Determination of vitamin K1 isomers in foods by liquid chromatography with C30 bonded-phase column

Vitamin K1 was determined in a variety of foods by using reversed-phase liquid chromatography with a C30 column followed by post-column reduction to the fluorescent hydroquinone derivatives. Lipids were removed by lipase digestion, followed by single extraction into hydrocarbon, and the protocol was extended to selected natural and processed foods. Biologically active trans- and inactive ci-vitamin K1 isomers were measured individually to evaluate the true nutritional status of the products. Method performance parameters confirmed the validity of the technique. The use of the triacontyl-bonded C30 phase for selective phylloquinone isomer measurement extends previously validated AOAC Method 999.15 for vitamin K1 in milk and infant formula to a wider range of foods important in the human diet. The cis-vitamin K1 isomer contributes up to about 15% of total phylloquinone in certain foods.     

An analysis of the effect of crosslinking on the Tg of polyester and other networks

The dynamic mechanical T_g of styrene-poly(diethylene succinate fumarate) networks has been quantitatively related to the crosslink density. The crosslinking constants, derived from this analysis, were compared with those obtained from other network systems. It is concluded that the crosslinking constant of Di Marzio is not a universal constant; however, a related parameter was found to be very nearly material independent.     

N-Directed fluorination of unactivated Csp3–H bonds

Site-selective fluorination of aliphatic C–H bonds remains synthetically challenging. While directed C–H fluorination represents the most promising approach, the limited work conducted to date has enabled just a few functional groups as the arbiters of direction. Leveraging insights gained from both computations and experimentation, we enabled the use of the ubiquitous amine functional group as a handle for the directed C–H fluorination of Csp³–H bonds. By converting primary amines to adamantoyl-based fluoroamides, site-selective C–H fluorination proceeds under the influence of a simple iron catalyst in 20 minutes. Computational studies revealed a unique reaction coordinate for the catalytic process and offer an explanation for the high site selectivity.

By converting primary amines to adamantoyl-based fluoroamides, site-selective C–H fluorination proceeds under the influence of a simple iron catalyst in 20 minutes.

