehensively covered is the section on the drug design strategies employed by drug discovery enthusiasts for furnishing PARP inhibitors. The impact of structural variations in the template of designed scaffolds on enzymatic and cellular activity (structure-activity relationship studies) has been discussed. The insights gained through the biological evaluation such as profiling of physicochemical properties andin vitroADME properties, PK assessments, and high-dose pharmacology are covered.

Flavone-based dual PARP-Tubulin inhibitor manifesting efficacy against endometrial cancer.

Structural tailoring of the flavone framework (position 7) via organopalladium-catalyzed C-C bond formation was attempted in this study. The impact of substituents with varied electronic effects (phenyl ring, position 2 of the benzopyran scaffold) on the antitumor properties was also assessed. Resultantly, the efforts yielded a furyl arm bearing benzopyran possessing a 4-fluoro phenyl ring (position 2) (14) that manifested a magnificent antitumor profile against the Ishikawa cell lines mediated through dual inhibition of PARP and tubulin [(IC50 (PARP1) = 74 nM, IC50 (PARP2) = 109 nM) and tubulin (IC50 = 1.4 µM)]. Further investigations confirmed the ability of 14 to induce apoptosis as well as autophagy and cause cell cycle arrest at the G2/M phase. Overall, the outcome of the study culminated in a tractable dual PARP-tubulin inhibitor endowed with an impressive activity profile against endometrial cancer.

Anticancer Study of a Novel Pan-HDAC Inhibitor MPT0G236 in Colorectal Cancer Cells.

Colorectal cancer (CRC) is one of the most commonly diagnosed malignancies and a leading cause of cancer worldwide. Histone deacetylases (HDACs), which regulate cell proliferation and survival, are associated with the development and progression of cancer. Moreover, HDAC inhibitors are promising therapeutic targets, with five HDAC inhibitors approved for cancer treatment to date. However, their safety profile necessitates the exploration of well-tolerated HDAC inhibitors that can be used in cancer therapeutic strategies. In this study, the pan-HDAC inhibitor MPT0G236 reduced the viability and inhibited the proliferation of human colorectal cancer cells, and normal human umbilical vein endothelial cells (HUVECs) showed reduced sensitivity. These findings indicated that MPT0G236 specifically targeted malignant tumor cells. Notably, MPT0G236 significantly inhibited the activities of HDAC1, HDAC2, and HDAC3, Class I HDACs, as well as HDAC6, a Class IIb HDAC, at low nanomolar concentrations. Additionally, it promoted the accumulation of acetyl-α-tubulin and acetyl-histone H3 in HCT-116 and HT-29 cells in a concentration-dependent manner. Furthermore, MPT0G236 treatment induced G2/M cell cycle arrest in CRC cells by initially regulating the levels of cell-cycle-related proteins, such as p-MPM2; specifically reducing p-cdc2 (Y15), cyclin B1, and cdc25C levels; and subsequently inducing apoptosis through the caspase-dependent pathways and PARP activation. Our findings demonstrate that MPT0G236 exhibits significant anticancer activity in human colorectal cancer cells.

Beyond the vaccines: a glance at the small molecule and peptide-based anti-COVID19 arsenal.

