Stereoselective synthesis of Gly-Gly-type (E)-methylalkene and (Z)-chloroalkene dipeptide isosteres and their application to 14-mer RGG peptidomimetics

Stereoselective and efficient synthesis of Gly-Gly-type (E)-methylalkene and (Z)-chloroalkene dipeptide isosteres is realized by organocuprate-mediated single electron transfer reduction. The synthetic isosteres can be used in Fmoc-based solid phase peptide synthesis, resulting in the preparation of the 14-mer RGG peptidomimetics containing an (E)-methylalkene or a (Z)-chloroalkene unit.

An efficient synthesis of Gly-Gly-type (E)-methylalkene and (Z)-chloroalkene dipeptide isosteres is realized by organocuprate-mediated single electron transfer reduction.

Glycylglycine (Gly-Gly) is the smallest dipeptide and has been synthesized by many approaches in the last 100 years.¹ Due to its versatile nature and ready availability, glycylglycine has been used as a chemical probe² and buffer³ in biochemical studies and also as a reagent which can enhance the solubility of overexpressed proteins.⁴ In addition to the utility of Gly-Gly itself, oligoglycine (oligo-Gly) motifs are notable for being more flexible and less-functionalized than any combination of other amino acids. These features are useful in bioconjugation strategies which link multiple biomolecules without interfering with the function of each biomolecule, allowing synthesis of bioconjugated artificial molecules including dimeric, multi-domain, and fusion proteins.⁵ The flexibility of oligo-Gly enables the formation of unusual secondary structures of peptides and proteins.⁶ Thus, the oligo-Gly motif can be found in the biologically important peptides and proteins such as Met/Leu-enkephalin ( and ), the C-terminus of ubiquitin , ctenidin,⁷ shepherin I,⁸ and DNA/RNA-binding proteins with repeated sequences related to the various physiological processes via protein–protein and protein–nucleic acids interactions.⁹ These proteins relate with gene expression, DNA damage signal and apoptosis, however, the detail effects of Gly-Gly with steric and electronic factors to these functions are unknown. Given the importance of oligo-Gly in various fields, non-hydrolyzable peptidomimetics of oligo-Gly could be attractive building blocks for the synthesis of novel bioconjugated molecules and complex peptidomimetics with improved chemical stability and functionality. For example, even the Gly-Gly dipeptide mimic with the tetra-substituted alkene unit replacing the Gly-Gly peptide bond has been shown to promote the β-hairpin formation, and is thus the smallest peptidomimetic that is known to control a peptide structure.¹⁰ There are two reports of the synthesis of Gly-Gly-type fluoroalkene dipeptide isosteres.¹¹ However, the poor synthetic access to such molecules has hindered their application to the peptidomimetics. Our long-standing interest in the drug discovery with amide-to-alkene isosteric switching prompted this investigation into the stereoselective synthesis of Gly-Gly-type (E)-methylalkene and (Z)-chloroalkene dipeptide isosteres.In this report, we describe the beginning of our oligo-Gly-based peptidomimetic study with the stereoselective synthesis of Gly-Gly-type (E)-methylalkene and (Z)-chloroalkene dipeptide isosteres and their use in Fmoc-based solid phase peptide synthesis (SPPS). In the first application of isosteres of this type to access complex peptidomimetics, we synthesize 14-mer RGG peptidomimetics containing (E)-methylalkene or (Z)-chloroalkene unit as surrogates for a Gly-Gly peptide bond. These two isosteres were selected because the potentials of both isosteres have not been fully uncovered, though there are several promising examples.