Due to the pervasiveness of fluorine atoms in industrially relevant small molecules, all practicing organic chemists appreciate the importance of this element. As a result of its unusual size and electronegativity, fluorine imparts unique physicochemical properties to pendant organic molecules.¹ For example, the strong C–F bond can prevent biological oxidation pathways, thereby thwarting rapid clearance and potentially improving pharmacokinetics of molecules.² Moreover, the installation of fluorine or trifluoromethyl groups, with their strong inductive effects,² can have a profound effect on the pK_a of nearby hydrogen atoms.³ These attributes, among others, have solidified the importance of fluorinated molecules in the medicinal,^1–4 material,⁵ and agrochemical⁶ industries. Yet, the same unique properties that make fluorine atoms attractive chemical modifiers also make their installation difficult. Consequently, new methods for site-selective fluorine incorporation remain highly desirable.⁷Methods to construct Csp²–F bonds traditionally make use of the Balz–Schiemann fluorodediazonization⁸ and halogen exchange (“Halex” process).⁹ Advances in transition metal-mediated fluorination have broadened access to Csp²–F-containing molecules,¹⁰ but methods to access aliphatic fluorides remain limited. Conventional methods to make Csp³–F bonds—such as nucleophilic displacement of alkyl halides¹¹ and deoxyfluorination¹²—can have limited functional group compatibility and unwanted side reactions. A more efficient route to form aliphatic C–F bonds would target the direct fluorination of Csp³–H bonds (Scheme 1).¹³Open in a separate windowScheme 1(a) Previous work on functional-group directed Csp³–H fluorination; (b) our approach to N-directed fluorination.Recent efforts with palladium catalysis employ conventional C–H-metallation strategies to target Csp³–H bonds for fluorination.¹⁴ Alternatively, radical H-atom abstraction can remove the transition metal from the C–H-cleavage step, thereby offering a promising approach for Csp³–H-bond functionalization.¹⁵ With undirected C–H fluorination,¹⁶ however, selectivity remains a challenge in molecules without strength-differentiated Csp³–H bonds.¹⁷ To overcome this, our group pioneered the directed fluorination of benzylic Csp³–H bonds through an iron-catalyzed process that involves 1,5 hydrogen-atom transfer (HAT) to cleave the desired Csp³–H bond.¹⁸ Since this work, other groups have demonstrated directed Csp³–H fluorination based on radical propagation that proceeds through an interrupted Hofmann–Löffler–Freytag (HLF)¹⁹ reaction (Scheme 1a). These examples employ various radical precursors such as enones,²⁰ ketones,²¹ hydroperoxides,²² and carboxamides²³ to direct fluorination to specific Csp³–H bonds. Since amines are ubiquitous in natural products and drugs, we sought to use amines as the building block of our directing group to achieve fluorination of unactivated Csp³–H bonds (Scheme 1b). By using amines as the starting point, one could use the approach in straightforward synthetic planning for the late-stage functionalization of remote C–H bonds.In the design phase of the project, we needed to devise a synthetically tractable N–F system that would enable 1,5-HAT and allow for fluorine transfer (Scheme 1b). To begin, we decided to examine common amine activating groups that would support 1,5-HAT while avoiding undesired radical reactions. The chosen activating group would provide the ideal steric and electronic properties to enable both N–F synthesis and N–F scission for 1,5-HAT. We first examined common acyl groups (e.g., acetyl-, benzoyl, and tosyl-based amides), but these proved unsatisfactory. For example, fluoroamide synthesis was either not achieved or low yielding, and the desired fluorine transfer proceeded with significant side reactions or returned starting material. We then turned our attention to more sterically hindered amides—which allow for higher yielding fluoroamide synthesis. For fluorine transfer, we hypothesized that the increased steric bulk could slow intermolecular H-atom transfer, thereby leading more efficient intramolecular 1,5-HAT. To that end, we were delighted that pivaloyl-based fluoroamide 1a proceeded in 64% yield to form product 2a (Scheme 2a). Interestingly, 7% of 1a underwent fluorination at the tert-butyl group of the pivaloyl—presumably through a 1,4-HAT reaction (2aa, Scheme 2a).²⁴ The problem is further exacerbated when the pivaloyl group is homologated by one methylene—providing only 7% yield of desired 2b with 32% of the fluorination taking place on the iso-pentyl group (2bb, Scheme 2a). In an attempt to “tie back” the pivaloyl group and prevent the undesired fluorination, we employed a cyclopropylmethyl-based fluoroamide but observed no improvement.Open in a separate windowScheme 2(a) The targeted 1,5-fluorination of unactivated aliphatic C–H bonds results in partial fluorination of the amine activating group; (b) DFT studies (uM06/cc-pVTZ(-f)-LACV3P**//uM06/LACVP** level of theory) identified the competing pathways responsible for alternate fluorination; (c) DFT (uM06/cc-pVTZ(-f)-LACV3P**//uM06/LACVP** level of theory) evaluation of adamantoylamides revealed higher transition state energy for 1,4-HAT due to restricted vibrational scissoring (d) adamantoyl-activated octylamine shows no fluorination of the activating group. ^{a 1}H-NMR yield using 1,3,5-trimethoxybenzene as an internal standard. ^{b 19}F-NMR yield using 4-fluorotoluene as an internal standard.At this point, 1a proved most promising for efficient fluorine transfer, as well as being the most synthetically accessible fluoroamide. The increased steric hindrance minimizes N-sulfonylation during fluorination with NFSI, a problem that plagued the synthesis of our previously targeted fluoroamides.¹⁸ Therefore, to further investigate how to improve fluorine transfer from 1a, we decided to model H-abstraction computationally.We hypothesized that the fluorinated side product 2aa was formed after 1,4-HAT. Since 1,4-HAT is rare,²⁴ we employed DFT (see ESI^† for details) to calculate the 5-membered and 6-memebered transition-states for 1,4- and 1,5-HAT, respectively. Surprisingly, we found that the barrier for 1,4 C–H abstraction in 1a was 18.7 kcal mol⁻¹, which was only 2.6 kcal mol⁻¹ higher in energy than the barrier calculated for 1,5 C–H abstraction in the same system (Scheme 2b). This suggested that both processes were competing at room temperature. We attributed the comparable barriers to the flexibility of the tert-butyl group, which undergoes vibrational scissoring to accommodate the C–H abstraction. The transition state distortion is modest and allows the molecule to maintain bond angles close to the ideal 109.5° (Scheme 2b). Based on this insight, we sought to limit the scissoring of the tert-butyl group and prevent the 1,4-HAT that leads to the undesired side product. After investigating several possible candidates, the underutilized adamantoyl group appeared promising. To evaluate the rigidity of adamantane, we calculated the barriers for 1,4- and 1,5-HAT for the adamantoyl-capped octylamine 1c (Scheme 2c). As expected, the barriers for 1,4- and 1,5-HAT differed significantly—with 1,4 C–H abstraction proceeding with a barrier of 25.1 kcal mol⁻¹ and the 1,5-HAT barely changed at 16.4 kcal mol⁻¹—an 8.7 kcal mol⁻¹ difference. Consequently, we synthesized 1c and subjected it to the reaction conditions. Excitingly, the adamantoyl-capped system produced desired product 2c in 75% yield with no fluorination of the adamantyl group (Scheme 2d).Using the newly devised adamantoyl-based fluoroamides, the reaction conditions were optimized. While a range of metal salts, ligands, and radical initiators were evaluated, Fe(OTf)₂ proved unique in catalyzing fluorine transfer with fluoroamides.¹⁸ Catalyst loading of 10 mol% allowed convenient setup and minor deviations above or below this loading had little effect on yield (see ESI^†). Increasing the temperature to 40 °C produced a slight increase in yield (entry 2, Table 1). Likewise, raising the temperature to 80 °C resulted in full conversion of the starting material in 20 minutes with 81% yield of the desired product (entry 3, Table 1). It should be noted that fluorine transfer occurs efficiently at a variety of temperatures with adjustments in reaction time (see ESI^†). Increasing the reaction concentration or changing the solvent resulted in decreased yield (entries 4 and 5, Table 1). Furthermore, the absence of Fe(OTf)₂ leads to no reaction and quantitative recovery of starting material, attesting to the stability of fluoroamides and the effectiveness of Fe(OTf)₂ (entry 6, Table 1).Optimization of pertinent reaction parameters

