Vladislav A. Litosh

Investigating the hydrogen-bonding model of urea denaturation.

The direct binding mechanism for urea-based denaturation of proteins was tested with a thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAM). Thermodynamic measurements of the polymers hydrophobic collapse were complemented by Fourier transform infrared (FTIR) spectroscopy, Stokes radius measurements, and methylated urea experiments. It was found that the lower critical solution temperature (LCST) of PNIPAM decreased as urea was added to the solution. Therefore, urea actually facilitated the hydrophobic collapse of the macromolecule. Moreover, these thermodynamic measurements were strongly correlated with amide I band data which indicated that the decrease in the LCST was coupled to the direct hydrogen bonding of urea to the amide moieties of the polymer. In addition, the hydrogen bonding was found to be highly cooperative, which is consistent with a cross-linking (bivalent binding) mechanism. Cross-linking was confirmed by Stokes radius measurements below the polymers LCST using gel filtration chromatography. Finally, phase transition measurements with methylurea, dimethylurea, and tetramethylurea indicated that these substituted compounds caused the LCST of PNIPAM to rise with increasing methyl group content. No evidence could be found for the direct binding of any of these methylated ureas to the polymer amide moieties by FTIR. These results are inconsistent with a direct hydrogen-bonding mechanism for the urea-induced denaturation of proteins.

Methyl Groups of Trimethylamine N-Oxide Orient Away from Hydrophobic Interfaces

The molecular orientation of trimethylamine N-oxide (TMAO), a powerful protein stabilizer, was explored at aqueous/hydrophobic interfaces using vibrational sum frequency spectroscopy (VSFS). The systems studied included the octadecyltrichlorosilane (OTS)/water interface, which represents an aqueous solution in direct contact with a hydrophobic medium. Surprisingly, the measurements revealed that the methyl groups of TMAO pointed into the aqueous phase and away from the OTS. This orientation may arise from the more hydrophilic nature of methyl groups attached to a strongly electron-withdrawing atom such as a quaternary nitrogen. Additional studies were performed at the air/water interface. This interface showed a high degree of TMAO alignment, but the dangling OH from water was present even at 5 M TAMO. Moreover, the addition of this osmolyte modestly increased the surface tension of the interface. This meant that this species was somewhat depleted at the interface compared to the bulk solution. These findings may have implications for the stabilizing effect of TMAO on proteins. Specifically, the strong hydration required for the methyl groups as well as the oxide moiety should be responsible for the osmolytes depletion from hydrophobic/aqueous interfaces. Such depletion effects should help stabilize proteins in their folded and native conformations on entropic grounds, although orientational effects may play an additional role.

Termination of DNA synthesis by N6-alkylated, not 3′-O-alkylated, photocleavable 2′-deoxyadenosine triphosphates

The Human Genome Project has facilitated the sequencing of many species, yet the current Sanger method is too expensive, labor intensive and time consuming to accomplish medical resequencing of human genomes en masse. Of the ‘next-generation’ technologies, cyclic reversible termination (CRT) is a promising method with the goal of producing accurate sequence information at a fraction of the cost and effort. The foundation of this approach is the reversible terminator (RT), its chemical and biological properties of which directly impact the performance of the sequencing technology. Here, we have discovered a novel paradigm in RT chemistry, the attachment of a photocleavable, 2-nitrobenzyl group to the N6-position of 2′-deoxyadenosine triphosphate (dATP), which, upon incorporation, terminates DNA synthesis. The 3′-OH group of the N6-(2-nitrobenzyl)-dATP remains unblocked, providing favorable incorporation and termination properties for several commercially available DNA polymerases while maintaining good discrimination against mismatch incorporations. Upon removal of the 2-nitrobenzyl group with UV light, the natural nucleotide is restored without molecular scarring. A five-base experiment, illustrating the exquisite, stepwise addition through a homopolymer repeat, demonstrates the applicability of the N6-(2-nitrobenzyl)-dATP as an ideal RT for CRT sequencing.

Stereochemistry of benzylic carbon substitution coupled with ring modification of 2-nitrobenzyl groups as key determinants for fast-cleaving reversible terminators.

