Anilkumar R. Kore

Locked nucleic acid (LNA)-modified dinucleotide mRNA cap analogue: synthesis, enzymatic incorporation, and utilization.

There has been considerable therapeutic interest in the development of human vaccines against cancers and infectious diseases such as HIV and biowarfare agents by using transfected mRNAs for antigenic proteins of interest. The highest expression levels of these proteins are obtained when the transfected mRNA contains 5-capped ends. In the present study, the locked nucleic acid (LNA)-modified cap analogue 3, m(7(LNA))G[5]ppp[5]G, has been synthesized and its biological properties were examined. The LNA-modified cap analogue was an efficient substrate for T7 RNA polymerase, and the mRNA transcribed, with a poly(A) tail, was efficiently utilized in an in vitro translation process. The RNA with the 5-LNA-modified cap was found to be approximately 1.61- and 1.28-fold more stable than the RNA with the 5-standard 4 and ARCA cap, respectively, and approximately 4.23-fold more stable than the uncapped control RNA. The RNA capped with the m(7(LNA))G[5]ppp[5]G 3 cap analogue was translated the most efficiently, with approximately 3.2-fold more activity than the standard cap, m(7)G[5]ppp[5]G 4. Furthermore, we have developed a nonradioactive analytical HPLC assay to determine that the LNA-modified 3 cap analogue was incorporated solely into the forward orientation. Molecular modeling of the m(7(LNA))G[5]ppp[5]G 3 cap analogue with the cap binding protein elF4E complex indicates that the LNA-modified cap-protein complex is more stable by 47.28 kcal/mol as compared to the standard mCAP-protein complex. These findings suggest that the new antireverse cap analogue m(7(LNA))G[5]ppp[5]G 3 is a potential candidate for RNA-based therapeutic vaccine production as well as studying biochemical processes.

An Improved Protection-Free One-Pot Chemical Synthesis of 2′-Deoxynucleoside-5′-Triphosphates

A facile, straightforward, reliable, and an efficient method for the gram-scale chemical synthesis of both purine deoxynucleotides such as 2 ′-deoxyguanosine-5 ′-triphosphate (dGTP) and 2 ′-deoxyadenosine-5 ′-triphosphate (dATP) and pyrimidine deoxynucleotides such as 2 ′-deoxycytidine-5 ′-triphosphate (dCTP), thymidine-5 ′-triphosphate (TTP), and 2 ′-deoxyuridine-5 ′-triphosphate (dUTP) starting from the corresponding nucleoside is described. This improved “one-pot, three step” Ludwig synthetic strategy involves the monophosphorylation of nucleoside followed by reaction with tributylammonium pyrophosphate and hydrolysis of the resulting cyclic intermediate to provide the corresponding dNTP in good yields (65%–70%).

Synthesis and biological validation of N7-(4-chlorophenoxyethyl) substituted dinucleotide cap analogs for mRNA translation

Design, synthesis and biological validation of dinucleotide cap analogs, N(7)-(4-chlorophenoxyethyl)-G(5)ppp(5)G (5a) and N(7)-(4-chlorophenoxyethyl)-m(3-O)G(5)ppp(5)G (5b) are reported. The effect of N(7)-(4-chlorophenoxyethyl) substitution on cap analogs has been evaluated with respect to its in vitro transcription by using T7 RNA polymerase capping efficiency, and translational activity. The gel shift assay indicates that the new cap analogs (5a, 5b) showed 77% and 76% capping efficiency respectively, whereas the standard cap analog, m(7)G(5)ppp(5)G has a capping efficiency of 63%. The capping efficiency experiment clearly demonstrates that the N(7)-modified analogs are good substrate for T7 RNA polymerase. It is noteworthy that the mRNA poly(A) capped with N(7)-(4-chlorophenoxyethyl)-m(3-O)G(5)ppp(5)G (5b) was translated ∼1.64-fold more efficiently, while compound (5a) was translated ∼0.72-fold less efficiently than mRNA capped with standard cap analog. The observed low translation activity for (5a) could be due to stability in the form of dinucleotide cap analogs. Based on the substrate compatability of the N(7) modification in dinucleotide form, these new analogs may be used for structure function studies as well as protein production.

