Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND COMPOSITIONS FOR CONTROLLED POLYPEPTIDE
SYNTHESIS
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in part of non-provisional application
number
09/272,109, filed March 19,1999, which claimed priority under Section 119(e)
to
provisional application number 60/078,649, filed March 19, 1998. This
application
also claims priority under Section 119(e) to provisional application numbers
60/133,304, filed May 10,1999, 60/133,305 also filed May 10, 1999, 60/187,488
filed
March 7, 2000, and 60/193,054 filed March 29, 2000. The contents of the
foregoing
provisional and non-provisional applications are hereby incorporated by
reference.
STATEMENT REGARDING FEDERALLY FUNDED RESEARCH
This invention was made with Government support under Grant Nos. DMR
9632716, CHE 9701969, and 9701969 awarded by the National Science Foundation,
Grant No. N00014-96-0729 awarded by the Office of Naval Research, and Grant
No.
DAAH04-96-1-004 awarded by the Department of Defense. The Government has
certain rights in this invention.
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to the synthesis of amino-acid based polymers.
In particular, this invention relates to methods and compositions for the
synthesis of
amino-acid based polymers using catalysts under "living" conditions, that is
conditions free of termination and chain transfer.
Description of Related Art
Synthetic polypeptides have a number of advantages over peptides produced
in biological systems and have been used to make fundamental contributions to
both
the physical chemistry of macromolecules and the analysis of protein
structures.
See e.g. G.D. Fasman, Poly a-Amino Acids, Dekker, New York, (1967). Moreover,
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synthetic peptides are both more cost efficient and can possess a greater
range of
material properties than peptides produced in biological systems.
Small synthetic peptide sequences, typically less than 100 residues in length,
are conventionally prepared using stepwise solid-phase synthesis. Such solid
phase
synthesis makes use of an insoluble resin support for a growing oligomer. A
sequence of subunits, destined to comprise a desired polymer, are reacted
together
in sequence on the support. A terminal amino acid is attached to the solid
support in
an initial reaction, either directly or through a keying agent. The terminal
residue is
reacted, in sequence, with a series of further residues such as amino acids or
blocked amino acid moieties to yield a growing oligomer attached to the solid
support
through the terminal residue. At each stage in the synthetic scheme, unreacted
reactant materials are washed out or otherwise removed from contact with the
solid
phase. The cycle is continued with a pre-selected sequence of residues until
the
desired polymer has been completely synthesized, but remains attached to the
solid
support. The polymer is then cleaved from the solid support and purified for
use.
The foregoing general synthetic scheme was developed by R.B. Merrifield for
use in
the preparation of certain peptides. See e.g. See Merrifield's Nobel Prize
Lecture
"Solid Phase Synthesis", Science, Volume 232, pp. 341-347 (1986).
A major disadvantage of conventional solid phase synthetic methods for the
preparation of oligomeric materials results from the fact that the reactions
involved in
the scheme are imperfect; no reaction proceeds to 100% completion. As each new
subunit is added to the growing oligomeric chain a small, but measurable,
proportion
of the desired reaction fails to take place. The result of this is a series of
peptides,
nucleotides, or other oligomers having deletions in their sequence. The result
of the
foregoing imperfection in the synthetic scheme is that as desired chain length
increases, the effective yield of desired product decreases drastically, since
increased chances for deletion occur. Similar considerations attend other
types of
unwanted reactions, such as those resulting from imperfect blocking, side
reactions,
and the like. Of equal, if not greater, significance, is the fact that the
increasing
numbers of undesired polymeric species which result from the failed individual
reactions produce grave difficulties in purification. For example, if a
polypeptide is
desired having 100 amino acid residues, there may be as many as 99 separate
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peptides having one deleted amino acid residue and an even greater possible
number of undesired polymers having two or more deleted residues, side
reaction
products and the like.
Due to the above-mentioned problems associated with solid phase
methodologies, practitioners employ other protocols for peptide synthesis. For
example, synthetic copolymers of narrow molecular weight distribution,
controlled
molecular weight, and with block and star architectures can be prepared using
so
called living polymerization techniques. See e.g. O. Webster, Science, 251:887-
893
(1991). In these polymerizations, chains grow linearly by consecutive addition
of
monomers, and chain-breaking transfer and termination reactions are absent.
The
active end-groups of growing polymer chains do not deactivate (i.e. they
remain
"living") and chains continue to grow as long as monomer is present. Chain
length in
living polymerizations is controlled through adjustment of monomer to
initiator
stoichiometry. Under circumstances when all chains grow at the same rate,
living
polymers will possess a narrow distribution of chain lengths. Complex
sequences,
such as block copolymers, are then built up by stepwise addition of different
monomers to the growing chains. A. Noshay, et al., Block Copolymers, Academic
Press, New York, (1977).
The chemical synthesis of high molecular weight polypeptides is most directly
accomplished by the ring-opening polymerization of a-aminoacid-N-
carboxyanhydride (NCA) monomers (see equation 1 below). See e.g. H.R.
Kricheldorf, in Models of Biopolymers by Ring-Opening Polymerization, Penczek,
S.
Ed., CRC Press, Boca Raton, (1990). In general terms, NCA polymerizations can
be
classified into two categories based on the type of initiator used: either a
nucleophile
(typically a primary amine) or strong base (typically a sodium alkoxide) (see
equation
1 below). Nucleophile initiated polymerizations are believed to propagate
through a
primary amine end-group (see equation 2 below). These polymerizations display
complicated kinetics where an initial slow first order process is followed by
accelerated monomer consumption: indicative of multiple propagating species
with
different reactivities. See e.g. M. Idelson, et al., J. Am. Chem Soc., 80:2387-
2393
(1958). The prevalence of side reactions limit these initiators to the
formation of low
molecular weight polymers (10 kDa < Mn < 50 kDa) which typically contain a
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substantial fraction of molecules with degree of polymerization less than 10.
As
such, the polymers have very broad molecular weight distributions (Mw/Mn = 4 -
10).
See e.g. R.D. Lundberg, et al., J. Am. Chem Soc., 79:3961-3972 (1957).
R
Hue. R
n H~N~O ~N~ + n CO ,
nucleophile , n '
O O or base H O
NCA Polypeptide
t0 ~0 O H O
CO ,
~NH~ N\H ---~ aH~N~OH ~- ~N~NH~
H
R O R
R OP = polymer chain
Strong base initiated NCA polymerizations are much faster than amine
initiated reactions. These polymerizations are poorly understood but are
believed to
propagate through either NCA anion or carbamate reactive species (see
equations 3
and 4 below, respectively). See e.g. C.H. Bamford, et al., Synthetic
Polypeptides,
Academic Press, New York, (1956).
R_
M+ t0~0 R O H M+
O~ N + O NCB O N O 3
N, ~ N~ ~ (
O H attack
R O -~ R O
O
O H M r ~IO ~O carbamate O H _R O
N_ 'p- O (N' attack ~N~ ~ M+ (4
-CO P H p-
R O ~ R O
R
A significant limitation of NCA polymerizations employing conventional
initiators is due to the fact that they are plagued by chain-breaking transfer
and
termination reactions which prevent formation of block copolymers. See e.g.
H.R.
