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-TrpOEt, 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:
O
O
O
O
O
O
O
N P N
Cl
O
N P N
Cl
Bop Cl
O
O
O
O
O
N P N
Cl
1
2
1.Phosgene /
Dimethyl carbonate
2. BuLi POCl3
HO
1.Phosgene /
Dimethyl carbonate
2. BuLi POCl3
NH2
O
HO
O
O
O
O
O
N P N
Cl
3
O
1.Phosgene /
Dimethyl carbonate
2.PCl5 H2O
NH2
OH
NH2
Amino propanol
Mandelonitrile
Norephedrine
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.
2.
3.
William J. Colucci and Daniel H. Rich J. Org. Chem. 1990,55, 2895.
Roger D. Tung and Daniel H. Rich J. Am. Chem. SOC. 1985, 107, 4343.
Roger D. Tung, and Daniel H. Rich. J. Org. Chem.1986, 17, 3351
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 nelectron 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 carboncarbon 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 bioavailable, 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
trp-lys-ala-leu-lys
trp-lys-thr-leu
ala-leu-trp-lys-thr-leu-leu-lys-lys-val-leu-lys-ala
lys-thr-lys trp
leu-trp-lys-thr
ala-leu-trp-lys-thr-leu-leu-lys-lys-val-leu-lys-ala-ala-ala-lys
Peptide
Rm1
Rm2
Rm3
Rm4
Rm5
Rm6
The results pointed out on two promising tetrapeptide hits Rm 4 and Rm 5 possessing the
inhibiting activity against Escherichia coli. :
Rm-6
Rm-5
Rm-4
Rm-3
Rm-2
Rm-1
NOT
ACTIVE
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.
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 DNAcomplexed 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
N
Fe O
O
N
R1
O
NH
O
R
HN
Reference:
1. Arnoux, P.; Haser, R.; Izadi, N.; Lecroisey, A.; Delepierre, M.; Wandersman, C.;
Czjzek, M. Nature Structural Biology 1999, 6, 516-520.