Unprecedented efforts of the researchers have been witnessed in the recent past towards the development of vaccine platforms for the control of the COVID-19 pandemic. Albeit, vaccination stands as a practical strategy to prevent SARS-CoV-2 infection, supplementing the anti-COVID19 arsenal with therapeutic options such as small molecules/peptides and antibodies is being conceived as a prudent strategy to tackle the emerging SARS-CoV-2 variants. Noteworthy to mention that collective efforts from numerous teams have led to the generation of a voluminous library composed of chemically and mechanistically diverse small molecules as anti-COVID19 scaffolds. This review article presents an overview of medicinal chemistry campaigns and drug repurposing programs that culminated in the identification of a plethora of small molecule-based anti-COVID19 drugs mediating their antiviral effects through inhibition of proteases, S protein, RdRp, ACE2, TMPRSS2, cathepsin and other targets. In light of the evidence ascertaining the potential of small molecule drugs to approach conserved proteins required for the viral replication of all coronaviruses, accelerated FDA approvals are anticipated for small molecules for the treatment of COVID19 shortly. Though the recent attempts invested in this direction in pursuit of enrichment of the anti-COVID-19 armoury (chemical tools) are praiseworthy, some strategies need to be implemented to extract conclusive benefits of the recently reported small molecule viz. (i) detailed preclinical investigation of the generated anti-COVID19 scaffolds (ii) in-vitro profiling of the inhibitors against the emerging SARS-CoV-2 variants (iii) development of assays enabling rapid screening of the libraries of anti-COVID19 scaffold (iv) leveraging the applications of machine learning based predictive models to expedite the anti-COVID19 drug discovery campaign (v) design of antibody-drug conjugates.

Recent developments in epigenetic cancer therapeutics: clinical advancement and emerging trends.

Epigenetic drug discovery field has evidenced significant advancement in the recent times. A plethora of small molecule inhibitors have progressed to clinical stage investigations and are being explored exhaustively to ascertain conclusive benefits in diverse malignancies. Literature precedents indicates that substantial amount of efforts were directed towards the use of epigenetic tools in monotherapy as well as in combination regimens at the clinical level, however, the preclinical/preliminary explorations were inclined towards the identification of prudent approaches that can leverage the anticancer potential of small molecule epigenetic inhibitors as single agents only. This review article presents an update of FDA approved epigenetic drugs along with the epigenetic inhibitors undergoing clinical stage investigations in different cancer types. A detailed discussion of the pragmatic strategies that are expected to steer the progress of the epigenetic therapy through the implementation of emerging approaches such as PROTACS and CRISPR/Cas9 along with logical ways for scaffold fabrication to selectively approach the enzyme isoforms in pursuit of garnering amplified antitumor effects has been covered. In addition, the compilation also presents the rational strategies for the construction of multi-targeting scaffold assemblages employing previously identified pharmacophores as potential alternatives to the combination therapy.

Effect of 3-subsitution of quinolinehydroxamic acids on selectivity of histone deacetylase isoforms.

A series of 3-subsituted quinolinehydroxamic acids has been synthesised and evaluated for their effect on human lung cancer cell line (A549), human colorectal cancer cell line (HCT116) and HDAC isoforms 1, 2, 6, and 8. The results indicated that substitution at C3 of quinoline is favoured for HDAC6 selectivity. Two compounds (25 and 26) were also found to be potent anti-proliferative compounds with IC50 values ranging from 1.29 to 2.13 µM against A549 and HCT116 cells. These compounds displayed remarkable selectivity for HDAC6 over other HDAC isoforms with nanomolar IC50 values. Western blot analysis revealed that compounds of this series activate apoptotic caspase pathway as indicated by cleavage of caspase 3, 8, and 9 and also increase phosphorylated H2AX thus inducing DNA double strand fragmentation in a concentration dependent manner. Flow cytometric analysis also displayed a dose dependent increase of cell population in sub G1 phase.

HDAC inhibitor protects chronic cerebral hypoperfusion and oxygen-glucose deprivation injuries via H3K14 and H4K5 acetylation-mediated BDNF expression.