Kricheldorf, a-Aminoacid-N-Carboxyanhydrides and Related Materials, Springer-
Verlag, New York, (1987). Consequently, the mechanisms of NCA polymerization
have been under intensive study so that problematic side reactions could be
eliminated. See e.g. H.R. Kricheldorf, in Models of Biopolymers by Ring-
Opening
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Polymerization, Penczek, S. Ed., CRC Press, Boca Raton, (1990). These
investigations have been severely hindered by the complexity of the
polymerizations,
which can proceed through multiple pathways. Moreover, the high sensitivity of
NCA
polymerizations to reaction conditions and impurities has also led to
contradictory
data in the literature resulting in controversy over the different
hypothetical
mechanisms. H. Sekiguchi, Pure andAppl. Chem., 53:1689-1714 (1981); H.
Sekiguchi, et al., J. Poly. Sci. Symp., 52:157-171 (1975).
The significant problems with existing peptide synthesis methodologies create
a variety of problems for practitioners. For example, the chain breaking
transfer
reactions that occur in the NCA polymerizations preclude the systematic
control of
peptide molecular weight. Moreover, block copolymers cannot be prepared using
such methods.
Block copolymers of amino acids have been less well studied, largely
because our synthetic methods do not yet have fine enough control to produce
well-
defined structures. F. Cardinauz, et al., 8iopolymers, 16:2005-2028 (1977).
The
same is true of the synthesis of block copolypeptides for use as biomaterials
or as
selective membranes - the potential advantages of the protein-like
architectures
have remained unrealized for want of adequate synthetic building blocks and
tools.
For example, biomedical applications, such as drug delivery typically require
water-soluble components to enhance their ability for circulation in vivo. The
problem with common water-soluble polypeptides (e.g., poly-L-lysine and poly-L-
aspartate) is that they are polyelectrolytes that display pH-dependent
solubility and
limited circulation lifetime due to aggregation with oppositely charged
biopolymers.
Nonionic, water-soluble polypeptides are desired for biomedical applications
since
they avoid these problems, and can also display the stable secondary
structures of
proteins that influence biological properties. However, all high molecular
weight
nonionic homopolypeptides (>25 residues) derived from naturally occurring
amino
acids are notoriously insoluble in water.
One approach to producing nonionic water-soluble polypeptides employs
polyethylene glycol (PEG), which is typically grafted onto polypeptides or
other
polymers to improve their properties in vivo. PEG is nonionic, water-soluble,
and
most importantly not recognized by immune systems. It is believed that PEG
imparts
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biocompatibility through formation of a hydrated "steric barrier" at the
surface of
material that cannot be penetrated or recognized by biological molecules, such
as
proteolytic enzymes. As such, block or graft copolymer drug carriers
containing PEG
are able to circulate for long periods in the bloodstream without degradation.
Despite its attractive properties, a drawback to grafting PEG onto
polypeptides is the need for expensive amino- or carboxylato-functionalized
molecules for coupling, which typically must be short (<5,000 Da) to ensure
high
functionalization. Accordingly, there remains considerable interest in
developing
alternative methods for producing nonionic water-soluble polypeptide building
blocks
that also incorporate the attractive properties of biochemical stability, self-
assembly
and water solubility into polypeptides.
Polypeptides are being considered for a variety of biomedical problems such
as tissue engineering and drug delivery. Another consideration for these
applications
is the incorporation of endgroup functionality onto the chains, which is
essential for
targeting of the drug delivery complexes as well as substrate specific
anchoring of
these materials. These, and other features would be useful for controlling
both the
structure and the properties of polypeptide materials. Consequently, there is
a need
for novel methods and compositions which allow for the facile generation of
peptides
tailored to have specific desirable properties.
SUMfVIARY OF THE INVENTION
The present invention discloses novel methods and compositions which
address the need for advanced tools to generate polypeptides having varied
material
properties. The methods and initiator compositions for NCA polymerization
disclosed herein allow the precise control of such polypeptide synthesis. In
particular, the methods of the invention allow successful peptide synthesis by
utilizing the versatile chemistry of transition metals to mediate the addition
of
monomers to the active polymer chain-ends, and therefore eliminate chain-
breaking
side reactions in favor of the chain-growth process. In this way, the
disclosed
methods allow the formation of block copolymers. Moreover, by binding the
active
end-group of the growing polymer to a metal center, its reactivity toward
monomers
can be precisely controlled through variation of the metal and ancillary
ligands bound
to the metal. The wide range of selective chemical transformations and
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polymerizations which are catalyzed by transition metal complexes attests to
the
versatility of this approach.
One embodiment of the invention provides a method of making an amido-
containing metallacycle comprising combining an amount of an a-aminoacid-N-
carboxyanhydride monomer with an initiator molecule comprising a low valent
transition metal-Lewis Base ligand complex so that an amido-containing
metallacycle
is formed.
An alternative embodiment of the invention provides a method of making an
initiator molecule, which includes the step of combining an allyloxycarbonyl
(alloc)
protected amino acid amide and a low valent transition metal-Lewis base ligand
complex so that an amido-amidate metallacycle is formed having the following
general formula:
R1
N
c~) ~'~'\
N
R3
wherein M is a low valent transition metal, L is a Lewis base ligand; one of
R1
and R2 is an amino acid side group and the other is hydrogen; and R3 is any
functional end group capable of being attached to a primary amine group. The
R3
end group will typically be used to "tag" or functionalize the polypeptide
chains, and
is the main advantage associated with using this method. Typically, this group
will
be a peptide, oligosaccharide, oligonucleotide, fluorescent molecule, polymer
chain,
small molecule therapeutic, chemical linker to attach the polypeptide to a
substrate,
chemical linker to act as a sensing moiety, or reactive linker to couple the
polypeptide to larger molecules such as proteins, polysaccharides or
polynucleotides
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Another embodiment of the invention provides compositions consisting of five
or six membered amido-containing metallacycles comprising molecules of the
general formula:
I1
R2 1 2
R3
1 'R R3 I 1 R R3
N N N
(L~ / (L)n O (L~ / (L~
O R _, O O ~ O
~4 R5 R4 R7
wherein M is a low valent transition metal;
L is a Lewis Base ligand;
each of R1, R2, R3, R5 and R6 (independently) is a moiety selected from the
group consisting of the side chains of alanine, arginine, asparagine, aspartic
acid,
cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine,
lysine,
methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or
valine;
R4 is a hydrogen moiety or a polyaminoacid chain; and R7 is a functional end
group.
In preferred embodiments of these compositions, the metal is a transition
metal selected from the group consisting of nickel, palladium, platinum,
cobalt,
rhodium, iridium and iron and the Lewis Base ligand is selected from the group
consisting of pyridyl ligands, diimine ligands, bisoxazoline ligands, alkyl
phosphine
ligands, aryl phosphine ligands, tertiary amine ligands, isocyanide ligands
and
cyanide ligands.
A related embodiment of the invention consists of a method of adding an
aminoacid-N-carboxyanhydride (NCA) to a polyaminoacid chain having an amido
containing metallacycle end group by combining the NCA with the polyaminoacid
chain so that the NCA is added to the polyaminoacid chain.
