Synthesis of modified coupling reagents based on Bop Cl for low level racemization. David Malka*#, Gary Gellerman*, Yitzhak Mastai# and Shimon Shatzmiler*

*Department of Biological Chemistry, Judea & Samaria College, Ariel, Israel; # Department of Chemistry, Bar-Ilan University, Ramat Gan, Israel

The wide range of coupling reagents for peptide chemistry with the large disparity of functions played by each coupling reagent 1 made it strongly necessary to synthesize a super reagent that will couple any amino acid with high yield and minimum racemization. The most attractive candidate based on Bop Cl 2,3 which known as a rough reagent for the preparation of amides and esters showed high yield and high optical purity when used as a coupling agent in N-methylated amino acids in solution. Our preliminary results showed 1-2% of racemization when we used Bop Cl for coupling Fmoc-Phe to H-Trp-OEt, while the same system with DCC in the presence of HOBT showed 10% racemization. These promising results encourage us to try Bop Cl in our further studies in SPPS. Using standard rink amide resin and Fmoc chemistry, Bop Cl was compared with various coupling reagents for the preparation of the model dipeptide Phe-Phe. The results showed racemization at different values as describe below: HBPyU- 0-1% TCTU - 2-3% PyBrop-10% Bop Cl-10% These results emphasize the need for optical modification on Bop Cl to get lower levels of racemization: Both candidates 1 and 2 display one or two chiral centers that can be used for preparing an optical active reagent in order to examine the mechanism of loosing the chiral center in the coupling of two amino acids. Another aspect is testing the bulkiness of the groups or the optical purity of the reagent. The efficiency of the Bop Cl and its optical modifications on preserving the optical center is currently investigated on more problematic amino acids like His and Cys. References:

1. William J. Colucci and Daniel H. Rich J. Org. Chem. 1990,55, 2895. 2. Roger D. Tung and Daniel H. Rich J. Am. Chem. SOC. 1985, 107, 4343. 3. Roger D. Tung, and Daniel H. Rich. J. Org. Chem.1986, 17, 3351

N P N

O

Cl

OO

O O

N P N

O

Cl

OO

O O

NH2HO

Mandelonitrile

1.Phosgene / Dimethyl carbonate

2. BuLi POCl3

N P N

O

Cl

OO

O O

NH2HO

Norephedrine

1.Phosgene / Dimethyl carbonate

2. BuLi POCl3

N P N

O

Cl

1.Phosgene / Dimethyl carbonate2.PCl5 H2O

OO

O O

OH NH2

Amino propanol

Bop Cl

12

3

A Study of the Alkylation Reactions of Anions Derived from Oxime Ethers. Gaisin Vladimir*#, Hoz Shmaryahu#, Gellerman Gary* and Shatzmiller Shimon*

*Department of Biological Chemistry, Judea & Samaria College, Ariel, Israel; # Department of Chemistry, Bar-Ilan University, Ramat Gan, Israel

The α-carbon of the oxime ether function might support a negative charge by n-electron delocalization from the oxime group. These reactive species could be synthetically equivalent to the known α-acyl intermediates, but as compared with them oxime ethers have two principal advantages:

a) The geometry of the oxime function allows control of the region of activation on the α-carbons either alone or through the choice of kinetic or thermodynamic control;

b) Polar and chelation effects from the electronegative N and O atoms may directly affect the nature of reactive intermediates, leading to novel characteristics for the C=O analogues with respect to new bond formation reactions.

So far the oxime ethers were studied in 3 levels:

a) The regioselectivity as well as the stereoselectivity were examined in methylation and deuteration reactions of open chain oxime ethers on the α-carbons1,2.

b) Cyclic oxime ethers as compounds with latent functionality3,4. c) Synthetic uses of oxime ethers5.

