Synthesis of Peptides Containing a Combination of Free and 2-trans-Cyclooctene Carbamate Protected Lysine Residues.

The allylic trans-cyclooctene (TCO) functionality facilitates powerful control over the spatiotemporal activity of bio-active molecules, enabling precision targeting of druglike and imaging modalities. However, the introduction of this function onto molecules remains chemically challenging, particularly for peptides. Modification with TCOs of this important class of biomolecules remains a challenge, primarily due to the sensitivity of the TCO group to the strong acids typically used in global deprotection during solid phase peptide synthesis. Here, we present a novel synthetic approach to site-selectively introduce TCO-groups in peptides. Our approach utilizes azide groups to mask amine functions, enabling selective introduction of the TCO on a single lysine residue. Staudinger reduction of the azides back to the corresponding amines proceeds without disturbing the sensitive TCO. We show that using our method, we can produce TCO-inactivated antigenic peptides of previously unseen complexity.

Tracking amyloid oligomerization with monomer resolution using a 13-amino acid peptide with a backbone-fixed spin label.

Amyloid oligomers are suspected as toxic agents in neurodegenerative disease, and are transient and often heterogeneous, making them difficult to detect. Here we show an approach to track the development of amyloid oligomers in situ by room temperature, continuous wave (cw) 9 and 95 GHz EPR. Three amyloid peptides with the 2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid (TOAC) spin label were synthesized by solid phase peptide synthesis: T0EZ (TKVKVLGDVIEVGG) with TOAC (T) at the N-terminus, T5EZ with TOAC in the middle (KVKVTGDVIEVG) and T12EZ with TOAC at the C-terminus (KVKVLGDVIEVTG). These sequences are derived from the K11V (KVKVLGDVIEV) amyloid peptide, which self-aggregates to oligomers with a ß-sheet configuration (A. Laganowsky, et al., Science, 2012, 335, 1228-1231). To monitor oligomerization, the rotational correlation time (τr) is measured by cw-EPR. For the backbone-fixed TOAC label that is devoid of local mobility τr should reflect the rotation and thereby the size of the peptide, resp. oligomer. For T5EZ a good match between the measured τr and the size of the peptide is obtained, showing the validity of the approach. One of the three peptides (T0EZ) aggregates (circular dichroism), whereas the other two do not. Since also the respective MTSL (S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate) labelled peptides fail to aggregate, molecular crowding due to the label, rather than the helix-inducing properties of TOAC, seems to be responsible. Following in situ oligomer formation of T0EZ by the change in rotational correlation time, two oligomers are observed, a 5-6 mer and a 15-18 mer. The EPR approach, particularly 95 GHz EPR, enables following oligomerization of one monomer at a time, suggesting that the cw-EPR approach presented is a novel tool to follow amyloid oligomerization with high resolution.

Light fluorous synthesis of glucosylated glycerol teichoic acids.

We here describe the synthesis of glucosylated teichoic acid (TA) fragments using two complementary fluorous scaffolds. The use of a perfluorooctylpropylsulfonylethyl (F-Pse) linker in combination with (glucosyl)glycerol phosphoramidite building blocks allows for the assembly of TA fragments with a terminal phosphate mono-ester, whereas the use of a perfluorooctylsuccinyl spacer delivers TA oligomers featuring a terminal alcohol functionality. These complementary linker systems have been developed because the nature of the TA chain terminus can play a role in the biological activity of the synthetic TAs. A novel α-glucosylated glycerolphosphoramidite building block is introduced to allow for a robust light fluorous synthetic protocol.

[Clinico-statistical analysis of arterial hypertension complicated with hypertensive crisis in Moscow in 2005-2009].

Clinicostatistical analysis of arterial hypertension complicated with hypertensive crisis using data of Moscow A.S.Puchkov Station of Urgent and Emergent Medical Aid revealed 14% rise in number of hypertensive crises during the period from 2005 to 2009. Number of hypertensive crises increased among persons of young age (18-35 years). Frequency of cerebrovascular complications of hypertensive crises was age dependent with maximal values among men aged 36-74 years and women older than 75 years.