Another embodiment of the invention disclosed herein entails a method of
polymerizing aminoacid-N-carboxyanhydride monomers by combining a NCA
monomer with an initiator molecule complex comprised of a low valent
transition
metal-Lewis Base ligand. A specific embodiment of the invention disclosed
herein
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entails a method of polymerizing aminoacid-N-carboxyanhydride monomers having
a
ring with a O-C5 and a O-C2 anhydride bond which consists of combining a first
NCA
monomer with an initiator molecule complex comprised of a low valent metal
capable
of undergoing an oxidative addition reaction wherein the oxidative addition
reaction
formally increases the oxidation state by two electrons; and an electron donor
comprising a Lewis base. The initiator molecule is then allowed to open the
ring of
the first NCA through oxidative addition across either the O-C5 or O-Cz
anhydride
bond and then combine with a second NCA monomer, to form an amido-containing
metallacycle. A third NCA monomer is then allowed to combine with the amido
containing metallacyle so that the amido nitrogen of the amido containing
metallacyle
attacks the carbonyl carbon of the NCA . Thus, the NCA is added to the
polyaminoacid chain and the amido containing metallacyle is regenerated for
further
polymerization. In a preferred embodiment of the invention, the efficiency of
the
initiator is controlled by allowing the reaction to proceed in a solvent
selected for its
ability to influence the reaction. In a specific embodiment of the invention,
the
solvent is selected from the group consisting of ethyl acetate, toluene,
dioxane,
acetonitrile, THF and DMF.
Another embodiment of the invention provides a method of making a block
copolypeptide consisting of combining an amount of a first aminoacid-N-
carboxyanhydride (NCA) monomer with an initiator molecule comprising a low
valent
transition metal-Lewis Base ligand complex so that a polyaminoacid chain is
generated and then combining an amount of a second aminoacid-N-
carboxyanhydride monomer with the polyaminoacid chain so that the second
aminoacid-N-carboxyanhydride monomer is added to the polyaminoacid chain. In a
preferred embodiment of this method, the initiator molecule combines with the
first
aminoacid-N-carboxyanhydride monomer to form an amido containing metallacycle
intermediate of the general formula:
R1
R2
N R3
N O
R4
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wherein M is the low valent transition metal;
L is the Lewis Base ligand;
each of R1, R2 and R3 independently is a moiety selected from the group
consisting of the side chains of alanine, arginine, asparagine, aspartic acid,
cysteine,
glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine,
methionine,
phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine; and
R4 is the polyaminoacid chain.
In yet another embodiment, the invention provides block copolypeptide
compositions having characteristics which have been previously unattainable
through conventional techniques. A specific embodiment of this invention
consists of
a polypeptide composition comprising a block polypeptide having a number of
overall
monomer units that are greater than about 100 amino acid residues and a
distribution of chain-lengths at least about 1.01 < Mw/Mn < 1.25. In a related
embodiment, the polypeptide has a number of overall monomer units that are
greater
than about 250 amino acid residues. In a specific embodiment, the
copolypeptide
consists of a least 3 blocks of consecutive identical amino acid monomer
units. In a
specific embodiment of this invention, at least one of the block's components
is g-
benzyl-L-glutamate.
The present invention also discloses novel methods and compositions, which
address the need for biocompatible materials having improved properties of
biochemical stability, water solubility, and self assembly. The methods of
making
amphiphilic block copolypeptides disclosed herein allow the synthesis and
assembly
of compositions containing well-defined vesicular structures, which are
potentially
valuable for biomedical applications, such as drug delivery.
One embodiment of the invention provides a method of making an amphiphilic
block copolypeptide, which includes the steps of (1 ) generating a soluble
block
polypeptide by combining an amount of an oligo (ethyleneglycol) functionalized
aminoacid-N-carboxyanhydride (EG-aa-NCA) monomer with an initiator molecule;
and (2) attaching an insoluble block by combining the soluble block with a
composition comprising at least one other amino acid NCA monomer. In preferred
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embodiments of this method, the amino acid component of the EG-aa-NCA
monomer is lysine, serine, cysteine, or tyrosine, whereas the insoluble block
can
contain a mixture of amino acids, which includes one or more naturally
occurring
amino acids.
A related embodiment of the invention consists of a method of adding an
aminoacid-N-carboxyanhydride (NCA) to a soluble block polypeptide having one
or
more oligo(ethyleneglycol)-terminated amino acid residues by combining the NCA
with the polypeptide so that the NCA is added to the polypeptide.
In yet another embodiment, the invention provides amphiphilic block
copolypeptide compositions, which have improved characteristics of solubility,
biochemical stability and biocompatibility. The amphiphilic block
copolypeptide
includes a soluble block polypeptide having one or more oligo(ethyleneglycol)-
terminated amino acid residues and an insoluble block comprised substantially
of
nonionic amino acid residues. A specific embodiment of this invention is a
polypeptide composition comprising: (1 ) a soluble block polypeptide having EG-
lysine residues, and (2) an insoluble block polypeptide containing a mixture
of two to
three different kinds of amino acid components in a statistically random
sequence.
In another specific embodiment, the copolypeptide consists of a least 3
blocks,
wherein one or more of the blocks is a soluble block polypeptide and another
block is
an insoluble block polypeptide.
The amphiphilic nature of the block copolypeptides provides yet another
embodiment, which is a method of forming vesicles. This method consists of
suspending the amphiphilic block copolypeptides in an aqueous solution so that
the
copolypeptides spontaneously self assemble into vesicles. In a specific
embodiment, smaller vesicles having a diameter of about 50 nm to about 500 nm
can be formed by sonicating the suspension of larger vesicles.
In a related embodiment, the invention provides vesicle-containing
compositions comprised of the amphiphilic block copolypeptides of the present
invention and water.
In another related embodiment, the invention provides methods for making
EG-functionalized amino acid monomers, which includes the step of combining an
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ethyleneglycol (EG) derivative with an amino acid having a reactive side
group, e.g.,
lysine, serine, cysteine, and tyrosine.
The methods and compositions for making amphiphilic block copolypeptides
are particularly attractive since the EG-amino acid domains will emulate
certain
desirable features of poly (ethyleneglycol), PEG. For example, PEG is well
known for
its bioinvisibility meaning that it is not recognized by immunological defense
mechanisms in the body, and thus has found many useful applications in drug
delivery, enzyme stabilization, tissue engineering, and implant surface
modification.
As examples of preferred embodiments of the invention, a series of initiators
for the polymerization of amino acid-N-carboxyanhydrides (NCAs) into block
copolypeptides based on a variety of metals and ligands are described. These
initiators are substantially different in nature from all known conventional
initiators
used to polymerize NCAs and are also unique in being able to control these
polymerizations so that block copolymers of amino acids can be prepared.
Specifically, these initiators eliminate chain transfer and chain termination
side
reactions from these polymerizations resulting in narrow molecular weight
distributions, molecular weight control, and the ability to prepare copolymers
of
defined block sequence and composition. All of these traits have previously
been
unobtainable using conventional initiator systems. Furthermore, the initiators
described herein are readily prepared in a single step from commercially
available
materials.