^12,13 Since the carbonyl oxygen equivalents of those isosteres are similar in their size¹⁴ but differ in their electronic properties,¹⁵ comparative studies of these isosteres would have the advantage of exploring the role of peptide bonds in terms of their steric and electronic natures. This work also uncovered the unique ability of the Gly-Gly-type (Z)-chloroalkene isostere to induce β-turn structures in the almost unfoled peptides (Fig. 1).Open in a separate windowFig. 1Gly-Gly peptide and its alkene-type peptidomimetics.The main challenges in this study include the stereoselective formation of the (E)-methylalkene and (Z)-chloroalkene moieties as the surrogates of the Gly-Gly trans-peptide bond, together with the control of the olefine isomerization under the condition of the isostere synthesis and Fmoc-based SPPS. There are several synthetic approaches toward tri-substituted alkene-type isosteres, including the Overman rearrangement,¹³ the S_N2′-type opening of alkenylaziridines,^12a Cu-mediated CF₃-coupling of vinyl iodide,¹⁶ and cross-couplings of vinyl stannane.¹⁷ Organocuprate-mediated reactions of α,β-unsaturated carbonyl compounds with γ-leaving group(s) are also powerful methods to produce tri-substituted alkene-type isosteres, and are particularly suitable for the stereoselective synthesis of (Z)-fluoroalkene¹⁸ and (Z)-chloroalkene isosteres.¹⁹The preparation of Gly-Gly-type alkene dipeptide isosteres was facilitated by the organocuprate-mediated SET reduction that was used in the stereoselective formation of the (E)-methylalkene and (Z)-chloroalkene moieties. Scheme 1 shows the synthesis of (E)-methylalkene isosteres (5). A Witting reaction of methacrolein (1) with ethyl (triphenylphosphoranylidene)acetate followed by epoxidation with m-CPBA afforded the alkenyl oxirane (2) which, treated with Gillman reagent (n-Bu₂CuLi), produced the allylic alcohol (3) with high E-selectivity. A Mitsunobu reaction of 3 with Ns(Boc)NH and deprotection provided the Gly-Gly-type (E)-methylalkene dipeptide isostere 5 that can be applied to the Fmoc-based SPPS. All reactions leading to the synthesis of 5 were performed on a gram scale.Open in a separate windowScheme 1Synthesis of Gly-Gly-type (E)-methylalkene dipeptide isosteres (5).Synthesis of chloroalkene isostere (14) is shown in Scheme 2. Based on previous results for the successful, efficient synthesis of Val-Xaa-type chloroalkene isosteres,^19c we assumed that a similar protocol would allow for the synthesis of 14. However, our attempts revealed that the Gly substrate used has different reactivity and selectivity compared to the other substrates, particularly in the SET reduction step. Consequently, Gly-specific reaction conditions are necessary. The nucleophilic addition of the lithium enolate of methyl dichloroacetate to the N-sulfinylaldimine (7), prepared from (±)-tert-butylsulfinamide (6) and paraformaldehyde and m-CPBA oxidation of 6 gave the N-tert-butylsulfonyl (Bus)-protected α,α-dichloro-β-amino ester (8). Precise control of the amount of DIBAL-H at low temperatures enables the partial reduction of 8, and this is followed by a Horner–Wadsworth–Emmons reaction to produce the corresponding (E)-enoate (9). Initial efforts to apply our established conditions for SET reduction with Me₂CuLi identified the poor Z-selectivity of the reaction and also its propensity to form the α-methylated side products 11 and 12 (Open in a separate windowScheme 2Synthesis of Gly-Gly-type (Z)-chloroalkene dipeptide isosteres (14).Reactivity of (E)-enoate (9) with organocuprates^a