Next-generation sequencing (NGS) technologies have facilitated important biomedical discoveries, yet high error rates and slow cycle times warrant further improvements in the chemistry.1a Such technologies that employ the cyclic reversible termination (CRT) method1a,b typically utilize 3′-O-blocked reversible terminators.2a–c Recently, we described a novel 3′-OH-unblocked reversible terminator based on 2-nitrobenzyl-modified 5-hydroxymethyl-2′-deoxyuridine (HOMedU) 5′-triphosphate.3 Our study revealed that the proximity of the 2-nitrobenzyl group to the nucleobase and the size of the alkyl group attached to its α-methylene carbon are important structural features that confer the unique properties of single-base termination, efficient incorporation, and high nucleotide selectivity (i.e., high fidelity) to these 3′-OH-unblocked nucleotides.3 These properties have the potential to improve accuracy and read-lengths in the CRT method. As HOMedU is a naturally found hypermodified nucleoside,4a we set out to identify other such examples. 5-Hydroxymethyl-2′-deoxycytidine (HOMedC) is found naturally in the genomes of T-even bacteriophages4a,b and mammals.5 Pyrrolopyrimidine (7-deazapurine) is also found naturally in nucleoside antibiotics6 and tRNAs.7 Thus, various analogues of 2-nitrobenzyl-modified 7-deaza-7-hydroxymethyl-2′-deoxyadenosine (C7-HOMedA),8 HOMedC, 7-deaza-7-hydroxymethyl-2′-deoxyguanosine (C7-HOMedG),9 and HOMedU were synthesized with the goal of developing a complete set of reversible terminators (Figure 1).

Improved nucleotide selectivity and termination of 3′-OH unblocked reversible terminators by molecular tuning of 2-nitrobenzyl alkylated HOMedU triphosphates

We describe a novel 3′-OH unblocked reversible terminator with the potential to improve accuracy and read-lengths in next-generation sequencing (NGS) technologies. This terminator is based on 5-hydroxymethyl-2′-deoxyuridine triphosphate (HOMedUTP), a hypermodified nucleotide found naturally in the genomes of numerous bacteriophages and lower eukaryotes. A series of 5-(2-nitrobenzyloxy)methyl-dUTP analogs (dU.I–dU.V) were synthesized based on our previous work with photochemically cleavable terminators. These 2-nitrobenzyl alkylated HOMedUTP analogs were characterized with respect to incorporation, single-base termination, nucleotide selectivity and photochemical cleavage properties. Substitution at the α-methylene carbon of 2-nitrobenzyl with alkyl groups of increasing size was discovered as a key structural feature that provided for the molecular tuning of enzymatic properties such as single-base termination and improved nucleotide selectivity over that of natural nucleotides. 5-[(S)-α-tert-Butyl-2-nitrobenzyloxy]methyl-dUTP (dU.V) was identified as an efficient reversible terminator, whereby, sequencing feasibility was demonstrated in a cyclic reversible termination (CRT) experiment using a homopolymer repeat of ten complementary template bases without detectable UV damage during photochemical cleavage steps. These results validate our overall strategy of creating 3′-OH unblocked reversible terminator reagents that, upon photochemical cleavage, transform back into a natural state. Modified nucleotides based on 5-hydroxymethyl-pyrimidines and 7-deaza-7-hydroxymethyl-purines lay the foundation for development of a complete set of four reversible terminators for application in NGS technologies.

ν1 and ν1+ν5 of DCCN: Determination of the ν5 energy and the quasilinearity of DCCN

The high resolution infrared spectrum of DCCN in the region of 2385–2530 cm−1 has been investigated using infrared kinetic spectroscopy. The CD stretching fundamental, ν1,together with the closely related hot bands, ν1+ν5−ν5,ν1+2ν5±2−2ν5±2, and ν1+2ν50−2ν50 have been observed and assigned. In addition the combination band, ν1+ν5, was observed. When the information provided by this perpendicular band is combined with that provided by ν1+ν5−ν5, the vibrational energy of ν5 is accurately determined to be 74.845(2) cm−1. This measurement, when combined with the ab initio and semirigid bender calculations of Malmquist et al., gives a barrier to linearity of 280 cm−1. Generally, fine structure splittings are not resolved for any levels except those belonging to the upper l- or K-type doubling components of ν1+ν5. This fine structure splitting is most strongly exhibited in the Q-branch of the ν1+ν5 band where it is 0.039 cm−1.

Design, synthesis, and optimization of novel epoxide incorporating peptidomimetics as selective calpain inhibitors.

Hyperactivation of the calcium-dependent cysteine protease calpain 1 (Cal1) is implicated as a primary or secondary pathological event in a wide range of illnesses and in neurodegenerative states, including Alzheimers disease (AD). E-64 is an epoxide-containing natural product identified as a potent nonselective, calpain inhibitor, with demonstrated efficacy in animal models of AD. By use of E-64 as a lead, three successive generations of calpain inhibitors were developed using computationally assisted design to increase selectivity for Cal1. First generation analogues were potent inhibitors, effecting covalent modification of recombinant Cal1 catalytic domain (Cal1cat), demonstrated using LC-MS/MS. Refinement yielded second generation inhibitors with improved selectivity. Further library expansion and ligand refinement gave three Cal1 inhibitors, one of which was designed as an activity-based protein profiling probe. These were determined to be irreversible and selective inhibitors by kinetics studies comparing full length Cal1 with the general cysteine protease papain.