The discovery of this new class of initiators and methods for their use allows
for the elimination of side reactions from NCA polymerizations and further
allows the
preparation of well-defined block copolypeptides. Formation of an illustrative
example of our initiator results from the oxidative-addition reaction of an
NCA
monomer to a zerovalent nickel complex, bipyNi(COD); bipy = 2,2'-bipyridyl,
COD =
1,5-cyclooctadiene. This reaction is similar to the known oxidative-addition
of cyclic
anhydrides to zerovalent nickel to yield acyl-carboxylato divalent nickel
complexes
(see equation 5 below).
0 0
y 1 _
(L)~Ni~ I + O C~ (L),Ni~ ~ (L)~Ni~
O O
(L), = phosphine, diimine O O
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While this reaction is similar to these known oxidative-addition reactions,
the
reaction occurring in the formation of the molecules disclosed herein is
without
precedent.
The methods and initiator compositions disclosed herein allow the preparation
of complex polypeptide biomaterials which have potential applications in
biology,
chemistry, physics, and materials engineering. Potential applications include
medicine (drug delivery, tissue engineering), "smart" hydrogels
(environmentally
responsive organic materials), and in organic/inorganic biomimetic composites
(artificial bone, high performance coatings).
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 compares the abilities of different initiators to control molecular
weight of PBLG as a function of initiator concentration in polymerizations of
Glu-
NCA: A, phenethylamine initiator; B, bipyNi(COD) initiator; C, sodium tent-
butoxide
initiator; D, theoretical molecular weight calculated from [M]o/[I]o. All
polymerizations
were run in anhydrous DMF at 25°C for 1 day in sealed tubes. Molecular
weight (Mn
was determined by tandem GPC/light scattering in 0.1 M Liar in DMF at 60
°C.
Figure 2 is a chromatogram of a PBLGo.7s-b-PZLLo.22 diblock copolymer
prepared by sequential addition of Lys-NCA and Glu-NCA to bipyNi(COD)
initiator in
DMF. The polymer was injected directly into the GPC, eluted using 0.1 M Liar
in
DMF at 60 °C through 10~A and 103A Phenomenex 5Nm columns, and
detected with
a Wyatt DAWN DSP light scattering detector and Wyatt Optilab DSP.
Figure 3 shows 2 chemical reaction schemes associated with amino acid
derived nickelacycles, intermediates in nickel initiator mediated polypeptide
synthesis.
Figure 4 shows 4 chemical reaction equations associated with amino acid
derived nickelacycles, intermediates in nickel initiator mediated polypeptide
synthesis.
Figure 5 shows chemical structures of some ligands used in NCA
polymerization reactions
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Figure 6 shows the formation of an amido-containing metallacycle by reaction
of NCAs with a metal initiator.
Figure 7 shows MALDI-MS of leucyl isoamylamide-C-terminated
oligo(phenylglycine)s. A partial expansion of the spectrum is shown in the
upper
right. Mass series were observed for (a) leucine-OH terminated oligomers
resulting
from the hydrolysis of the terminal amide in TFA, (b) Leucine isoamylamide
terminated oligomers resulting from intact end-functionalized chains, and (c)
non-
functionalized chains. For example, 9a indicates the MH+ ion o f the
nona(phenylglycine) of the a series, the b and c series are labeled similarly.
The
ions of the various series also formed adducts with O atoms and CO2, and are
labeled as such.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
The term "block copolypeptide" as used herein refers to polypeptides
containing at least two covalently linked domains ("blocks"), one block having
amino
acid residues that differ in composition from the composition of amino acid
residues
of another block. The number, length, order, and composition of these blocks
can
vary to include all possible amino acids in any number of repeats. Preferably
the
total number of overall monomer units (residues) in the block copolypeptide is
greater than 100 and the distribution of chain-lengths in the block copolymer
is about
1.01 < Mw/Mn < 1.25, where Mw/Mn = weight average molecular weight divided by
number average molecular weight.
The terms "protection" and "side-chain protecting group" as used herein refer
to chemical substituents placed on reactive functional groups, typically
nucleophiles
or sources of protons, to render them unreactive as protic sources or
nucleophiles.
The choice and placement of these substituents was according to literature
procedures. M. Bodanszky, et al., The practice of Peptide Synthesis, 2"d Ed.,
Springer, Berlin/Heidelberg, (1994).
II. Overview
The ideal living polymerization is characterized by fast initiation and an
absence of the termination and chain transfer steps that in most
polymerization
systems compete with propagation of the growing chain. When these conditions
are
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realized, all polymer chains begin growing at about the same time and continue
to
grow until the monomer has been exhausted. The average number of monomer
residues per chain is then simply the molar ratio of monomer to initiator, and
the
distribution of chain lengths is described by Poisson statistics. M. Szwarc,
Carbanions, Living Polymers, and Electron-Transfer Processes, Wiley, New York
(1968). As disclosed herein, these conditions have been met in the
polymerization
of NCAs by bipyNi (COD). The distribution of chain lengths is narrow,
consistent
with Poission statistics, and the rate of polymerization is proportional to
monomer
concentration, indicating that the number of active chain ends remains
constant
throughout the reaction.
It is the absence of termination and transfer that makes living polymerization
so powerful for synthesizing block copolymers. Because the growing chains
remain
active even after the monomer has been exhausted, adding a second monomer at
that stage results in the growth of a second block distinct in composition
from the
first. Proper choice of monomers allows one to engineer the kinds of
combinations
of properties described above: rubbery glassy; hydrophilic and hydrophobic;
conducting and insulating; and so on.
By providing examples of NCAs polymerized by zero-valent nickel catalysts
under'living' polymerization conditions; that is, conditions free of
termination and
chain transfer, the disclosed methods and compositions allow for the
generation and
manipulation of peptides in manners that have not previously been possible.
Living
polymerizations allow the synthesis of polymers of predetermined molecular
weights
and narrow molecular-weight distribution; and, perhaps more importantly, the
preparation of well-defined block copolymers in which long sequences of each
of the
individual monomer residues are linked together at a single site. The
advantages of
living polymerizations, which once were reserved for a small subset of
polymerizable
monomers, can now be extended to NCAs and to the preparation of high-molecular-
weight polypeptides and block copolypeptides with unusual and useful
properties.
The methods and compositions disclosed herein teach new ways to
polymerize amino acids and to add amino acids to polyamino acid chains.
Further,
the initiators and amido-containing metallacyle compositions disclosed herein
allow
the synthesis of block copolypeptides by eliminating of side reactions in
favor of the
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16
chain-growth process (i.e. living polymerization), thus allowing multiple
monomer
additions to polyaminoacid chains. While the specific methods and initiator
and
amido-containing metallacyle compositions disclosed represent preferred
embodiments of this invention, as discussed below, other embodiments are also
contemplated.
In the examples below, general features for the formation of active metal
initiators are discussed as well a means to determine initiator efficiency
(see e.g.
Example 3). Moreover disclosed herein are parameters for generating effective
initiators as well assays to assess the activity of different initiator
complexes and
their ability to function in the disclosed methods. Moreover, disclosed herein
are a
number of different initiator compositions which were evaluated for their
ability to
work in this system. In addition, the Examples illustrate the effects of
different
solvents on the various polypeptide addition reactions. Using these protocols,
one
skilled in the art may construct and then assess the ability of a new
potential initiator
molecule to function in the disclosed methods. Using the protocols disclosed
herein,
one may also assess the activity of different amido-containing metallacycles
and
their ability to function in the disclosed methods.