Oxime-ethers can be efficiently metaleted at the α-carbon with n-BuLi to give α lithiated oxime-ethers. The resulting anions can then participate in a variety of useful carbon-carbon bond forming reactions. In oxime ethers derived from acyclic ketones, C=N-O-R geometry controls the site of activation. In the present research we are studying the kinetics of the reactions with oxime ethers in the following aspects:

1) The rate of lithiation reaction of the oxime-ethers was explored by quenching the reaction at fixed time intervals by adding D2O and then testing the incorporation rate of the deuterium atoms into the starting materials.

2) The reactions were held under temperature -53°C, and the rate constant of deprotonation for this temperature was determined.

3) Resulting anions have undergone the alkylation with different electrophiles. The products of reactions were isolated and identified by spectroscopic methods.

The influence of other temperatures on the kinetics is under investigation. References:

1. Spencer, T.A.; Leong, S. Tetrahedron Lett., 1975, 3889. 2. Karabatsos, G.J.; Hsi, H. Tetrahedron, 1967, 23, 1079. 3. Gypax, P.; Das Gupta, T.; Eshenmoser, A., Helv. Chim. Acta, 1972, 55,

2205. 4. Hardegger, B.; Shatzmiller, S., Helv. Chim. Acta, 1976, 59, 2765. 5. Lidor, R. Synthetic Uses of Oxime Ethers; Thesis submitted for the degree

"Doctor of Philosophy". Tel-Aviv, 1984.

Synthesis of new anti bacterial agents based on the natural peptide Dermaseptin S4. Rony Malka*#, David Malka*#, Gary Gellerman*, Yitzhak Mastai# and Shimon Shatzmiller*

*Department of Biological Chemistry, Judea & Samaria College, Ariel, Israel; # Department of Chemistry, Bar-Ilan University, Ramat Gan, Israel

Dermaseptin is a family of 28-36 amino acid peptides extracted from the skin of a native frog Phyllomedusa sauvagii which grows at South America and showed activity against various microbes. The shortest active sequence so far derived from the native Dermaseptin contains 16 amino acids: ALWKTLLKKVLKA. Mor et al 1,2 has investigated the mechanism of the action suggesting that positive charge created by multiple Lysine residues interact with the microbe membrane and able to punch it. Apparently, this long peptide has low chances to be bio-available, so the need of revealing shorter active analogs is clear. Our work focuses on synthesizing small peptide segments that mimic the native peptide activity against Staphylococcus aureus and Escherichia coli. The synthesized compounds are subsequently submitted to the preliminary screening on Petri plates using standard screening protocol. The active sequences are discovered by using Epitope Mapping and Positional Scanning techniques. The resulting active peptide sequences are as follows:

Amino acid sequence Peptide trp-lys-ala-leu-lys Rm1

trp-lys-thr-leu Rm2 ala-leu-trp-lys-thr-leu-leu-lys-lys-val-leu-lys-ala Rm3

lys-thr-lys trp Rm4 leu-trp-lys-thr Rm5

ala-leu-trp-lys-thr-leu-leu-lys-lys-val-leu-lys-ala-ala-ala-lys Rm6 The results pointed out on two promising tetrapeptide hits Rm 4 and Rm 5 possessing the inhibiting activity against Escherichia coli. :

Currently we are working on optimizing Rm 4 and Rm 5 as well as on developing fast and reliable biological screening in 96 well plate format.

References:

1. S. Navon, R. Feder , A. Mor, Antimicrobial Agents and Chemotherapy, 2002.46.689. 2. I. Kustanovich, D. Shalev, A.Mor .The Journal of Biological Chemistry. 2002.277.16941.