Vascular dementia (VaD) is the second most common cause of dementia, but the treatment is still lacking. Although many studies have reported that histone deacetylase inhibitors (HDACis) confer protective effects against ischemic and hypoxic injuries, their role in VaD is still uncertain. Previous studies shown, one HDACi protected against cognitive decline in animals with chronic cerebral hypoperfusion (CCH). However, the underlying mechanisms remain elusive. In this study, we tested several 10,11-dihydro-5H-dibenzo[b,f]azepine hydroxamates, which act as HDACis in the CCH model (in vivo), and SH-SY5Y (neuroblastoma cells) with oxygen-glucose deprivation (OGD, in vitro). We identified a compound 13, which exhibited the best cell viability under OGD. The compound 13 could increase, in part, the protein levels of brain-derived neurotrophic factor (BDNF). It increased acetylation status on lysine 14 residue of histone 3 (H3K14) and lysine 5 of histone 4 (H4K5). We further clarified which promoters (I, II, III, IV or IX) could be affected by histone acetylation altered by compound 13. The results of chromatin immunoprecipitation and Q-PCR analysis indicate that an increase in H3K14 acetylation leads to an increase in the expression of BDNF promoter II, while an increase in H4K5 acetylation results in an increase in the activity of BDNF promoter II and III. Afterwards, these cause an increase in the expression of BDNF exon II, III and coding exon IX. In summary, the HDACi compound 13 may increase BDNF specific isoforms expression to rescue the ischemic and hypoxic injuries through changes of acetylation on histones.

Synthesis and biological evaluation of 2-quinolineacrylamides.

A series of C6-substituted N-hydroxy-2-quinolineacrylamides (3-15), with four types of bridging groups have been synthesized. Most of these compounds exhibit antiproliferative activity against A549 and HCT116 cells and Western blot analysis revealed that they are able to inhibit HDAC. Measurement of the HDAC isoform activity of ether-containing compounds showed that compound 9 has distinct HDAC6 selectivity, more than 300-fold over other isoforms. This paper describes the development of 6-aryloxy-N-hydroxy-2-quinolineacrylamides as potential HDAC6 inhibitors.

Amide-tethered quinoline-resorcinol conjugates as a new class of HSP90 inhibitors suppressing the growth of prostate cancer cells.

The study is focused on the design and synthesis of amide tethered quinoline-resorcinol hybrid constructs as a new class of HSP90 inhibitor. In-vitro studies of the synthetic compounds led to the identification of compound 11, which possesses potent cell growth inhibitory effects against HCT116, Hep3B and PC-3 cell lines, exerted through HSP90 inhibition. Compound 11 triggers degradation of HSP90 client proteins along with concomitant induction of HSP70, demonstrates apoptosis inducing ability and causes G2M phase cell cycle arrest in PC-3 cells. Molecular modeling was used to dock compound 11 into the HSP90 active site and key interactions with the amino acid residues of the HSP90 chaperone protein were determined.

Novel microtubule inhibitor MPT0B098 inhibits hypoxia-induced epithelial-to-mesenchymal transition in head and neck squamous cell carcinoma.

BACKGROUND: Tumor hypoxia-induced epithelial-mesenchymal transition (EMT) is critical in promoting cancer metastasis. We recently discovered a novel microtubule inhibitor, MPT0B098, that employs a novel antitumor mechanism. It destabilizes hypoxia-inducible factor (HIF)-1α mRNA by blocking the function of human antigen R. Thus, we proposed that MPT0B098 modulates hypoxia-induced EMT. METHODS: In vitro IC50 values were determined through the methylene blue dye assay. To investigate molecular events, reverse transcriptase-polymerase chain reaction, Western blotting, immunofluorescence staining, and wound healing assay were employed. RESULTS: MPT0B098 significantly inhibited HIF-1α expression, epithelial-to-mesenchymal morphology changes, and migratory ability in the human head and neck squamous cell carcinoma cell line OEC-M1. Furthermore, after MPT0B098 treatment, the expression of two mesenchymal markers, vimentin and N-cadherin, was downregulated under hypoxic conditions. Moreover, MPT0B098 suppressed hypoxia-induced EMT in part by inhibiting EMT-activating transcription factors, Twist and SNAI2/Slug. In addition, the inhibition of hypoxia-induced F-actin rearrangement and focal adhesion kinase phosphorylation may have contributed to suppression of EMT by MPT0B098in OEC-M1 cells. MPT0B098 significantly inhibited transforming growth factor(TGF)-ß-induced phosphorylation of receptor-associated Smad2/3 by downregulating TGF-ß mRNA and protein expression. CONCLUSIONS: Taken together, this study provides a novel insight into the role of MPT0B098 in inhibiting hypoxia-induced EMT, suggesting its potential use for treating head and neck cancers.