Synthesis and evaluation of 2′-O-allyl substituted dinucleotide cap analog for mRNA translation

The first example of the synthesis and biological evaluation of a new analog containing 2-OH modification on m(7)G moiety, that is, m(7,2-)(O-Allyl)GpppG is reported. The effect of the 2-O-allyl substitution on cap analog has been evaluated with respect to its in vitro transcription by using T7 RNA polymerase, capping efficiency, and translational activity. The gel shift assay indicates that the new cap analog has 59% capping efficiency whereas the standard cap analog, m(7)GpppG has a capping efficiency of 70%. The capping efficiency experiment clearly demonstrates that the new analog was a substrate for T7 RNA polymerase. The nature of the orientation has been determined by HPLC that reveals that the new analog incorporates exclusively in the forward orientation. It is noteworthy that the mRNA poly(A) capped with 2-O-allyl substituted cap analog was translated ∼1.7-fold more efficiently than the mRNA capped with standard cap analog. Based on the higher translational data compared to the standard cap analog, it is likely that the new analog may find application that utilize mRNA transfection such as protein production, anti-cancer immunization, and gene therapy.

An Efficient Synthesis of Pyrimidine Specific 2′-Deoxynucleoside-5′-Tetraphosphates

An efficient chemical synthesis of pyrimidine specific 2′-deoxynucleoside-5′-tetraphosphates, such as 2′-deoxycytidine-5′-tetraphosphate (dC4P) and thymidine-5′-tetraphosphate (T4P) is described. The present three-step synthetic strategy involves monophosphorylation of 2′-deoxynucleoside using phosphorous oxychloride, conversion of 5′-monophosphate into the corresponding imidazolide salt, followed by reaction with tris[tributylammonium] triphosphate leading to the 2′-deoxynucleoside-5′-tetraphosphate in good yields.

Facile Protection-Free One-Pot Chemical Synthesis of Nucleoside-5′-Tetraphosphates

A new, straightforward, reliable, and convenient protection-free one-pot method for the synthesis of 2′-deoxynucleoside-5′-tetraphosphate and ribonucleoside-5′-tetraphosphate is reported. The present synthetic strategy involves the monophosphorylation of a nucleoside followed by reaction with tris-(tri-n-butylammonium) triphosphate and subsequent hydrolysis of the putative cyclic tetrametaphosphate intermediate to provide nucleoside-5′-tetraphosphate in moderate yield with high purity. A plausible mechanism is proposed to account for the formation of product.

Synthesis and application of MeOSuc-Ala-Ala-Pro-Phe-CH2Cl as potent proteinase K inhibitor.

The synthesis and proteolytic inhibitor function of two new tetrapeptides, methoxysuccinyl-Ala-Ala-Pro-Phe-chloromethyl ketone (MeOSuc-AAPF-CH(2)Cl) and methoxysuccinyl-Ala-Pro-Ala-Phe-chloromethyl ketone (MeOSuc-APAF-CH(2)Cl) are described. The efficacy of these two new analogs in inhibiting the proteolytic activity of proteinase K has been compared with the previously-documented proteainase K inhibitor, methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone (MeOSuc-AAPV-CH(2)Cl). An examination of inhibitory activity using a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay in the presence of proteinase K reveals that the AAPF inhibitor (MeOSuc-AAPF-CH(2)Cl) at a concentration of 0.05mM allows a signal to be obtained for an exogenous target (Xeno RNA) at 30cycles (i.e. Ct=30), whereas the MeOSuc-AAPV-CH(2)Cl control requires a 10-fold higher concentration (0.5mM) to produce the same Ct. Interestingly, the other new analog, with the rearranged amino acid sequence APAF (MeO Suc-APAF-CH(2)Cl), provides no proteinase K inhibition under the same experimental conditions. These results suggest that when P1 is phenylalanine, alanine at P2 and proline at P3 is not tolerated as a good proteinase K inhibitor. A plausible explanation for the higher efficiency of MeOSuc-AAPF-CH(2)Cl over control is proposed based on the molecular modeling studies.

