Igor L. Rodionov

Cyclic dipeptides as building blocks for combinatorial libraries. Part 2: Synthesis of bifunctional diketopiperazines

Abstract Twenty-four bifunctional diketopiperazines, cyclo(-Aax-Bbx-) consisting of glutamic or aspartic acid (Aax) and lysine, ornithine or diaminobutyric (Dab) acid (Bbx) were synthesized. d -Dab moiety was incorporated as d -Gln followed by Hoffman-type rearrangement induced by PhI[OC(O)CF3]2. Cyclization conditions for the related linear dipeptide precursors allowing racemization to be kept at a minimum were determined.

The Biological Function of a Fragment of the Neurotrophic Factor from Pigment Epithelium: Structural and Functional Homology with the Differentiation Factor of the HL-60 Cell Line

It was shown that the full-size neurotrophic factor from pigment epithelium (PEDF) induces the cell differentiation of the human promyelocyte leukemia cell line HL-60. A structural analysis of PEDF revealed in itsC-terminal region a six-membered peptide fragment PEDF-(352-357) (PEDF-6) whose sequence is highly homologous to the 41–46 fragment of the active site of the human leukocyte differentiation factor HLDF (HLDF-6). The biological effect of PEDF and synthetic peptides PEDF-6 and HLDF-6 on the HL-60 cells and the early gastrula ectoderm ofXenopus laevis embryos was studied. On the basis of the structural and functional homologies of HLDF, PEDF, and their homologous peptides and the computer models of the spatial structures of the full-size PEDF and the PEDF with theC-terminal fragment split off tby the cleavage of the Leu380-Thr381 bond in the serpin loop, a hypothesis on the functional role of the serpin loop in PEDF was put forward.

Hexapeptides HLDF-6 and PEDF-6 restore memory in rats after chronic intracerebroventricular treatment with β-amyloid peptide Aβ(25–35)

Effects of homologous peptides HLDF-6 and PEDF-6 on behavior of animals with experimental Alzheimer’s disease induced by chronic intracerebroventricular administration of β-amyloid peptide Aβ(25–35) were studied in the zoosocial recognition test and Morris water maze. Peptides HLDF-6 and PEDF-6 possessed neuroprotective activity and counteracted the toxic effect of Aβ(25–35). Peptides HLDF-6 and PEDF-6 mainly improved long-term memory and working memory, respectively.

Different Mechanisms of Protective and Differentiative Activities of Homological Peptides TGENHR and TQVEHR

Previously we identified a six-membered fragment 354TQVEHR359 of the C-terminal part of the PEDF (Pigment Epithelium-Derived Factor) differentiation factor molecule that shares homology with fragment 41TGENHR46 of the HLDF (Human Leukemia Differentiation Factor) differentiation factor molecule, which is responsible for its differentiation activity. HLDF has been isolated from the culture medium of human promyelocytic leukemia cell line HL-60. Hexapeptides HLDF-6 (TGENHR) and PEDF-6 (TQVEHR) corresponding to these HLDF and PEDF molecule fragments, which were previously shown to induce cell differentiation (Kostanyan et al. (2000) Russian Journal of Bioorganic Chemistry, 26, 505-511), also have neuroprotective properties. Both peptides prevent degeneration of Purkinje cells of rat cerebellar vermis upon chemical hypoxia induced by sodium azide in vivo; this effect is also observed on a behavioral level. Peptide HLDF-6 but not PEDF-6 promotes survival of HL-60 cells upon chemical hypoxia. Peptides HLDF-6 and PEDF-6 affect different second messenger biosynthesis systems in HL-60 cells. HLDF-6 diminishes cyclic AMP level in those cells due to adenylate cyclase inhibition, while PEDF-6 inhibits phosphatidylinositol-specific phospholipase C stimulated by aluminum tetrafluoride anions.

CONTINUOUS MONITORING OF THE SWELLING BEHAVIOUR OF GEL-TYPE REACTIVE POLYMERS AND CHROMATOGRAPHIC MEDIA * SOME PRACTICAL APPLICATIONS OF THE TECHNIQUE

Abstract A new technique allowing monitoring of the swelling behaviour of gelatinous polymeric reagents and chromatographic supports is described. It is based up on the application of a continuous flow ‘zero dead volume operating’ reaction system of original design equipped with a potentiometric pickup kinematically attached to the movable piston of the reactor. The latter transduces the polymer bed volume change into an electrical signal that can be conveniently registered on a common laboratory chart recorder. The proposed method provides rapid access to swelling data of the polymer and, moreover, for the first time allows for real-time investigation of swelling dynamics of reactive polymers that are involved in chemical and/or physical process. The practical scope of the technique is illustrated by the results of its application in solid phase peptide synthesis, ion exchange and studies of swelling behaviour of resins and gel-type polymeric reagents.