In providing new means to polymerize amino acids and to add amino acids to
polyamino acid chains, the disclosed methods and compositions overcome a
number
of problems associated with complex polypeptide synthesis. Successful block
copolypeptide synthesis requires elimination of side reactions in favor of the
chain-
growth process (i.e. living polymerization), thus allowing multiple monomer
additions
to each chain. L.J. Fetters, "Monodisperse Polymers" in Encyclopedia of
Polymer
Science and Engineering 2nd Ed., Wiley-Interscience, New York, 10:19-25
(1987);
O. Webster, "Living Polymerization Methods" Science, 251:887-893 (1991). This
problem was addressed by utilizing the versatile chemistry of transition
metals to
mediate the addition of monomers to the active polymer chain-ends. T.J.
Deming,
"Polypeptide Materials: New Synthetic Methods and Applications" Adv.
Materials,
9:299-311 (1997). The wide range of selective chemical transformations and
polymerizations that are catalyzed by transition metal complexes attests to
the
potential of this approach. J. P. Collman, et al., Principles and Applications
of
Organotransition Metal Chemistry 2nd Ed., University Science, Mill Valley,
(1987).
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17
The oxidative addition of cyclic carboxylic acid anhydrides to nickel(0) was
first reported by Uhlig and coworkers. E. Uhlig, et al., Z. Anorg. Allg.
Chem.,
465:141-146 (1980). When succinic anhydride is added to LZNi(COD) a six-
membered acyl-carboxylato nickelacycle is initially formed which
decarbonylates
above ambient temperature to form a stable five-membered alkyl-carboxylato
complex. L2 = donor ligand(s); COD = 1,5-cyclooctadiene; bipy = 2,2'-
bipyridyl. With
unsymmetric anhydrides, the regioselectivity of oxidative addition was found
to vary
with the donor ligand (L2) and solvent. A. M. Castario, et al.,
Organometallics,
13:2262-2268 (1994). When an NCA oxidatively adds to nickel(0) across the
unsymmetric anhydride linkage, regioselectivity of addition is important in
determining the nature and reactivity of the products. With both initial
products,
decarbonylation would be expected to be favored over decarboxylation due to
the
greater stability of the resulting five-membered metallacycles (see scheme 1
of
Figure 3). E. Uhlig, et al., Z. Anorg. Allg. Chem., 465:141-146 (1980). The
addition
of NCAs to nickel(0) is of interest because the resulting metal-amido or metal-
carbamato complexes might prove useful as reactive, chiral synthetic
intermediates.
The reaction chemistry of a-amino acid-N-carboxyanhydrides (NCAs) has
been under study since these molecules are potential precursors to sequence
specific peptides, polypeptides, and other amino acid containing compounds.
H.R.
Kricheldorf, a-Aminoacid-N-Carboxyanhydrides and Related Materials, Springer-
Verlag, New York, (1987); H.R. Kricheldorf, in Models of Biopolymers by Ring-
Opening Polymerization, Penczek, S. Ed., CRC Press, Boca Raton, (1990). NCAs
are attractive peptide building blocks since they are readily prepared from
amino
acids and since they show no racemization at the chiral a-carbon either during
preparation or in subsequent reactions. W.E. Hanby, et al., Nature, 161:132
(1948);
A. Berger, et al., J. Am. Chem Soc., 73:4084-4088 (1951). Utilization of NCAs,
however, has been limited because of their complicated reactivity and tendency
to
uncontrollably polymerize.
Attempts to use metal coordination complexes of conventional amine initiators
to control the polymerizations have been described in the art. T.J. Deming,
"Transition Metal-Amine Initiators for Preparation of Well-Defined Poly(g-
benzyl-L-
glutamate)" J. Am. Chem. Soc., 1997, 119:2759-2760 (1997). Use of metal-amine
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18
complexes for polymerization of g-benzyl-L-glutamate N-carboxyanhydride, Glu-
NCA
as described herein, allowed the preparation of poly(g-benzyl-L-glutamate),
PBLG,
with narrow molecular weight distribution (Mw/Mn = 1.05 - 1.10) and some
control
over molecular weight. However, typical problems inherent in primary amine
initiated
polymerizations (i.e. slow propagation and chain transfer reactions) prevented
use of
these initiators for preparation of block copolypeptides.
The living polymerization of NCAs and synthesis of block copolypeptides
using nickel initiators has been reported. T.J. Deming, Nature, 390:386-389
(1997)
This reference discloses stoichiometric reactions where NCAs oxidatively add
regioselectively to sources of zerovalent nickel to yield complexes which
subsequently rearrange to unprecedented amido-containing metallacycles. When
complexed with donor ligands, the nickelacycles are efficient NCA
polymerization
initiators.
III. Methods and Compositions of the Invention
Unlike the initiators known in the art, the molecules described herein are a
new class of initiators based on low valent metal-Lewis base complexes which
are
able to eliminate significant competing termination and transfer steps from
NCA
polymerizations and allow preparation of well-defined block copolypeptides.
Donor liqand/transition metal complexes
A variety of illustrative initiator complexes useful in the generation of
block
copolypeptides are described herein such as those generated using bis-1,5-
cyclooctadiene nickel (Ni(COD)2) as the nickel source and 2,2'-bipyridyl
(bipy) as the
donor ligand component in tetrahydrofuran (THF) solvent. As discussed below
and
as shown in Tables 7 and 8, the use of other sources of zerovalent nickel
(e.g.
nickel-olefin complexes, nickel-carbonyl complexes, nickel-isocyanide or
cyanide
complexes, and other specific ligands such as PR3 [R = Me, Et, Bu, cyclohexyl,
phenyl], R2PCH2CHZPR2 [R = Me, phenyl], a, a'-diimine ligands [1,10-
phenanthroline, neocuproine], diamine ligands [tetramethylethylene diamine],
and
isocyanide ligands [tert-butyl isocyanide and related nickel nitrogen or
phosphorous
donor ligand complexes) can work in the complexes of the present invention to
initiate these polymerizations.
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19
As shown in Example 4 below, in addition to bis-1,5-cyclooctadiene nickel
(Ni(COD)2), other sources of zerovalent nickel (e.g. Ni(CO)4) as well as other
low
valent metals in the initiator complexes have been used successfully in these
methods. Illustrative metals useful in the generation of initiators are "low
valent"
transition metals, in particular the metals of Group VIII of the Periodic
Table and
illustrative examples of initiators using such metals is provided in Table 8.
This
group includes the metals, Fe, Ru, Os, Co, Rh, Ir, Ni, Pd and Pt. The "low
valent"
forms of the metals implies that the metals are in low oxidation states. For
Ni, Pd,
Pt, Co, and Fe this means the zerovalent (0) oxidation state. For Ir and Rh,
this
means the monovalent (+1 ) oxidation state. For Ru and Os, this means the
divalent
(+2) oxidation state. See the complexes in Table 8 for relevant examples. The
term,
"Low valent metal" also extends to other metals in an oxidation state such the
metal
may undergo a 2 electron oxidation.
