Propan-1,3-diol (PD) and propan-1,2-diol (propylene glycol, PG) are very similar compounds because their structures, safety data, and anti-microbial activities are almost the same. Actually, both compounds are made up of three carbon atoms and two hydroxyl groups. Regarding their safety, they do not have serious hazard data for animals, and LD50 values (in rats) of both are similar. As for the anti-microbial activity, minimum inhibitory concentration (MIC) values of both PD and PG are approximately 10% (v/v). In this study, we used the preservatives-effectiveness test (PET) to evaluate the anti-microbial activities of PD and PG, because both compounds are used in cosmetics as preservatives. The results indicated that PD was more effective as an anti-microbial agent compared with PG, and the effect of PD was marked against Escherichia coli and Pseudomonas aeruginosa. Scanning electron microscopy (SEM) images showed that the membrane of Escherichia coli was injured by PD and PG, but the damage by PD was more marked. The damage of the cell membrane may be the cause of high anti-microbial activity of PD in PET. These results suggest that PD has greater potential as a preservative, and PD should be recommended as an additive for food and medicine.

Benzalkonium chloride (BAC) is a useful preservative for ophthalmic solutions but has some disadvantageous effects on corneal epithelium, especially keratinocytes. Therefore, patients requiring the chronic administration of ophthalmic solutions may suffer from damage due to BAC, and ophthalmic solutions with a new preservative instead of BAC are desired. To resolve the above situation, we focused on 1,3-didecyl-2-methyl imidazolium chloride (DiMI). As a preservative for ophthalmic solutions, we evaluated the physical and chemical properties (absorption to a sterile filter, solubility, heat stress stability, and light/UV stress stability), and also the anti-microbial activity. The results indicated that DiMI was soluble enough to prepare ophthalmic solutions, and was stable under severe heat and light/UV conditions. In addition, the anti-microbial effect of DiMI as a preservative was considered to be stronger than BAC. Moreover, our in vitro toxicity tests suggested that DiMI is safer to humans than BAC. Considering the test results, DiMI may be an excellent candidate for a new preservative to replace BAC. If we can overcome manufacturing process issues (soluble time and flushing volume) and the insufficiency of toxicological information, DiMI may be widely adopted as a safe preservative, and immediately contribute to the increased well-being of all patients.

The burkholdines are a family of cyclic lipopeptides reported to exhibit antifungal activity. We synthesized a series of 18 burkholdine analogues in good yield by conventional Fmoc-SPPS followed by cyclization with DIPCI/HOBt in the solution phase. Although none of the synthesized peptides exhibited antifungal activity, several did potentiate the antibiotic effect of the antibiotic G418, including the Thr-bearing Bk analogue (4b) and the tartaramide-bearing Bk analogue (5b). This work exemplifies the potential of burkholdine analogues as potentiating agents.

The synthesis of xylose-bearing cyclic octalipopeptides burkholdine-1213 analogues is described. Sugar-containing lipopeptides are generally highly amphiphilic and often exhibit potent antifungal activities, but a synthesis of a xylose-bearing burkholdine analogue has never been reported. We prepared burkholdine-1213 analogues by solution-phase macrolactamization of xylose-bearing linear peptides obtained by incorporating the bespoke Fmoc-Ser(β-O-xyloseAc3)-OH building block into a Fmoc-solid phase peptide synthesis protocol. All analogues synthesized exhibited similar properties and moderate antifungal activities.

Helicobacter pylori lacks the genes involved in the de novo synthesis of thiamin, and is therefore a thiamin auxotroph. The PnuT transporter, a member of the Pnu transporter family, mediates the uptake of thiamin across the membrane. In the genome of H. pylori, the pnuT gene is clustered with the thiamin pyrophosphokinase gene thi80. In this study, we found that [H-3]thiamin is incorporated into the H. pylori SS1 strain via facilitated diffusion with a K-m value of 28 mu M. The incorporation of radioactive thiamin was inhibited to some extent by 2-methyl-4-amino-5-hydroxymethylpyrimidine or pyrithiamine, but was largely unaffected by thiamin phosphate or thiamin pyrophosphate. RT-PCR analysis demonstrated that the pnuT and thi80 genes are cotranscribed as a single transcript. The estimated Km value for thiamin in the thiamin pyrophosphokinase activity exerted by the recombinant Thi80 protein was 0.40 mu M, which is much lower than the K-m value of thiamin transport in H. pylori cells. These findings suggested that the incorporated thiamin from the environment is efficiently trapped by pyrophosphorylation to make the transport directional. In addition, the thiamin transport activity in the pnuT-deficient H. pylori strain was less than 20% of that in the wild-type strain at extracellular thiamin concentration of 1 mu M, but the incorporated scintillation signals of the pnuT-deficient strain with 100 nM [H-3] thiamin were nearly at the background level. We also found that the pnuT-deficient strain required 100-times more thiamin to achieve growth equal to that of the wild-type. These findings reflect the presence of multiple routes for entry of thiamin into H. pylori, and PnuT is likely responsible for the highaffinity thiamin transport and serves as a target for antimicrobial agents against H. pylori.