Comparative study of the neuroprotective and nootropic activities of the carboxylate and amide forms of the HLDF-6 peptide in animal models of Alzheimer’s disease

A comparative study of the neuroprotective and nootropic activities of two pharmaceutical substances, the HLDF-6 peptide (HLDF-6-OH) and its amide form (HLDF-6-NH2), was conducted. The study was performed in male rats using two models of a neurodegenerative disorder. Cognitive deficit in rats was induced by injection of the beta-amyloid fragment 25–35 (βA 25–35) into the giant-cell nucleus basalis of Meynert or by coinjection of βA 25–35 and ibotenic acid into the hippocampus. To evaluate cognitive functions in animals, three tests were used: the novel object recognition test, the conditioned passive avoidance task and the Morris maze. Comparative analysis of the data demonstrated that the neuroprotective activity of HLDF-6-NH2, evaluated by improvement of cognitive functions in animals, surpassed that of the native HLDF-6-OH peptide. The greater cognitive/ behavioral effects can be attributed to improved kinetic properties of the amide form of the peptide, such as the character of biodegradation and the half-life time. The effects of HLDF-6-NH2 are comparable to, or exceed, those of the reference compounds. Importantly, HLDF-6-NH2 exerts its effects at much lower doses than the reference compounds.

AT THE CROSSROADS OF HETEROCYCLIC AND PEPTIDE CHEMISTRIES. THE AMINOACYL INCORPORATION REACTION IN THE SYNTHESIS OF MEDIUM-SIZED RING HETEROCYCLES (REVIEW)

The aminoacyl incorporation reaction of peptides is considered in the context of synthetic heterocyclic chemistry with particular emphasis on the scope, mechanism, and major by-products of the reaction. Medium-sized mono- and bicyclic molecules available via aminoacyl incorporation reaction are unique building blocks with significant promise in biomolecular design.

Epitope mapping of human Fas using peptide phage display

Sandwich EIA for measurement of soluble Fas was developed on the basis of SA-7 and SA-8 monoclonal antibodies to full-length human Fas. The threshold sensitivity of the test system is 0.3 ng/ml. Several isoforms of soluble Fas were identified. The structure of SA-7 and SA-8 antibody epitopes was determined using the peptide phage library. It was shown that SA-7 antibody epitope is determined by amino acid residues 129-134 (CKPNFF), while SA-8 antibody epitope is determined by amino acid residues 94-99 (KAHFSS) of full-length Fas. Hence, sandwich EIA on the basis of SA-7 and SA-8 monoclonal antibodies for detection of soluble Fas in human serum is to detect the following Fas isoforms: FasExo6Del, FasExo4Del, FasExo4,6Del, FasExo4,7Del, and FasExo8Del.

Formation of truncated peptide by‐products via sequence‐specific formyl group transfer from Trp(For) residues to Nα in the course of Boc‐SPPS

(NIn)‐Formyl protective group of tryptophan has been introduced as a base/nucleophile‐labile protective group. It has long been known that a free Nα‐amino group of the peptide can serve as a nucleophile: an irreversible formyl NIn → NH2 transfer is consistently observed when deformylation is performed last on an otherwise deprotected peptide that possesses free Nα‐amino group. Obviously, this particular side reaction should be expected any time free amino group is exposed to Trp(For), but, at the best of our knowledge, has never been reported in the course of Boc‐SPPS. In the present communication, we describe a set of appropriately designed model experiments that permitted to detect the title side reaction both in solution and in solid‐phase reactions. We observed intermolecular formyl group transfer with a model compound, Trp(For)‐NH2. Importantly, we also observed this migration on solid support with the rate roughly estimated to be up to 1% of residues per minute. We also observed that the formyl‐group transfer reaction occurred in a sequence‐dependent manner and was suppressed to a non‐detectable level using ‘in situ neutralization’ technique. Because this side reaction is sequence dependent, there might be situations when the rate of the formation of Nα‐formyl termination by‐products is significant. In other cases, the Nα‐For truncated by‐products would not contaminate the final peptide significantly but still could be a source of microheterogeneity. Copyright

The nuclease activity of the endogenous differentiation factor of the HL-60 cell line

A structural homology between the endogenous differentiation factor of the HL-60 cell line of promyelocyte leukemia (HLDF) and several DNA/RNA-binding and DNA/RNA-hydrolyzing proteins was revealed, and expression of thehldf gene in prokaryotic systems was studied. On the basis of these experiments, the amino acid sequence of an 8-membered fragment of HLDF with potential nuclease activity was identified. The synthetic octapeptide RRWHRLKE was shown to be capable of the cleavage of RNA, linear DNA from phage λ, and all forms of plasmid DNA. We established that treatment of the HL-60 cell culture with this peptide (10−6 M) results in an increase in the number of apoptotic cells and suggested that HLDF is involved in processes of apoptosis.