As shown in Example 4 below, in addition to using 2,2'-bipyridyl (bipy) as the
donor ligand component, a variety of other donor ligands can be used in the
initiator
complexes. As shown in Table 7, ligands which can be used to bind to the
initiator
metals complexes need to comprise a non nucleophilic electron donor comprising
a
Lewis base and can consists of a variety of groups which have this property
including those that are pyridyl based (see e.g. entry 2-144, Table 7),
diimine (see
e.g. DIPRIM, 2-148), bisoxazoline (see e.g. DPOX, 3-2), alkyl phosphine (see
e.g.
dmpe, 2-148), aryl phosphine (see e.g. PPh3, 2-151 ), tertiary amine (see e.g.
tmeda,
3-10), isocyanide or cyanide (see e.g. 3-34), or combinations of these
ligands.
Generally, the ligands are bidentate (coordinate through 2 atoms) or are
composed
of 2 equivalents of monodentate ligands. Tridentate ligands can also be used
(e.g.
terpyridine). The ligands generally are bound to the metal through N, P, or C
atoms
of the molecule. Other N or P donor ligands, similar to those mentioned above
(i.e.
neutral, non-nucleophilic, aprotic) can also support these initiators.
NCA monomers
As illustrated in the references cited above, NCA monomers are well known in
the art (see e.g. H.R. Kricheldorf, a-Aminoacid-N-Carboxyanhydrides and
Related
Materials, Springer-Verlag, New York, (1987)). Moreover, the use of a variety
of
NCA monomers in methods of polypeptide synthesis is well known in the art. For
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example the stepwise synthesis of polypeptides using NCAs (or derivatives
thereof)
is disclosed in U. S. Patent No. 3,846,399 (incorporated by reference herein).
In
addition, U.S. Patent No. 4,267,344 discloses N-substituted N-carboanhydrides
of a
amino acids and their application in the preparation of peptides (incorporated
by
reference herein).
Another embodiment of the present invention involves the synthesis of
unique oligo (ethyleneglycol) functionalized amino acids and their subsequent
polymerization into oligo (ethyleneglycol) functionalized polypeptides. The
method of
making these monomers includes the step of combining an ethyleneglycol (EG)
derivative with an amino acid having a reactive side group, e.g., lysine,
serine,
cysteine, and tyrosine, to form an EG-functionalized amino acid. The EG
derivative
has the general formula (CH30CH2CH2)~X, where n amounts to about 1 to 3 EG
repeats and X is a reactive group, such as chloroformate, N-
hydroxysuccidimydyl
acetate, or a halide. The EG functionalized amino acids can then be converted
to an
NCA monomers for use in the synthesis of oligo (EG) functionalized
polypeptides.
A generalized scheme for synthesizing oligo(EG) functionalized serine,
tyrosine and cysteine is shown in Scheme I (below).
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21
0 0-
0 0
~ ~v ~~Y9
a
0 / 0 0-
/0 ~~ ~0 ~i~ + V / v '0 / g
~/0 ~0 S
NH ~ ~0 ~ V 'NH ~
UUjIv
0 0-
0 ~ 0 0 \~
~0 ~ NH ~
x = -Br
~o b' ci r
~o ~o ~o o ~ o o -
-off \ ~ °
r=
NH ~
- SH
/ OH
Scheme I
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22
More detailed synthetic routes are illustrated in Scheme II and in Example 5
(below).
NReoc NR eoc
NaN 0 " ' ,0
R 0 \,"..~0 ~ 0 ~Rt DMf ~° ~ COI(/
R H
BOC = tert-butoxvcatbonyl 1
0
NR t NN~
NCI ° O LOCI t °0
1 NOdc ~0 \', ~ THE
R 3
hpyNIIC001 NH
O or ~0 0
fame ~I~Cp ° Z
4
COCI
1'0H ~f t 0 ~O "CI
R° ~ I NRZ ~ ~e o
NaOR O i NRZ
H
Z=carboborltvlDx51 R
0
CI 1CRDCR ~ ~° ° ° / I R N
Z
CR iCl ~
1
NaOUa ~° 0 MR
_ Z
T ~f _ ~0 I w "... 0
a
NR t
NH ~
~ / 0
H S D NaNCO 0 S D
\,..... H'0 R
R 9 O
AN~
R LOCI 1 ~° D SS
Z
Tll f
~IIR
Na0tH0 ° S O
10 °
THE
11
SCHEME II
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23
In addition, a preferred method of synthesizing oligo(EG) functionalized
lysine
is shown in Scheme III (below).
Q o 0
0
DCC \ ~
O~n v OH y HO-N -1.~ ~O~n0~0-N
1
n=lor''_ O O
=i Z
O NHZ
O NaHC03 _
1 + H=N~''~ ' THr/H,O ~O~~~N~'''.. O
OH H
OH
C
O HN \
CI~CHOCH~ ~ ~ ~O' ~ O
~O~n ~N~''~.
CH=Cl: H I
O
3
O
bpy'-'~i(COD) ~ ~ ~.O\ ~
or 1 O "I n ~N~~~~~ O
(Fye:i;CO H
n
SCHEME III
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24
These amino acid derivatives and polymers are new compositions of matter,
and the polymers possess unique properties which make them potentially
valuable
for biomedical/biotechnological applications. Oligo-EG functionalized NCA
monomers can be used to make polypeptide chains where the side chain of every
residue is capped by a ethylene glycol oligomer; in effect, "PEG coated
polypeptides". Poly(ethyleneglycol), PEG, is well known for its
"bioinvisibility",
meaning that it is not recognized by immunological defense mechanisms in the
body
(non-antigenic), and thus has found many useful applications in drug delivery,
enzyme stabilization, tissue engineering, and implant surface modification.
Such polymers are unusual in possessing excellent ~niater solw~bility over
broad
pH ranges (2-13) and salt concentrations. Furthermore, the PEG "coating"
strongly
stabilizes the secondary structure of the polypeptides (beta-sheets and alpha-
helices) such that the polymers possess stable secondary structures over broad
pH
and temperature ranges. These new polymers are attractive since they display
most
of the same properties of PEG (water solubility, biocompatibility), but
possess
completely different chain structures. The serine and cysteine-derived
polymers
adopt beta-sheet structures and represent the first examples of water soluble
polypeptides that form stable beta-sheet structures. As such their solution
and
mechanical properties are markedly different from PEG and thus provide an
interesting alternative to PEG in biomedical materials.
Making amido-containing metallacycles
The initiator complexes of the present invention can be synthesized by two
different approaches, both of which entail the use of a low valent transition
metal-
Lewis Base ligand complex and result in the formation of an amido-containing
metallacycle.
Transition metal/Donor ligand + NCA monomer
One embodiment of the invention provides a method of making an amido-
containing metallacycle comprising combining an amount of an a-aminoacid-N-
carboxyanhydride monomer with an initiator molecule comprising a low valent
transition metal-Lewis Base ligand complex so that an amido-containing
metallacycle
is formed. Formation of these initiators results from the unprecedented
reaction of
CA 02372526 2001-11-07
WO 00/68252 PCT/US00/12770
an NCA monomer with a low valent metal-Lewis base complex such as a zerovalent
nickel complex bipyNi(COD); bipy = 2,2'-bipyridyl, COD = 1,5-cyclooctadiene.
