Astatine-211 labeling of protein using TCP as a bi-functional linker: synthesis and preliminary evaluation in vivo and in vitro

With 2,3,5,6-tetrafluorophenyl 3-(nodo-carboranyl) propionate (TCP) as a new potential bi-functional linker, bovine serum albumin (BSA) was conjugated with ²¹¹At, and the conjugated model protein (²¹¹At-TCP-BSA) was preliminarily evaluated in vitro and in vivo by comparison with ²¹¹At-SAB-BSA and ²¹¹At-SAPC-BSA, which conjugated with ²¹¹At via aryl derivatives ATE (N-succinimidyl-3-(tri-n-butylstannyl) benzoate) or SPC (N-succinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate). The radiolabeled intermediate ²¹¹At-TCP was coupled to BSA in yields ranging from 35 to 45% with radiochemical purity of more than 98%. The conjugated ²¹¹At-TCP-BSA exhibited considerable stability in vitro in 0.1 mol/L PBS (pH 7.6) at room temperature (RT), similar to ²¹¹At-SAPC-BSA and ²¹¹At-SAB-BSA. Biodistribution of the ²¹¹At conjugated protein was investigated in NIH strain mice by I.V injection. The results showed that ²¹¹At-TCP-BSA was constantly stable in vivo as well as in vitro, but more stable than ²¹¹At-SAPC-BSA and ²¹¹At-SAB-BSA. These results implied that radioastatinated carboranes based on B–At bonds are higher stability than radioastatinated aryl derivatives based on C–At to in vivo deastatination. In other word, TCP should be a promising bi-functional linker for ²¹¹At conjugation of proteins or antibodies.     

The preparation and cytotoxic properties of211at labelled concanavalin a bound to cell membranes

A method of labelling biologically active proteins with the alpha emmitting halogen²¹¹At is presented. The labelling procedure is discussed with reference to the chemistry of astatine. Proteins which have been labelled retain approximately 50% of their original biological activity. Using cell specific labelled proteins, dose response curves are given indicating that such reagents are extremely cytotoxic, D₃₇ human CML cells=5 atmos²¹¹At/cell. The research potential of²¹¹At labelled biologically active proteins is briefly discussed.     

Synthesis and stability of sulfur-35 labelled 4-ethylbicyclothiophosphate

Different synthetic routes to the preparation of 4-ethyl-2,6,7-trioxa-1-phosphabicyclo[2,2,2]octane-1-thioxide labelled with sulfur-35 have been investigated since it is of considerable interest as radioligand for neurochemistry. The thiophosphate was successfully synthesized by sulfuration of the corresponding bicyclophosphite by elemental sulfur-35, however the radiochemical purity of the material obtained in this way proves to be low. Convenient methods for synthesis of the thioxide from [³⁵S] thiourea, based on the results of the autoradiolysis of the starting [³⁵S] thiourea, has been discovered giving rise to thioxide with high radiochemical purity. The autoradiolytic stability of the labelled thiophosphate has been studied.M. V. Lomonosov Moscow State University, Moscow 119899, Russia Translated from Khimiya Geterotsiklicheskikh Soedinenii, No 8, pp. 1107–1111, August, 1999.     

Synthesis of isotopically labelled SGLT inhibitors and their metabolites

Isotopically labelled analogues of two structurally very similar SGLT inhibitors AVE2268 (1a) and AVE8887 (1b) have been synthesized by various routes. The radioactive labelled [¹⁴C]-AVE2268 was prepared in five steps including a Friedel-Crafts acylation as the key step for the ¹⁴C-label introduction. For [¹⁴C]-AVE8887 the same synthetic approach was not successful and therefore an alternative thiophene metallation/Weinreb amide sequence was developed. This pathway was also applied to obtain stable isotopically labelled analogues of both AVE2268 and AVE8887. Finally, the synthesis of two metabolites, sulfate 12 and glucuronide 13 were achieved by applying interesting protecting group and oxidation strategies.     