The plant alkaloids, iso-6-spectaline and spectaline, isolated from the Cassia or Senna genera contain a characteristic 2,6-disubstituted piperidin-3-ol scaffold. Although both natural products are reported to exhibit a variety of interesting biological activities, few stereo-selective schemes for the construction of the 2,6-disubstituted scaffold have been reported. Following our previous studies regarding the synthesis of (+)-spectaline, herein we report the first convergent synthesis of (-)-iso-6-spectaline using a cross-metathesis under thermal conditions where the cis-2,6-disubstituted piperidin-3-ol scaffold is condensed with a long alkyl chain containing a terminal olefin. The cis-2,6-disubstituted piperidin-3-ol used in the synthesis was prepared simply via Pd(II)-catalyzed diastereoselective cyclization. It was confirmed that (+)-spectaline, an epimer of (-)-iso-6-spectaline, was selectively synthesized by the cross-metathesis reaction under less intense thermal conditions starting from the same cis-2,6-disubstituted piperidin-3-ol derivative.

Thiamin pyrophosphate is an essential cofactor in all living systems. In its biosynthesis, the thiamin structure is initially formed as thiamin phosphate from a thiazole and a pyrimidine moiety, and then thiamin pyrophosphate is synthesized from thiamin phosphate. Many eubacterial cells directly synthesize thiamin pyrophosphate by the phosphorylation of thiamin phosphate by thiamin phosphate kinase (ThiL), whereas this final step occurs in two stages in eukaryotic cells and some eubacterial cells: hydrolysis of thiamin phosphate to free thiamin and its pyrophosphorylation by thiamin pyrophosphokinase. In addition, some eubacteria have thiamin kinase, a salvage enzyme that converts the incorporated thiamin from the environment to thiamin phosphate. This final step in thiamin biosynthesis has never been experimentally investigated in archaea, although the putative thiL genes are found in their genome database. In this study, we observed thiamin phosphate kinase activity in the soluble fraction of the hyperthermophilic archaeon Pyrobaculum calidifontis. On the other hand, neither thiamin pyrophosphokinase nor thiamin kinase activity was detected, suggesting that in this archaeon the phosphorylation of thiamin phosphate is only way to synthesize thiamin pyrophosphate and it cannot use exogenous thiamin for the salvage synthesis of thiamin pyrophosphate. We also investigated the kinetic properties of thiamin phosphate kinase activity using the recombinant ThiL protein from P. calidifontis. Furthermore, the results obtained by site-directed mutagenesis suggest that the Ser196 of ThiL protein plays a pivotal role in the catalytic process.

Cyclic and linear lipopeptides, burkholdine analogues, were synthesized by conventional Fmoc-SPPS and cyclisation with DIPC/HOBt in the solution phase. Synthesized peptides were evaluated for antifungal activities with MIC values against Saccharomyces cerevisiae and Aspergillus oryzae. As a result, the stereochemistry of the amino acid residues and sequences of burkholdine analogues exerted a significant influence on antifungal activities. In addition, we found a linear burkholdine analogue with moderate antifungal activities.

Thiamine pyrophosphokinase (TPK) produces thiamine pyrophosphate, a cofactor for a number of enzymes, including pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase. Episodic encephalopathy type thiamine metabolism dysfunction (OMIM 614458) due to TPK1 mutations is a recently described rare disorder. The mechanism of the disease, its phenotype and treatment are not entirely clear. We present two patients with novel homozygous TPK1 mutations (Patient 1 with p.Ser160Leu and Patient 2 with p.Asp222His). Unlike the previously described phenotype, Patient 2 presented with a Leigh syndrome like non-episodic early-onset global developmental delay, thus extending the phenotypic spectrum of the disorder. We, therefore, propose that TPK deficiency may be a better name for the condition. The two cases help to further refine the neuroradiological features of TPK deficiency and show that MRI changes can be either fleeting or progressive and can affect either white or gray matter. We also show that in some cases lactic acidosis can be absent and 2-ketoglutaric aciduria may be the only biochemical marker. Furthermore, we have established the assays for TPK enzyme activity measurement and thiamine pyrophosphate quantification in frozen muscle and blood. These tests will help to diagnose or confirm the diagnosis of TPK deficiency in a clinical setting. Early thiamine supplementation prevented encephalopathic episodes and improved developmental progression of Patient 1, emphasizing the importance of early diagnosis and treatment of TPK deficiency. We present evidence suggesting that thiamine supplementation may rescue TPK enzyme activity. Lastly, in silico protein structural analysis shows that the p.Ser160Leu mutation is predicted to interfere with TPK dimerization, which may be a novel mechanism for the disease.