Correction: 2-Aroylquinoline-5,8-diones as potent anticancer agents displaying tubulin and heat shock protein 90 (HSP90) inhibition.

Correction for '2-Aroylquinoline-5,8-diones as potent anticancer agents displaying tubulin and heat shock protein 90 (HSP90) inhibition' by Kunal Nepali et al., Org. Biomol. Chem., 2016, 14, 716-723.

MPT0B002, a novel microtubule inhibitor, downregulates T315I mutant Bcr-Abl and induces apoptosis of imatinib-resistant chronic myeloid leukemia cells.

Chronic myeloid leukemia (CML) is a hematopoietic malignancy caused by the constitutive activation of Bcr-Abl tyrosine kinase. The Bcr-Abl inhibitor imatinib and other second-generation tyrosine kinase inhibitors such as dasatinib and nilotinib have remarkable efficacy in CML treatment. However, gene mutation-mediated drug resistance remains a critical problem. Among point mutations, the Bcr-Abl T315I mutation confers resistance to these Bcr-Abl inhibitors. Previously, we have synthesized the compound (1-methyl-1H-indol-5-yl)-(3,4,5-trimethoxy-phenyl)-methanone (MPT0B002) as a novel microtubule inhibitor. In this study, we evaluated its effects on the proliferation, cell cycle, and apoptosis of K562 CML cells and BaF3 cells expressing either wild-type Bcr-Abl (BaF3/p210) or T315I-mutated Bcr-Abl (BaF3/T315I). MPT0B002 inhibited cell viability in a dose-dependent manner in these cells but did not affect the proliferation of human umbilical vein endothelial cells. It disrupted tubulin polymerization and arrested cell cycle at the G2/M phase. Treatment with MPT0B002 induced apoptosis, and this induction was associated with increased levels of cleaved caspase-3 and cleaved PARP. Furthermore, MPT0B002 can downregulate both Bcr-Abl and Bcr-Abl-T315I mRNA expressions and protein levels and the downstream signaling pathways. Taken together, our findings suggest that MPT0B002 may be considered a promising compound to downregulate not only wild type Bcr-Abl but also the T315I mutant to overcome Bcr-Abl-T315I mutation-mediated resistance in CML cells.

Total Synthesis of Two Glycosylated Stilbenes, Oxyresveratrol 2-O-ß-d-Glucopyranoside and 2,3,5,4'-Tetrahydroxystilbene 2-O-ß-d-Glucopyranoside.

Glycosylated stilbenes are biologically active secondary metabolites of plants and have the potential to alleviate a broad range of human diseases. However, some of these compounds are not naturally abundant, and thus the synthesis of such molecules is desirable. This paper reports the first synthesis of oxyresveratrol 2-O-ß-d-glucopyranoside (1) and 2,3,5,4'-tetrahydroxystilbene 2-O-ß-d-glucopyranoside (1'), which are stilbene glycosides obtained from the rhizomes of Schoenocaulon officinale and Polygonum multiflorum, respectively. A facile four-step synthesis of 1 involved selective protection of the hydroxy groups and Wittig olefination to generate the compound in 8% overall yield. For compound 1', a 10-step synthesis utilized selective protection of the hydroxy groups, Baeyer-Villiger oxidation, modified Duff formylation, and Wittig olefination to generate the compound in 6.9% overall yield.

Pharmacokinetics and protein binding of MPT0B292.