[Possibilities of the use of parenteral form of α- and ß-adrenoblocker for the treatment of hypertensive crises at prehospital stage].

Butylaminohydroxypropoxyphenoxymethyl methyloxadiazole was used in prehospital treatment of 40 patients (15 women, 25 men aged 63+/-17.1 years) with complicated hypertensive crises (HC) characterized by acute involvement or high risk of progression of damage of internal organs. The drug was administered as intravenous bolus injection of 10-50 mg (1-5 ml of 1% solution) under control of arterial pressure (AP) and heart rate (HR). The patients noted alleviation of symptoms by 15th min and disappearance of main manifestations of HC by 30th min. Systolic and diastolic AP were significantly lowered from baseline 190.0+/-24.4 and 105.0+/-13.5 mm Hg to 153.3+/-20,1 and 85.0+/-12,6 mm Hg, respectively. Mean dynamics of lowering of systolic and diastolic AP was 19.5 and 19%, respectively. This was accompanied by 15.4% lowering of HR (from 90.3+/-12.0 to 76.4+/-9.4 bpm). Myocardial ischemia in those patients who had it diminished or disappeared. Thus in patients with complicated HC the study drug exerted effective antihypertensive and antiischemic actions. It was well tolerated and did not cause significant side effects.

[First experience with gel plasty of the anal canal in children with fecal incontinence].

Fecal incontinence in children after surgical correction of anorectal defects and trauma of the holding apparatus of the rectum in certain cases can be dependent on low basal pressure in the anal canal at the expense of incompetence of the interior sphincter muscle of the anus. In order to eliminate low pressure in the anal canal we introduced polyacrylamide gel "DAM +" into its submucous layer. The introduction of this volume-forming substance into the anal canal was an effective procedure. Fecal incontinence became less in all cases, and completely regressed in 50% of the patients.

[Evolution of diagnostics and treatment methods of closed isolated spleen injuries in children].

An analysis of treatment of 75 children of the age from 3 years to 17 admitted to the St. Petersburg Pediatric Medical Academy was made. All the children were admitted with the diagnoses of blunt (closed) injuries of the abdomen, ruptures of the spleen, intra-abdominal bleedings. The patients were divided into 3 groups according to the periods of hospitalization. In each group the patients were examined and treated which corresponded to the surgical strategy for rupture of the spleen at that period. Different methods of examinations and therapy were determined in the three groups of patients with similar pathology.

Monitoring Alzheimer Amyloid Peptide Aggregation by EPR.

Plaques containing the aggregated beta-Amyloid (Abeta) peptide in the brain are the main indicators of Alzheimer's disease. Fibrils, the building blocks of plaques, can also be produced in vitro and consist of a regular arrangement of the peptide. The initial steps of fibril formation are not well understood and could involve smaller aggregates (oligomers) of Abeta. Such oligomers have even been implicated as the toxic agents. Here, a method to study oligomers on the time scale of aggregation is suggested. We have labeled the 40 residue Abeta peptide variant containing an N-terminal cysteine (cys-Abeta) with the MTSL [1-oxyl-2,2,5,5-tetramethyl-Delta-pyrroline-3-methyl] methanethiosulfonate spin label (SL-Abeta). Fibril formation in solutions of pure SL-Abeta and of SL-Abeta mixed with Abeta was shown by Congo-red binding and electron microscopy. Continuous-wave 9 GHz electron paramagnetic resonance reveals three fractions of different spin-label mobility: one attributed to monomeric Abeta, one to a multimer (8-15 monomers), and the last one to larger aggregates or fibrils. The approach, in principle, allows detection of oligomers on the time scale of aggregation.

Solid-phase synthesis of polymyxin B1 and analogues via a safety-catch approach.