Preparation of several new polypeptides and proteins labelled with125I

Six¹²⁵I labelled polypeptides and proteins were prepared with high specific activity and good quality.The optimal iodination with Iodogen method has been studied and different Radio-HPLC methods for purification have been compared. In addition, the method of coating tube with Iodogen has been improved.     

Recombination proteins and telomere stability in plants

Repair of DNA damage is essential for the maintenance of the integrity and transmission of the genome in development and reproduction. Telomeres are nucleoprotein structures which protect the ends of (linear) eukaryotic chromosomes. Telomere dysfunction results in loss of this protection and the telomeres being recognised as DNA damage by the cellular DNA Damage Repair and Response (DDR) machinery, leading to senescence or cell death. Telomeric homeostasis is thus tightly controlled and many specific and non-specific proteins are involved in its regulation. Among these, DNA damage and Repair proteins contribute both to the recognition of telomere dysfunction and more surprisingly, are directly implicated in telomere homeostasis itself. Plants offer a great opportunity to study these mechanisms due to the fact that many key DNA repair and recombination proteins are non-essential in plants, in contrast to vertebrates. In the following text, after a brief summary of the current state of knowledge on telomere-specific proteins in plants, we review the DDR processes and the related proteins implicated in plant telomere stability. We focus specifically on telomere signalling and on recombination events induced by unprotected telomeres, at the origin of genome rearrangements and instability when telomere function is affected.     

211At标记蛋白质或多肽的方法研究

α核素211At具有良好的辐射生物学性质,以对肿瘤细胞具有高亲和性的单克隆抗体或多肽类配体为载体则是实现211At肿瘤靶向治疗的最理想方式之一。本文介绍了211At标记蛋白质或多肽的方法的现状与进展,对存在的一些问题及今后的发展方向进行了讨论。     

A stability switch for proteins

A paper published in the September 8 issue of Cell describes a generally applicable approach for chemical control of protein stability, with potential for broad use in chemical genetics.     

Incorporation of trifluoroisoleucine into proteins in vivo

Two fluorinated derivatives of isoleucine: d,l-2-amino-3-trifluoromethyl pentanoic acid (3TFI, 2) and d,l-2-amino-5,5,5-trifluoro-3-methyl pentanoic acid (5TFI, 3) were prepared. 5TFI was incorporated into a model target protein, murine dihydrofolate reductase (mDHFR), in an isoleucine auxotrophic Escherichia coli host strain suspended in 5TFI-supplemented minimal medium depleted of isoleucine. Incorporation of 5TFI was confirmed by tryptic peptide analysis and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) of the protein product. Amino acid analysis showed that more than 93% of the encoded isoleucine residues were replaced by 5TFI. Measurement of the rate of activation of 5TFI by the E. coli isoleucyl-tRNA synthetase (IleRS) yielded a specificity constant (k(cat)/K(m)) 134-fold lower than that for isoleucine. 5TFI was successfully introduced into the cytokine murine interleukin-2 (mIL-2) at the encoded isoleucine positions. The concentration of fluorinated protein that elicits 50% of the maximal proliferative response is 3.87 ng/mL, about 30% higher than that of wild-type mIL-2 (EC(50) = 2.70 ng/mL). The maximal responses are equivalent for the fluorinated and wild-type cytokines, indicating that fluorinated proteins can fold into stable and functional structures. 3TFI yielded no evidence for in vivo incorporation into recombinant proteins, and no evidence for activation by IleRS in vitro.     

A simple method for labelling proteins with211At via diazotized aromatic diamine

A simple and rapid method for labelling proteins with²¹¹At by means of a 1,4-diaminobenzene link is described. 1,4-diaminobenzene is transformed into the diazonium salt and subsequently reactions of both²¹¹At and proteins with the diazonium salt take place simultaneously. For possibly high yields of astatized protein an appropriate temperature of 273 K was found. The results demonstrate the difference between the reaction mechanisms of iodine and astatine with proteins.     