Rm-6 Rm-5 Rm-4 Rm-3 Rm-2 Rm-1

NOT

ACTIVE

Structural Analysis of a Covalent Intermediate of the DNA-repair Enzyme T4-DenV with AP site-containing DNA

Gali Golan1, Dmitry O. Zharkov2, Arthur P. Grollman3, M. L. Dodson4,

Amanda K. McCullough4,5, R. Stephen Lloyd4,5 and Gil Shoham1 1 Department of Inorganic Chemistry and the Laboratory for Structural Chemistry and Biology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel; 2 Institute of Chemical Biology and Fundamental Medicine, Siberian Division of the Russian Academy of Sciences, Novosibirsk 630090, Russia; 3 Laboratory of Chemical Biology, Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY 11794, USA; 4 Sealy Center for Molecular Science and Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555-1071, USA; 5 Center for Research on Occupational and Environmental Toxicology, Oregon Health and Science University, Portland, Oregon 97239, USA; Endonuclease-V from bacteriophage-T4 (T4-DenV, T4-PDG) is one of best characterized DNA glycosylases. It initiates the base excision repair pathway, mainly against abnormal pyrimidine dimers that are produced within duplex DNA by UV irradiation. In addition to the damaged base removal it was suggested that this bi-functional DNA-glycosylase/AP-lyase enzyme further processes the resulting empty (AP) site after the release of the target base through a β-elimination reaction. The crystal structure of T4-DenV in its free and DNA-complexed form were previously determined, representing two steps along the catalytic reaction, one before binding of DNA and the other after DNA binding and immediately before base removal. This enzyme is therefore an excellent DNA-repair system to study for both structural and mechanistic aspects, including the characterization of additional steps of its catalytic reaction. In the current poster we present the crystal structure of T4-DenV covalently trapped with its damaged DNA target (containing an AP-site). This structure allows us to elucidate the mode of action of T4-DenV towards an AP-site containing DNA and clarify the β-elimination step that follows the release of the damaged base. This covalent structure also serves as the first direct evidence for Thr2 being the critical catalytic residue, acting as a nucleophile in the initial part of the catalytic reaction. Several amino acids whose function was unclear before are now suggested to have specific roles in DNA binding and catalysis. Among these are Arg22, Arg26 and several residues involved in the binding of the flipped-out adenine opposite the damaged base. Combining this structure together with previously reported structural and biochemical data, we propose a detailed step-by-step mechanism for the catalytic reaction of T4-DenV. In this mechanistic scheme, part of which is relevant also to other AP endonucleases, a specific catalytic role is suggested to the previously unassigned residues Tyr21, Arg26, Glu23, Arg3, Arg117, as well as several structural water molecules.

Biomimetic Metallo-Porphyrin Receptors

Miry Shoshan, Galina Melman and Abraham Shanzer

Department of Organic Chemistry,The Weizmann Institute of Science, 76100 Rehovot, Israel

Iron is an essential trace element required practically by all living organisms. Pathogenic bacteria developed ingenious means to extract iron from their host. Recently, the first hemophore (heme-carrier), called HasA, has been isolated and characterized by X-ray diffraction studies1. The hemophore excreted by bacteria is capable of cannibalizing hemoglobin by removing its iron-heme core and transferring it to the bacterial cytoplasm through a specific outer membrane receptor (HasR). Despite the available X-ray structures and mutant studies, the way in which HasA functions is not well understood, and many questions regarding its mechanism remain unresolved. Novel design of monofunctional and bifunctional ligating systems mimicking the heme binding sites of the natural HasA will be presented. The methodology is based on an auxiliary structure that engulfs the iron-heme and bind to it via 5th and 6th coordination sites. A fused phenoloxazoline moiety is utilized as an analog for the natural tyrosine-histidine couple. The thermodinamics and kinetics of the synthesized mono-functional and bi-functional mimics toward Fe(III)(tetraphenylporphyrin) binding will be discussed.

HN

NO

R

O

O

NH

O

R1

HN

N Fe

Reference: 1. Arnoux, P.; Haser, R.; Izadi, N.; Lecroisey, A.; Delepierre, M.; Wandersman, C.; Czjzek, M. Nature Structural Biology 1999, 6, 516-520.