MPT0B292 was identified through screening of compounds able selectively to acetylate α-tubulins in cells and it exhibited potent anti-tumor, anti-angiogenesis and anti-metastatic effects in vitro and in vivo. Because of its poor water solubility, MPT0B292 is difficult to formulate with conventional approaches and hence difficulties are experienced in research practices. MPT0B292 was mixed with albumin in an aqueous solvent to form drug albumin nanoparticles with a size range around 333 nm. Unbound fractions of these nanoparticles were investigated in different or the same albumin concentration solutions. Unlike most drugs, the binding of MPT0B292 in human serum albumin increased with increasing drug concentrations. An analytical method was also developed and validated to determine MPT0B292 in rat plasma. This analytical method was applied successfully to the intravenous pharmacokinetic study of MPT0B292 in rats. A single dose study was regularly done to characterize the pharmacokinetic properties of the drug. Additionally, a novel i.v. infusion study was carried out to verify the extraction ratio of MPT0B292. The pharmacokinetic analysis revealed that MPT0B292 was a high extraction ratio drug with high systemic clearance, a high volume of distribution and a short half-life in rats. Copyright © 2017 John Wiley & Sons, Ltd.

A novel tubulin polymerization inhibitor, MPT0B206, downregulates Bcr-Abl expression and induces apoptosis in imatinib-sensitive and imatinib-resistant CML cells.

Imatinib, a Bcr-Abl-specific inhibitor, is effective for treating chronic myeloid leukemia (CML), but drug resistance has emerged for this disease. In this study, we synthesized a novel tubulin polymerization inhibitor, MPT0B206 (N-[1-(4-methoxy-benzenesulfonyl)-2,3-dihydro-1H-indol-7-yl]-formamide), and demonstrated its apoptotic effect and mechanism in imatinib-sensitive K562 and imatinib-resistant K562R CML cells. Western blotting and immunofluorescence microscopy showed that MPT0B206 induced microtubule depolymerization in K562 and K562R cells. MPT0B206 inhibited the growth of these cells in a concentration- and time-dependent manner. It did not affect the viability of normal human umbilical vein endothelial cells. MPT0B206 induced G2/M cell cycle arrest and the appearance of the mitotic marker MPM-2 in K562 and K562R cells, which is associated with the upregulation of cyclin B1 and the dephosphorylation of Cdc2. Treatment of K562 and K562R cells with MPT0B206 induced apoptosis and reduced the protein levels of procaspase-9 and procaspase-3 and increased caspase-3 activity and PARP cleavage. MPT0B206 also reduced the levels of the antiapoptotic proteins Mcl-1 and Bcl-2 and increased the level of the apoptotic protein Bax. Additional experiments showed that MPT0B206 markedly downregulated Bcr-Abl mRNA expression and total and phosphorylated Bcr-Abl protein levels and inhibited the phosphorylation of its downstream proteins STAT5, MAPK, and AKT, and the protein level of c-Myc in K562 and K562R cells. Furthermore, MPT0B206 triggered viability reduction and apoptosis in CML cells carrying T315I-mutated Bcr-Abl. Together, these results suggest that MPT0B206 is a promising alternative for treating imatinib-resistant CML.

MPT0B169, a novel tubulin inhibitor, induces apoptosis in taxol-resistant acute myeloid leukemia cells through mitochondrial dysfunction and Mcl-1 downregulation.

Acute myeloid leukemia (AML) is a hematological malignant disorder. AML cells are not susceptible to chemotherapeutic drugs because of their multidrug resistance (MDR). Antitubulin agents are currently employed in cancer treatments; however, drug resistance results in treatment failures because of MDR1 expressing cancer cells. We previously synthesized a new tubulin inhibitor, 2-dimethylamino-N-[1-(4-methoxy-benzenesulfonyl)-2,3-dihydro-1H-indol-7-yl]-acetamide (MPT0B169), which inhibits AML cell proliferation by arresting cell cycle at the G2/M phase. In this study, we explored the effect of MPT0B169 on apoptosis in AML HL60 and NB4 cells and MDR1-mediated taxol-resistant HL60/TaxR cells and the underlying mechanism. MPT0B169 induced concentration- and time-dependent apoptosis in these cancer cells, as observed through annexin V/propidium iodide double staining and flow cytometry. Furthermore, DNA fragmentation analysis confirmed MPT0B169-induced apoptosis. MPT0B169 induced a loss of mitochondrial membrane potential, release of cytochrome c into the cytosol, cleavage and activation of caspase-9 and caspase-3, and consequently cleavage of poly (ADP ribose) polymerase. Western blot analysis showed that MPT0B169 markedly reduced Mcl-1 (an antiapoptotic protein) levels; however, it caused no changes in Bcl-2 or BAX (a proapoptotic protein). Knockdown of Mcl-1 using small interfering RNA (siRNA) slightly induced growth inhibition and apoptosis in the HL60 and HL60/TaxR cells. Further investigation revealed that Mcl-1 siRNA enhanced the sensitivity of HL60 and HL60/TaxR cells to MPT0B169-induced growth inhibition and apoptosis. Together, these results demonstrated that MPT0B169-induced apoptosis in nonresistant and MDR1-mediated taxol-resistant AML cells through Mcl-1 downregulation and a mitochondria-mediated pathway. MPT0B169 can overcome MDR1-mediated drug resistance in AML cells.