This
reaction is similar to the oxidative-addition of cyclic anhydrides to
zerovalent nickel
which yields divalent nickel metallacycles (see equation 6 below). E. Uhlig,
et al.,
~Reaktionen cyclischer Carbonsaeureanhydride mit (a,a'-Dipyridyl)-
(cyclooctadien-
1,5)-nickel~ Anorg. Allg. Chem., 465:141-146 (1980); K. Sano, et al.,
"Preparation of
Ni- or Pt-Containing Cyclic Esters by Oxidative Addition of Cyclic Carboxylic
Anhydrides and Their Properties" Bull. Chem. Soc. Jpn., 57:2741-2747 (1984);
A.M.
Castano, et al., "Reactivity of a Nickelacycle Derived from Aspartic Acid:
Alkylations,
Insertions, and Oxidations" Organometallics, 13:2262-2268 (1994).
0
- COD <L/~ 1 \~% -C~ L~
COD~Ii ~, ~ + ~ ~ZI~
~O
CL~2 = phosphine, diimine
(6)
Activation and polymerization of NCAs through oxidative ring opening of the
anhydride, however, is without precedent. Successful polymerization of L-
proline
NCA, which lacks a proton bound to nitrogen, using bipyNi(COD) supports the
hypothesis of oxidative addition across the anhydride bond, rather than
reaction at
the N-H bond. In this context, it is observed that the initial oxidative
addition to the
NCA can occur at either side of the anhydride bond (for example, O-C5 for
nickel,
cobalt and iron and both O-C5 and O-CZ for rhodium and iridium).
Since NCAs are unsymmetrical anhydrides, the oxidative-addition of NCAs
can yield two distinct isomeric products In practicing one embodiment of the
invention, it is found that the addition of NCAs to nickel was completely
regioselective for ring opening across the O-C5 bond. Reaction of bipyNi(COD)
with
'3C2-L-leucine NCA and '3C5-L-leucine NCA yielded oxidative addition products
and
bipyNi(CO)2 which were examined by'3C NMR and FTIR spectroscopy. Detection
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26
of bipyNi('2C0)2 (FTIR(THF): n(CO) = 1978, 1904 cm-') from the reaction of'3C2-
L-
leucine NCA, and bipyNi('3C0)2 (FTIR(THF): n(CO) = 1934, 1862 cm-'; '3C
NMR(DMF-d~): d 198 (Ni-CO)) from the reaction of'3C5-L-leucine NCA identified
the
regiochemistry of the product . In dimethylformamide (DMF), a good solvent for
polypeptides, this addition product was found to be completely active for
polymerization of additional NCA monomers.
Donor ligand/transition metal + alloc-amino acid amide
The amido-amidate metallacycles generated from low valent transition metal
precursors, as described above, are active intermediates in the controlled
polymerization of a-amino acid-N-carboxyanhydrides (NCAs). A limitation of
this
methodology is that the active propagating species are generated in situ and
thus do
not allow for controlled functionalization of the polypeptide chain ends. For
this
reason, we pursued alternative methods for the direct synthesis of these types
of
initiators.
Thus, another embodiment of the present invention entails new tandem
addition reactions that allow the general synthesis of amido-amidate
metallacycles
useful for preparation of polypeptides containing a variety of defined
endgroups.
This method of making an initiator molecule includes the step of combining an
allyloxycarbonyl (alloc) protected amino acid amide and a low valent
transition metal-
Lewis base ligand complex so that an amido-amidate metallacycle is formed
having
the following general formula:
R1
H
N
~~) ~'v'~
N ~O
R3
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27
wherein M is a low valent transition metal, L is a Lewis base ligand; one of
R1
and R2 is an amino acid side group and the other is hydrogen; and R3 is any
functional end group capable of being attached to a primary amine group.
The alloc-amino acid amide has the general formula Alloc-NH-
CH(R')C(O)NHR" where R' is an amino acid side group and R" is a functional end
group. The "alloc" group includes at least one allylic moieity, i.e., a carbon-
carbon
double bond bound to a saturated carbon. The backbone "allylic" system is a
key
element which is required for the reaction to work. The allylic backbone is,
in turn,
coupled via an oxygen to the carbonyl bound to the nitrogen of the amino acid.
The
R~ ~... :.,.. ..~.a~ t, ~:...,..~;~., r d r r ~~,.. 2~
~i Gi.iN Gai '' uc a Sm:-w ' ii r W i 'Wm gamy W ui ~ iGr ai iy' G~ a is a i
aW i ai cai i iflG acids
(L-form), their corresponding D-forms, or unnatural synthetic amino acid side-
chains,
providing that reactive functional groups (those that are either protic or
nucleophilic -
such as those of lysine, ornithine, cysteine, serine, histidine, arginine,
glutamic or
aspartic acid (i.e. amines, alcohols, sulfhydryls, imidazoles, guanidines,
carboxylates)) are suitably protected (using standard peptide functional group
protection) to eliminate their reactivity. Among unnatural side-chains, those
that
would react with the low-valent metal complexes (e.g. aryl-halides, allylic
esters,
isothiocyanates, or isocyanates) also cannot be used.
The R" group is crucial as this is the group that will typically be used to
"tag"
or functionalize the polypeptide chains, and is the reason and advantage for
using
this method. This group can be virtually anything, bearing in mind the
chemical
limitations of functional groups as listed above for R'. Typically, this group
will be a
peptide, oligosaccharide, oligonucleotide, fluorescent molecule, polymer
chain, small
molecule therapeutic, chemical linker to attach the polypeptide to a
substrate,
chemical linker to act as a sensing moiety, or reactive linker to couple the
polypeptide to larger molecules such as proteins, polysaccharides or
polynucleotides.
In preferred embodiments, Na-allyloxycarbonyl-amino acid amides were
reacted with zerovalent nickel complexes LNi(1,5-cyclooctadiene) (L = 2,2'-
bipyridine
(bpy), 1,10-phenanthroline (phen), 1,2-bis(dimethylphosphino)ethane (dmpe),
and
1,2-bis(diethylphosphino)ethane (depe)), to yield amido-amidate metallacycles
of the
general formula: LNiNHC(R')HC(O)NR" (see Table 1 below).
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28
As shown in Table 2 (below), these complexes were found to initiate
polymerization of a-amino acid-N-carboxyanhydrides (NCAs) yielding
polypeptides
or block copolypeptides with defined molecular weights, narrow molecular
weight
distributions, and with quantitative incorporation of the initiating ligand as
an end-
group. These initiators provide a facile method to synthesize complex
copolypeptides where the polymer chain carboxy-terminus can be quantitatively
functionalized with a wide range of substituents. These substituents can
include, but
are not limited to: polymers (polystyrene, polyethylene oxide)), peptides,
oligosachharides, oligonucleotides, or other organic moieties.
The key feature utilized to connect these substituents to a polypeptide chain,
and the only requirement of the substituents, is a primary amine group. This
feature
allows the preparation of complex polypeptide biomaterials which have great
potential applications in medicine (drug delivery, tissue engineering).
Specifically,
the ability to incorporate chain-end functional groups (e.g. other polymers,
fluorescent or radioactive tags, or biomolecules (peptide sequences,
oligonucleotides, or oligo sachharides)) imparts these materials with highly
desirable
qualities for numerous biotechnological applications. For example, this
methodology
can be used for labelling polypeptide chains to analyze chain
mobility/location in
vivolin vitro. Moreover, incorporation of endgroup functionalities, such as
signaling
or receptor groups, onto polypeptide chains is essential for targeting of drug
delivery
complexes as well as substrate specific anchoring of these materials.