Direct demonstration of the monomerization of thymine-containing dimers in U.V.-irradiated DNA by yeast photoreactivating enzyme and light

Abstract— Ultraviolet-irradiated E. coli DNA (³H-thymine-labelled) was mixed with un-irradiated E. coli DNA (¹⁴C-thymine-labelled) and exposed to light in the presence of purified yeast photoreactivating enzyme. As the ³H-thymine-containing cyclobutane dimers disappeared during the photoreactivation, there was a stoichiometric increase of monomeric ³H-thymine as determined from the ³H/¹⁴C ratio in thymine. This is the first direct demonstration that thymine-containing dimers in u.v.-irradiated DNA are monomerized by yeast photoreactivating enzyme in the presence of light.     

ULTRAVIOLET-INDUCED APOCHLOROSIS AND PHOTOREACTIVATION IN TWO STRAINS OF EUGLENA GRACILIS*

Abstract— Very low doses of ultraviolet irradiation result in a complete loss of the ability to synthesize chlorophyll in two closely related strains of Euglena gracilis (var. hacillaris and strain Z). Both strains are equally sensitive in this response under non-photoreactivating conditions. The ability to synthesize chlorophyll is completely photoreactivated by visible light in E. grncilis var. bacillaris , even after lethal doses of u.v.: the Z strain. however, while photoreactivable following low doses of u.v., remains bleached after doses adequate to kill only 10–20 per cent of the cells.     