One-step labelling of a novel small-molecule peptide with astatine-211: preliminary evaluation in vitro and in vivo

In this paper, VP2, a novel small molecule fusion peptide, was labelled with ²¹¹At through a one-step method (coupled with bifunctional intermediate SPC first and then labelled) with a radiochemical yield of about 45%. The radiochemical purity was still > 95% after 24 h at room temperature. Specificity studies in vitro indicated that ²¹¹At-SPC-VP2 has a high affinity for several tumour cells. Additionally, biodistribution in KM mice showed that ²¹¹At-SPC-VP2 has sufficient stability in vivo. This research suggested that ²¹¹At–SPC-VP2 produced by the convenient method has the potential to be a novel targeted drug for cancer radiotherapy.     

Plant proteins and their colloidal state

Plant proteins are characterized by a complex colloidal state in their physiological environment. The main reasons are related to the multiple functions of plant proteins as well as the different architectures encountered in the plant cells from various sources. During extraction process to produce ingredients, plant proteins reorganize in several native or denatured colloidal states depending on the energy and the physico–chemical changes applied to the system. In most cases, an equilibrium between the native (soluble monomers or oligomers) and denatured (mostly insoluble) oligomeric/aggregated states is reached. Further, processing of the plant protein ingredients during food production, introducing new hydrophobic phases (e.g., gas, oil), energy (pressure, temperature, shear), and physico–chemical conditions (pH, ionic salts) will lead to a final colloidal state, specific to the structural features of the considered final food product.     

Photooxidation of polyolefins and their light stability

It is shown that all peculiarities of polypropylene photooxidation and alteration of its mechanical properties in this process are a process of second-order termination reaction. The influence of natural conditions (varied temperature and light intensity) on this process are considered.     

Investigation of the stability of labelled nanoparticles for SE(R)RS measurements in cells

We explore the long-term stability of two different classes of labelled nanoparticles as intracellular SE(R)RS probes. Whilst chemisorbed labels gave stable responses inside cells for extended periods of time, signals from physisorbed labels could only be measured for short periods of time. These results help inform strategies for cellular imaging using vibrational spectroscopies.     

A class of human proteins that deliver functional proteins into mammalian cells in vitro and in vivo

We discovered a class of naturally occurring human proteins with unusually high net positive charge that can potently deliver proteins in functional form into mammalian cells both in?vitro and also in murine retina, pancreas, and white adipose tissues in?vivo. These findings represent diverse macromolecule delivery agents for in?vivo applications, and also raise the possibility that some of these human proteins may penetrate cells as part of their native biological functions.     

Structure and stability of closo-hexaboranes and their heteroanalogs

The molecular and electronic structures of closo-hexaboranes B₆H₆ ^2–, B₆H₇ ^–, and B₆H₈ and closo-heterohexaboranes XYB₄H₄ (X = Y = CH, N; X = BH, Y = CH^–, N^–, NH, O) were studed by the ab initio (MP2(full)/6-311+G**) and density functional (B3LYP/6-311+G**) methods. The bridging H atoms in closo-hexaboranes B₆H₇ ^– and B₆H₈ can undergo facile low-barrier migrations around the boron cage (the barrier heights are about 10—15 kcal mol^–1). All heteroboranes having octahedron-like structures with hypercoordinated N and O atoms are rather stable and can be the subject of synthetic research efforts.     

Solution stability and variability in a simple model of globular proteins

It is well known among molecular biologists that proteins with a common ancestor and that perform the same function in similar organisms, can have rather different amino-acid sequences. Mutations have altered the amino-acid sequences without affecting the function. A simple model of a protein in which the interactions are encoded by sequences of bits is introduced, and used to study how mutations can change these bits, and hence the interactions, while maintaining the stability of the protein solution. This stability is a simple minimal requirement on our model proteins which mimics part of the requirement on a real protein to be functional. The properties of our model protein, such as its second virial coefficient, are found to vary significantly from one model protein to another. It is suggested that this may also be the case for real proteins in vivo.