Pathways for the thermally induced dehydrogenation of C60H2

Abstract The thermolysis of C 60 H 2 to yield C 60 and H 2 was studied by hybrid density functional theory (B3LYP/6-311G**//B3LYP/3-21G). The concerted loss of dihydrogen requires an activation energy of 92 kcal mol −1 at T =452 K. An alternative radical mechanism, which is first order in the C 60 H 2 concentration, has an activation energy at 452 K of only 61 kcal mol −1 . Monitoring of the C 60 H 2 decomposition in 1,2-dichloro-[D 4 ]-benzene solution by NMR spectroscopy indicates a pseudo first-order reaction with an activation energy of 61.38±2.35 kcal mol −1 .

Base-Modified Nucleosides as Chemotherapeutic Agents: Past and Future

Nucleoside and nucleobase antimetabolites have substantially impacted treatment of cancer and infections. Their close resemblance to natural analogs gives them the power to interfere with a variety of intracellular targets, which on one hand gives them high potency, but on the other hand incurs severe side effects, especially of the chemotherapeutics used against malignancies. Therefore, the development of novel nucleoside analogs with widened therapeutic windows represents an attractive target to synthetic organic and medicinal chemists. This review discusses the current antimetabolite drugs: 5- fluorouracil, 6-mercaptopurine, 6-thioguanine, Cladribine, Vidaza, Decitabine, Emtricitabine, Abacavir, Sorivudine, Clofarabine, Fludarabine, and Nelarabine; gives insight into the nucleoside drug candidates that are being developed; and outlines the approaches to nucleobase modifications that may help discover novel bioactive nucleoside analogs with the mechanism of action focused on termination of DNA synthesis, which is expected to diminish the off-target toxicity in non-proliferating human cells.

A novel aspirin prodrug inhibits NFκB activity and breast cancer stem cell properties

IntroductionActivation of cyclooxygenase (COX)/prostaglandin and nuclear factor κB (NFκB) pathways can promote breast tumor initiation, growth, and progression to drug resistance and metastasis. Thus, anti-inflammatory drugs have been widely explored as chemopreventive and antineoplastic agents. Aspirin (ASA), in particular, is associated with reduced breast cancer incidence but gastrointestinal toxicity has limited its usefulness. To improve potency and minimize toxicity, ASA ester prodrugs have been developed, in which the carboxylic acid of ASA is masked and ancillary pharmacophores can be incorporated. To date, the effects of ASA and ASA prodrugs have been largely attributed to COX inhibition and reduced prostaglandin production. However, ASA has also been reported to inhibit the NFκB pathway at very high doses. Whether ASA prodrugs can inhibit NFκB signaling remains relatively unexplored.MethodsA library of ASA prodrugs was synthesized and screened for inhibition of NFκB activity and cancer stem-like cell (CSC) properties, an important PGE2-and NFκB-dependent phenotype of aggressive breast cancers. Inhibition of NFκB activity was determined by dual luciferase assay, RT-QPCR, p65 DNA binding activity and Western blots. Inhibition of CSC properties was determined by mammosphere growth, CD44+CD24−immunophenotype and tumorigenicity at limiting dilution.ResultsWhile we identified multiple ASA prodrugs that are capable of inhibiting the NFκB pathway, several were associated with cytotoxicity. Of particular interest was GTCpFE, an ASA prodrug with fumarate as the ancillary pharmacophore. This prodrug potently inhibits NFκB activity without innate cytotoxicity. In addition, GTCpFE exhibited selective anti-CSC activity by reducing mammosphere growth and the CD44+CD24−immunophenotype. Moreover, GTCpFE pre-treated cells were less tumorigenic and, when tumors did form, latency was increased and growth rate was reduced. Structure-activity relationships for GTCpFE indicate that fumarate, within the context of an ASA prodrug, is essential for anti-NFκB activity, whereas both the ASA and fumarate moieties contributed to attenuated mammosphere growth.ConclusionsThese results establish GTCpFE as a prototype for novel ASA-and fumarate-based anti-inflammatory drugs that: (i) are capable of targeting CSCs, and (ii) may be developed as chemopreventive or therapeutic agents in breast cancer.