An optimum minimal basis of exponential atomic orbitals

An analysis of some of the energetic properties of the conventional minimal STO basis is used to suggest a new optimum set of exponential functions for use in molecular calculations.     

A structural, spectroscopic and theoretical study of the triphenylphosphine chalcogenide complexes of tungsten carbonyl, [W(XPPh3)(CO)5], X=O, S, Se

The series [W(XPPh₃)(CO)₅], X=O, S, Se has been structurally determined by X-ray crystallography and fully characterised spectroscopically to provide data for comparing the bonding of the Ph₃PX ligands to the metal. The P-X-W angles are 134.3°, 113.2° and 109.2°, respectively, for X=O, S, Se. The bonding has been analysed using EHMO calculations which suggest that lower P-X-W angles depend on the relative importance of σ-bonding, which in turn depends on the chalcogen in the order X=Se > S > O. The effect is enhanced by lower energies of the metal σ and π orbital energies.     

Porphyrazinediols: Synthesis, Characterization, and Complexation to Group IVB Metallocenes

The first metalated porphyrazinediols 11 have been prepared from (L)-(+)-dimethyl tartrate via conversion into the corresponding dispoke or 2,3-dimethoxy-2,3-butanediyl protected 2,3-dihydroxymaleonitrile, Linstead macrocyclization, transmetalation, and deprotection. Their stability is very dependent on the nature of the metal ion in the cavity of the porphyrazine. Reaction of these porphyrazinediols with metallocene dichlorides led to new solitaire porphyrazines 12 while DDQ oxidation followed by trapping with diaminomaleonitrile afforded new porphyrazine dinitriles 14.     

Design of active-site-directed fluorescent probes and their reactions with biopolymers

Fluorescent probes which are active-site-directed, reversible, competitive inhibitors of serum cholinesterase (ChE) enzymes have been designed and synthesized. Reversible inhibitors of enzyme active sites have a unique importance when they act as fluorescent probes, allowing fluorescence spectroscopic detection of conformation changes and activesite dynamics. 5-Dimethylamino-naphthalene-1-sulfonamido-N,N-dimethyl-n-propyl-amine and its aliphatic quaternary derivative are fluorescent probes for serum cholinesterase. The quaternary probe forms complexes with acetylcholinesterase (AChE). The dissociation constants K_d for the two probes with serum ChE are 6.0 × 10^?7 and 6.5 × 10^?7M. The inhibition constants K_i are 3.1 × 10^?6 and 6.3 × 10^?6M from the slopes of Lineweaver-Burk plots. The Michelis constant K_m for the enzyme was 8.8 × 10^?4M.     

Multicenter validation of PCR-based method for detection of Salmonella in chicken and pig samples

As part of a standardization project, an interlaboratory trial including 15 laboratories from 13 European countries was conducted to evaluate the performance of a noproprietary polymerase chain reaction (PCR)-based method for the detection of Salmonella on artificially contaminated chicken rinse and pig swab samples. The 3 levels were 1-10, 10-100, and 100-1000 colony-forming units (CFU)/100 mL. Sample preparations, including inoculation and pre-enrichment in buffered peptone water (BPW), were performed centrally in a German laboratory; the pre-PCR sample preparation (by a resin-based method) and PCR assay (gel electrophoresis detection) were performed by the receiving laboratories. Aliquots of BPW enrichment cultures were sent to the participants, who analyzed them using a thermal lysis procedure followed by a validated Salmonella-specific PCR assay. The results were reported as negative or positive. Outlier results caused, for example, by gross departures from the experimental protocol, were omitted from the analysis. For both the chicken rinse and the pig swab samples, the diagnostic sensitivity was 100%, with 100% accordance (repeatability) and concordance (reproducibility). The diagnostic specificity was 80.1% (with 85.7% accordance and 67.5% concordance) for chicken rinse, and 91.7% (with 100% accordance and 83.3% concordance) for pig swab. Thus, the interlaboratory variation due to personnel, reagents, thermal cyclers, etc., did not affect the performance of the method, which will be proposed as part of a developing international PCR standard.