Studies on thiamin biosynthesis have so far been achieved in eubacteria, yeast and plants, in which the thiamin structure is formed as thiamin phosphate from a thiazole and a pyrimidine moiety. This condensation reaction is catalyzed by thiamin phosphate synthase, which is encoded by the thiE gene or its orthologs. On the other hand, most archaea do not seem to have the thiE gene, but instead their thiD gene, coding for a 2-methyl-4-amino-5-hydroxymethylpyrimidine (HMP) kinase/HMP phosphate kinase, possesses an additional C-terminal domain designated thiN. These two proteins, ThiE and ThiN, do not share sequence similarity. In this study, using recombinant protein from the hyperthermophile archaea Pyrobaculum calidifontis, we demonstrated that the ThiN protein is an analog of the ThiE protein, catalyzing the formation of thiamin phosphate with the release of inorganic pyrophosphate from HMP pyrophosphate and 4-methyl-5-β-hydroxyethylthiazole phosphate (HET-P). In addition, we found that the ThiN protein can liberate an inorganic pyrophosphate from HMP pyrophosphate in the absence of HET-P. A structure model of the enzyme–product complex of P. calidifontis ThiN domain was proposed on the basis of the known three-dimensional structure of the ortholog of Pyrococcus furiosus. The significance of Arg320 and His341 residues for thiN-coded thiamin phosphate synthase activity was confirmed by site-directed mutagenesis. This is the first report of the experimental analysis of an archaeal thiamin synthesis enzyme.

The effects of additional substituents covering the prime-site of retro-inverso (RI)-modified HTLV-1 protease inhibitors containing a hydroxyethylamine isoster were clarified. Stereo-selective construction of the most potent isoster backbone was achieved by the Evans-aldol reaction. Addition of N-acetylated d-amino acid corresponding to the P2′ site gave an RI-modified inhibitor showing superior inhibitory activity to the previous inhibitor. Inhibitory activities of the newly synthesized inhibitors suggest that partially modified RI inhibitors would interact with HTLV-1 protease in the same manner as the parent hydroxyethylamine inhibitor.

A practical synthesis of the title compound (III) is achieved using regioselective Sharpless asymmetric dihydroxylation as a key step.

Synthesis and antifungal activity of cyclic octapeptide derivatives of burkholdines are described. To construct cyclic octapeptides, the combination of Fmoc-SPPS and cyclization with DIC/HOBt in the solution phase was employed. Synthesized peptides were evaluated for antifungal activity with MIC values against Saccharomyces cerevisiae, Aspergillus oryzae, and Candida viswanathii. As a result, the lipid side chain and the stereochemistry of each amino acid of Bk-1097 analogues significantly affected antifungal activity.

A practical synthesis of 10'R-acetoxy-11'-hydroxyumbelliprenin was achieved using regioselective Sharpless asymmetric dihydroxylation as a key reaction, in which the regioselectivity was achieved by using (DHQD)(2)AQN as a ligand instead of the conventional (DHQD)(2)PHAL. 10'R-Acetoxy-11'-hydroxyumbelliprenin was synthesized by the coupling of a coumarin unit (umbelliferone) and sesquiterpene unit in 11% overall yield through 5 steps.

We have investigated practical synthetic routes for the preparation of peptide aldehyde on a solid support. Peptide aldehyde was synthesized via efficient transformation of acetal/thioacetal structures.

In Saccharomyces cerevisiae, genes involved in thiamin pyrophosphate (TPP) synthesis (THI genes) and the pyruvate decarboxylase structural gene PDC5 are transcriptionally induced in response to thiamin starvation. Three positive regulatory factors (Thi2p, Thi3p, and Pdc2p) are involved in the expression of THI genes, whereas only Pdc2p is required for the expression of PDC5. Thi2p and Pdc2p serve as transcriptional activators and each factor can interact with Thi3p. The target consensus DNA sequence of Thi2p has been deduced. When TPP is not bound to Thi3p, the interactions between the regulatory factors are increased and THI gene expression is upregulated. In this study, we demonstrated that Pdc2p interacts with the upstream region of THI genes and PDC5. The association of Pdc2p or Thi2p with THI gene promoters was enhanced by thiamin starvation, suggesting that Pdc2p and Thi2p assist each other in their recruitment to the THI promoters via interaction with Thi3p. It is highly likely that, under thiamin-deprived conditions, a ternary Thi2p/Thi3p/Pdc2p complex is formed and transactivates THI genes in yeast cells. On the other hand, the association of Pdc2p with PDC5 was unaffected by thiamin. We also identified a DNA element in the upstream region of PDC5, which can bind to Pdc2p and is required for the expression of PDC5.