Gram-scale chemical synthesis of 2'-deoxynucleoside-5'-o-triphosphates.

A simple, straightforward, reliable, and efficient method for the chemical synthesis of sodium salt of 2′‐deoxynucleoside‐5′‐O‐triphosphates (dNTPs), starting from the corresponding nucleoside, is described. This improved “one‐pot, three‐step” synthetic strategy involves the monophosphorylation of nucleoside, followed by reaction with tributylammonium pyrophosphate and hydrolysis of the resulting cyclic intermediate to provide the corresponding dNTP in good yields (65% to 70%). It is noteworthy that the protocol holds good for both the purine deoxynucleotides, such as 2′‐deoxyguanosine‐5′‐O‐triphosphate (dGTP) and 2′‐deoxyadenosine‐5′‐O‐triphosphate (dATP), and pyrimidine deoxynucleotides, such as 2′‐deoxycytidine‐5′‐O‐triphosphate (dCTP), thymidine‐5′‐O‐triphosphate (TTP), and 2′‐deoxyuridine‐5′‐O‐triphosphate (dUTP). Curr. Protoc. Nucleic Acid Chem. 49:13.10.1‐13.10.12.

Synthesis and biological evaluation of modified pentapeptides as potent proteinase K inhibitors

This communication reports the first demonstration of synthesis and biological validation of modified pentapeptides, such as methoxysuccinyl-Ala-Ala-Ala-Pro-Leu-chloromethyl ketone 6b as a potent proteinase K inhibitor. The efficacy of MeOSuc-AAAPL-CH(2)Cl 6b analog in inhibiting the proteolytic activity of proteinase K was compared with the known MeOSuc-AAPV-CH(2)Cl analog. The examination of inhibitory activity using RT-PCR assay in the presence of proteinase K revealed that the MeOSuc-AAAPL-CH(2)Cl 6b inhibitor at a concentration of 0.05 mM allows a signal to be obtained for an exogenous target (Xeno RNA) at 30 cycles (i.e., Ct=30), whereas the control MeOSuc-AAPV-CH(2)Cl requires a fivefold higher concentration (0.25 mM) to produce the same Ct. A plausible explanation for the higher efficiency of MeOSuc-AAAPL-CH(2)Cl 6b over control is proposed based on the molecular modeling studies.

Synthesis and Activity of Modified Cytidine 5′-Monophosphate Probes for T4 RNA Ligase 1

We describe the synthesis of a series of unique base modified ligation probes such as p(5′)C-4-ethylenediamino 3, p(5′)C-4-biotin 4, and pre-adenylated form A(5′)pp(5′)C-4-biotin 6 and tested their biological activity with T4 RNA ligase 1 using a standard pCp probe 1 as a control. The intermolecular ligation assay was developed using a 5′-FAM labeled 24 mer single-stranded (ss) RNA and the average ligation efficiencies for pCp 1, p(5′)C-4-ethylenediamino 3, p(5′)C-4-biotin 4, and pre-adenylated form A(5′)pp(5′)C-4-biotin 6 were found to be 44%, 81%, 39% and 16% respectively, as determined using a denaturing gel analysis. Furthermore, confirmation of the ligation activity of the biotinylated probes to the RNA substrate was confirmed by streptavidin conjugation and analysis by nondenaturing gel electrophoresis. These results strongly suggest that the new probes are valid substrates for T4 RNA ligase 1 and therefore could be useful for developing a miRNA detection system that includes rapid isolation, efficient labeling and detection of miRNAs on sensitivity-enhanced microarrays.