Suberoylanilide hydroxamic acid represses glioma stem-like cells.

BACKGROUND: Glioma stem-like cells (GSCs) are proposed to be responsible for high resistance in glioblastoma multiforme (GBM) treatment. In order to find new strategies aimed at reducing GSC stemness and improving GBM patient survival, we investigated the effects and mechanism of a histone deacetylases (HDACs) inhibitor, suberoylanilide hydroxamic acid (SAHA), since HDAC activity has been linked to cancer stem-like cell (CSC) abundance and properties. METHODS: Human GBM cell lines were plated in serum-free suspension cultures allowed for sphere forming and CSC enrichment. Subsequently, upon SAHA treatment, the stemness markers, cell proliferation, and viability of GSCs as well as cellular apoptosis and senescence were examined in order to clarify whether inhibition of GSCs occurs. RESULTS: We demonstrated that SAHA attenuated cell proliferation and diminished the expression stemness-related markers (CD133 and Bmi1) in GSCs. Furthermore, at high concentrations (more than 5 µM), SAHA triggered apoptosis of GSCs accompanied by increases in both activation of caspase 8- and caspase 9-mediated pathways. Interestingly, we found that a lower dose of SAHA (1 µM and 2.5 µM) inhibited GSCs via cell cycle arrest and induced premature senescence through p53 up-regulation and p38 activation. CONCLUSION: SAHA induces apoptosis and functions as a potent modulator of senescence via the p38-p53 pathway in GSCs. Our results provide a perspective on targeting GSCs via SAHA treatment, and suggest that SAHA could be used as a potent agent to overcome drug resistance in GBM patients.

2-Aroylquinoline-5,8-diones as potent anticancer agents displaying tubulin and heat shock protein 90 (HSP90) inhibition.

This study reports the synthesis of a series of 2-aroylquinoline-5,8-diones (11-23) on the basis of scaffold hopping. The presence of a methoxy group at C6 assists the highly regioselective incorporation with various amines, and simplifies the structural identification process. Among the synthetic compounds, 6-dimethylamino-2-(3,4,5-trimethoxybenzoyl)-quinoline-5,8-dione (12) and 7-pyrrolidin-1-yl-2-(3,4,5-trimethoxybenzoyl)-quinoline-5,8-dione (23) exhibit remarkable anti-proliferative activity against the cancer cell lines tested with mean IC50 values of 0.14 and 0.27 µM, respectively. Compound 23 showed moderate inhibitory activity against tubulin polymerization with an IC50 value of 5.9 µM. In a western blot analysis, 23 caused induction of HSP70 and degradation of Akt, revealing that it possesses HSP90 inhibitory activity.

Total Synthesis of Denbinobin.

A total synthesis of denbinobin (1) in seven steps with an overall yield of 10% is reported. This synthesis used an FeCl3-assisted cyclization of stilbene to form a phenanthrene. The poor yields of the decarboxylation and methoxylation steps were improved upon to become essentially quantitative. This scalable methodology was carried out using ordinary laboratory reagents.