Electrochemical sensory detection of Sus scrofa mtDNA for food adulteration using hybrid ferrocenylnaphthalene diimide intercalator as a hybridization indicator

In this study, an electrochemical DNA biosensor was developed based on the fabrication of silicon nanowires/platinum nanoparticles (SiNWs/PtNPs) on a screen-printed carbon electrode (SPCE) for the detection of Sus scrofa mitochondrial DNA (mtDNA) in food utilizing a new hybrid indicator, ferrocenylnaphthalene diimide (FND). The morphology and elemental composition of the SiNWs/PtNPs-modified SPCE was analyzed by field emission scanning electron microscopy (FESEM) combined with energy dispersive X-ray spectroscopy (EDX). Cyclic voltammetry (CV) was used to study the electrical contact between the PtNPs and the screen-printed working electrode through SiNWs, while electrochemical impedance spectroscopy (EIS) was used to measure the charge transfer resistance of the modified electrode. The results clearly showed that the SiNWs/PtNPs were successfully coated onto the electrode and the effective surface area for the SiNWs/PtNPs-modified SPCE was increased 16.8 times as compared with that of the bare SPCE. Differential pulse voltammetry used for the detection of porcine DNA with FND as an intercalator confirmed its specific binding to the double-stranded DNA (dsDNA) sequences. The developed biosensor showed a selective response towards complementary target DNA and was able to distinguish non-complementary and mismatched DNA oligonucleotides. The SiNWs/PtNPs-modified SPCE that was fortified with DNA hybridization demonstrated good linearity in the range of 3 × 10⁻⁹ M to 3 × 10⁻⁵ M (R² = 0.96) with a detection limit of 2.4 × 10⁻⁹ M. A cross-reactivity study against various types of meat and processed food showed good reliability for porcine samples.

An electrochemical DNA biosensor was developed based on the fabrication of silicon nanowires/platinum nanoparticles on a screen-printed carbon electrode for the detection of Sus scrofa mitochondrial DNA in food.     

Laparoscopy-assisted repair with Ventralex ST Hernia Patch for a small subcostal hernia: A case report

A 55-year-old woman with a history of right hepatic lobectomy via a Benz incision presented for evaluation of a new abdominal bulge in the right upper quadrant. We diagnosed an incisional hernia, but because we could neither reduce the hernia contents nor locate the orifice, we performed a laparoscopic evaluation. Laparoscopy revealed subcostal herniation of the greater omentum via a 2-cm defect on the caudal side of the right ribs, which we repaired using a Ventralex ST Hernia Patch. Laparoscopic placement of this mesh with straps allowed for reliable deployment, fixation, and confirmation of defect closure, including the cranial aspect—often a major challenge in subcostal hernia repair.     

Efficacy,safety, and pharmacokinetics of oral valganciclovir in patients with congenital cytomegalovirus infection

ObjectivesValganciclovir (VGCV) has been shown to improve sensorineural hearing loss (SNHL) and neurological outcomes in patients with neonatal symptomatic congenital cytomegalovirus (cCMV) infection. However, reports on the pharmacokinetics, efficacy and safety of oral VGCV are limited. The aim of this study is to evaluate the pharmacokinetics of VGCV for use in the treatment of cCMV.MethodsThis was a single-center, retrospective observational study conducted at Saitama Children's Medical Center in Japan between 2012 and 2017. CMV DNA copy number, maximum plasma VGCV concentration (Cmax), and adverse events (ADEs) during treatment were evaluated.ResultsA total of 26 patients with cCMV who received VGCV were included in this study. The median age at VGCV initiation was 9.5 months (range 0–46). Twenty-one patients (81%) had SNHL at baseline. Of these, five patients (19%) presented with improved SNHL, and none experienced worsened SNHL during treatment. The mean VGCV Cmax was 3.5 μg/mL (range 2–5.3), with no significant variation among individual values, and the values were maintained during treatment. Furthermore, there were no correlations between the Cmax values and age, sex, SNHL improvement or ADEs. Neutropenia (<1000/mm³) was observed in six patients (23%); however, no serious ADEs occurred.ConclusionsVGCV prevented the progression of SNHL without serious ADEs due to its stable pharmacokinetics. This study provides safety and tolerability of VGCV for the treatment of cCMV patients.     

Change in antimicrobial susceptibility of pathogens isolated from surgical site infections over the past decade in Japanese nation-wide surveillance study