As part of a program towards the development of novel antibiotics, a convenient method for solid-phase synthesis of the cyclic cationic peptide polymyxin B1 and analogues thereof is described. The methodology, based on cleavage-by-cyclization using Kenner's safety-catch linker, yields crude products with purities ranging from 37-67%. Antibacterial assays revealed that analogues 23-26, in which the (S)-6-methyloctanoic acid moiety is replaced with shorter acyl chains, exhibit distinct antimicrobial activity. The results suggest that the length of the acyl chain is rather critical for antimicrobial activity. On the other hand, substitution of the hydrophobic ring-segment D-Phe-6/Leu-7 in polymyxin B1 with dipeptide mimics (i.e. analogues 27-33) resulted in almost complete loss of antimicrobial activity.

Binding and retention of polycationic peptides and dendrimers in the vascular wall.

Extracellular matrix (ECM) of tissues, vascular tissue in particular, contains a high concentration of negatively charged glycosaminoglycans (GAGs), which are involved in the regulation of cell motility, cell proliferation and the regulation of enzyme activities. Previously, we have shown that the vascular ECM is capable of binding an extremely high concentration of positively charged molecules, such as polylysine. Vascular ECM can be used therefore as a substrate for binding and retention of drugs delivered intravascularly, if these drugs are endowed with an ability to bind to the vascular ECM. In this study, we evaluated a number of positively charged molecules as potential affinity vehicles for delivery of drugs to the vascular ECM. We labelled the molecules of interest with fluorescence and compared them ex vivo in terms of binding and retention in the de-endothelialised rat carotid artery after intravascular delivery under pressure. High molecular weight polylysine (84 kDa) and polyamidoamine (PAMAM) dendrimers accumulated in the wall of the artery up to a concentration of 10 mg/ml and were not washed away significantly after 4 h of perfusion of the artery. A 24-mer peptide containing a consensus sequence for binding to GAGs (ARRRAARA)(3), 2.7 kDa, was comparable to high molecular weight polylysine and dendrimers in terms of binding and retention. A 14-mer GAG-binding peptide from vitronectin and low molecular weight polylysine, 3 kDa, accumulated in the vascular wall up to about 3 mg/ml and was washed away after 30 min of perfusion. A 10-mer consensus GAG-binding peptide did not bind significantly to the vascular tissue. We conclude that the consensus 24-mer GAG-binding peptide is by far superior to polylysine of a similar molecular weight in terms of binding to vascular tissue, and can provide high accumulation and long-term retention of a low molecular weight compound (fluorescein, as a model molecule) in the vascular wall. Rationally designed GAG-binding peptides can be useful as affinity vehicles for targeting drugs to the vascular ECM.

Secondary chemical shifts in immobilized peptides and proteins: a qualitative basis for structure refinement under magic angle spinning.

Resonance assignments recently obtained on immobilized polypeptides and a membrane protein aggregate under Magic Angle Spinning are compared to random coil values in the liquid state. The resulting chemical shift differences (secondary chemical shifts) are evaluated in light of the backbone torsion angle psi previously reported using X-ray crystallography. In all cases, a remarkable correlation is found suggesting that the concept of secondary chemical shifts, well established in the liquid state, can be of similar importance in the context of multiple-labelled polypeptides studied under MAS conditions.

[Synthesis of nucleoside analogs containing a cis-2-pentene fragment and their triphosphates]. / Sintez analogov nukleozidov, soderzhashchikh tsis-2-pentenovyi fragment, i ikh trifosfatov.

Acyclic analogs of thymidine, adenine, and cytosine where the ribose moiety is replaced with cis-5-hydroxypenthene, were obtained via condensation of cis-5-acetoxy-1-bromo-2-pentene with nucleic acid bases. Triphosphate derivatives of the new nucleosides were also synthesized to evaluate their action as terminators of DNA synthesis catalyzed by DNA polymerases.