Inappropriate antimicrobial therapy for surgical site infections (SSIs) can lead to poor outcomes and an increased risk of antibiotic resistance. A nationwide survey was conducted in Japan from 2018 to 2019 to investigate the antimicrobial susceptibility of pathogens isolated from SSIs. The data were compared with those obtained in 2010 and 2014–2015 surveillance studies. Although the rate of detection of extended-spectrum β-lactamase producing strains of Escherichia coli was increased from 9.5% in 2010 to 23% in 2014–2015, the incidence decreased to 8.7% in 2018–2019. Although high susceptibility rates were detected to piperacillin/tazobactam (TAZ), the geometric mean MICs were substantially higher than to meropenem (2.67 vs 0.08 μg/mL). By contrast, relatively low geometric mean MICs (0.397 μg/mL) were demonstrated for ceftolozane/TAZ. Although the MRSA incidence rate decreased from 72% in the first surveillance to 53% in the second, no further decrease was detected in 2018–2019. For the Bacteroides fragilis group species, low levels of susceptibility were observed for moxifloxacin (65.3%), cefoxitin (65.3%), and clindamycin (CLDM) (38.9%). In particular, low susceptibility against cefoxitin was demonstrated in non-fragilis Bacteroides, especially B. thetaiotaomicron. By contrast, low susceptibility rates against CLDM were demonstrated in both B. fragilis and non-fragilis Bacteroides species, and a steady decrease in susceptibility throughout was observed (59.3% in 2010, 46.9% in 2014–2015, and 38.9% in 2018–2019). In conclusion, Japanese surveillance data revealed no significant lowering of antibiotic susceptibility over the past decade in organisms commonly associated from SSIs, with the exception of the B. fragilis group.     

Assessing Blood Flow in an Intracranial Stent: A Feasibility Study of MR Angiography Using a Silent Scan after Stent-Assisted Coil Embolization for Anterior Circulation Aneurysms

BACKGROUND AND PURPOSE:Blood flow in an intracranial stent cannot be visualized with 3D time-of-flight MR angiography owing to magnetic susceptibility and radiofrequency shielding. As a novel follow-up tool after stent-assisted coil embolization, we applied MRA by using a Silent Scan algorithm that contains an ultrashort TE combined with an arterial spin-labeling technique (Silent MRA). The purpose of this study was to determine whether Silent MRA could visualize flow in an intracranial stent placed in the anterior circulation.MATERIALS AND METHODS:Nine patients treated with stent-assisted coil embolization for anterior circulation aneurysms underwent MRAs (Silent MRA and TOF MRA) and x-ray digital subtraction angiography. MRAs were performed in the same session on a 3T unit. Two neuroradiologists independently reviewed the MRA images and subjectively scored flow in a stent as 1 (not visible) to 4 (excellent) by referring to the latest x-ray digital subtraction angiography image as a criterion standard.RESULTS:Both observers gave MRA higher scores than TOF MRA for flow in a stent in all cases. The mean score for Silent MRA was 3.44 ± 0.53, and for TOF MRA, it was 1.44 ± 0.46 (P < .001).CONCLUSIONS:Silent MRA was able to visualize flow in an intracranial stent more effectively than TOF MRA. Silent MRA might be useful for follow-up imaging after stent-assisted coil embolization, though these study results may be only preliminary due to some limitations.

Endovascular therapy for intracranial aneurysms has been widely used since the International Subarachnoid Aneurysm Trial.¹ The number of cases of coil embolization for aneurysms is increasing, and the stent-protection technique has widened the applicability to cases that had been otherwise difficult to treat with conventional coil embolization.² Nevertheless, there is a risk of coil compaction or in-stent restenosis after stent-assisted coil embolization. X-ray digital subtraction angiography is the optimal technique used to examine these adverse events, and it is commonly used as a follow-up tool after using an intracranial stent. However, DSA presents some unavoidable risks related to the catheter procedure, radiation, and contrast media.^3–53D time-of-flight MR angiography is widely used for the assessment of cerebral vascular diseases and has also been examined as a noninvasive substitute for DSA.^6–9 These studies generally reported difficulty in visualizing flow in a stent with TOF MRA because of magnetic susceptibility and radiofrequency shielding, though some beneficial aspects were observed in assessing the residual lumen of aneurysms. As a novel follow-up tool after stent-assisted coil embolization, we applied MRA by using a Silent Scan algorithm (GE Healthcare, Milwaukee, Wisconsin) that contains an ultrashort TE combined with an arterial spin-labeling technique (Silent MRA). In this situation, visualizing flow means visualizing arterial geometry and patency. It does not mean directly visualizing blood flow rate. The purpose of this study was to determine whether Silent MRA can visualize flow in an intracranial stent placed at the anterior circulation.