To form the desired amido-amidate nickelacycles, we conducted a reaction
where N~-Alloc-amino acid allyl amides were used as substrates (eq 7)
0 0
O ~j~ ~ V i(CODI= ~f O~V
Y 'V ~i'i i~ R
bPY .~
G R ~, U
~H
/ / .... ~/~
r,
H H
ai N
boy~li~' bPY~t~ (7)
O ~~O
H
not formed 1
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29
This reaction was not expected to be highly successful since the oxidative
addition of
allylic amides to nickel is without precedent (Collman, J. P.; Hegedus, L. S.;
Norton,
J. R.; Finke, R. G. Principles and Applications of Organotransition Metal
Chemistry
2nd Ed., University Science, Mill Valley, 1987). A surprising result was
therefore
obtained when the reaction of bpyNi(COD) with Na-Alloc-L-leucine allyl amide
was
found to produce an amido-amidate species in good yield (60% isolated). The
product, however, was not the expected one, as evidenced by the lack of
byproduct
1,5-hexadiene. The product nickelacycle was found to result from initial
addition
across the Alloc C-O bond, followed by a second addition across the N-H bond
of the
amide, not the allylic N-C bond (eq 7). As a result, the product metallacycle,
1,
retained the allyl substituent on nitrogen, as determined by FAB/MS,'H NMR of
the
hydrolysis product from reaction with HCI, and'3C labeling studies. The N-H
addition was also verified by use of a Na-2-hexenyloxycarbonyl-amino acid
allyl
amide in the reaction which resulted in the formation of byproduct hexenes
that were
identified by'3C ~'H} NMR.
The reaction of readily synthesized Na-Alloc-amino acid allyl amides with
zerovalent nickel was found to be general for different substituents (R' and
R") and
donor ligands, allowing the use of many combinations of amino acids and
primary
amines in the construction of initiator complexes (Table 1 ). This method is
therefore
amenable to incorporation of a wide variety of end-group functionalities onto
polypeptides through amide linkages.
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O H R'
Ni(CODy~ N
AIIocNH~N~R" ~ ~Ni
H L 1L ~ ~N + 2 COD
R, I O
R"
n
Initiator ~ ~ R' R" Yield
1 bpy ~ ~ 69(95)
phen ~ 72(94)
/ \
depe -CH3 42(98)
3 / \
depe
39(97)
O
5 phen -H ~ N ~ 59(94)
H
O
g phen H' v \ 62(96)
Table 1 Example initiators synthesized from different N-Alloc-a-amino amides,
bidentate ligands and Ni(COD)2. a = isolated yield of initiator. Yield of
crude product,
as determined by FTIR spectroscopy, is given in parentheses.
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At this point, it was necessary to determine if these functional groups, once
attached to the initiating complex, were then quantitatively incorporated as
end-
groups on polypeptide chains. Polymerizations of ~y-benzyl-L-glutamate NCA
(Glu
NCA) using nickel complexes containing different bidentate donor ligands
revealed
that alkyl phosphine ligands (dmpe and depe) promoted the most efficient
initiation.
These initiators were able to prepare block copolypeptides of defined sequence
and
composition (Table 2).
First Diblock Copolymer
segment
First MonomersSecond MonomersM~ MW/M~ M~ MW/M~ Yield
(%)d
25 Glu-NCA none 5190 1.37 --~[ -- 88
50 Glu-NCA nono 13450# 1.31 -- -- 83
200 Glu-NCA none 49340 1.24 -- -- 90
25 Glu-NCA 71 Lys-NCA 5190 1.37 28880 1.18 75
25 Lys-NCA 87 Glu-NCA 8760 1.06 25600 1.13 77
Table 2 Synthesis of polypeptides and block copolypeptides using 4 (see Table
1 ) in
DMF at 20 °C. Lys NCA = E-CBZ-L-lysine-N-carboxyanhydride. a =
First and
second monomers added stepwise to the initiator; number indicates equivalents
of
monomer per 4. b = Molecular weight and polydispersity index after
polymerization
of the first monomer. c = Molecular weight and polydispersity index after
polymerization of the second monomer. d = Total isolated yield of polypeptide
or
block copolypeptide.
It was also found that the initiators could be used for polymerization without
isolation from the crude reaction mixture. This feature greatly simplifies the
use of
these complexes, which are near-quantitative yield, but can be tedious to
isolate
from the reaction solvent. Hence, polymerizations were conducted using either
isolated or in situ initiators with no noticabte differences in results.
Concerning the degree of functionalization of the polymers, reaction of
initiator
4 (see Table 1) with one equivalent of cis-5-norbornene-endo-2,3-dicarboxylic
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32
anhydride, which should add to the initiator like an NCA monomer but not form
polymer, followed by hydrolysis of the product with HCI, resulted in complete
consumption of 4 to yield the addition product (eq 8). The anhydride IR
stretch of the
starting material (1780 cm-') was observed to completely disappear over the
course
of the reaction. The only amino acid containing compound present after
hydrolysis
was the coupled product (FAB MS: MH+: 323.8 calc'd, 323 found). No unreacted
monopeptide from hydrolysis of unreacted 4 was detected, showing that all
metal
centers were active.
Ec Ec H O
\/
~/ 1) DW O
i N' O --~ H
v + ".~ _ H30. \ ~~/
CP ~~
/\ O ~ = ~ = H
E~ ~ O HOBO O /~
(8)
Furthermore, polymerization studies using initiator 3 (see Table 1 ) gave
polypeptides with a 1-naphthyl end-group. These end-groups were then
quantitated
using fluorescence spectroscopy, which showed that the number of end-groups
increased commensurately with the number of polymer chains. These fluorescent
tags are useful for monitoring polypeptide location and mobility, desirable
for
applications such as the monitoring of drug delivery complexes in vitro
(Singhal, A.;
Huang, L. in Gene Therapeutics: Methods and Applications of Direct Gene
Transfer,
J. A. Wolff Ed., Birkhauser, Boston, 1994).
Finally, MALDI-MS analysis of phenylglycine oligomers prepared using a
nickel complex containing a leucine isoamylamide initiating group revealed
that
nearly all chains were end-functionalized with the leucine residue of the
initiator.
(See Figure 7) Only very small peaks were observed for non-functionalized
oligo(phenylglycines), indicating that the degree or' chain functionalization
was
greater than 98%.
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Amido-containing metallacycles
Another embodiment of the invention entails a five or six membered amido-
containing metallacycle comprising molecules of the general formula:
1
I1
R2
1 'R R3 I 1 'R R3
R3 _
N N N
( ~ / (L)n O (Lh / (L
O R O O ~ O
~a R5 R4 R7
wherein "M" is a low valent transition metal capable of undergoing an
oxidative addition reaction, "L" is an electron donor such as a Lewis base and
"R#"
comprises any organic substituent not bearing free amine, hydroxyl, carboxylic
acid,
sulfhydryl, isocyanate, imidazole, or other highly protic or nucleophilic
functionality.
These functionalities may be present, however, if suitably chemically
protected to
render them unreactive as erotic sources or nucleophiles. Effective R
substituents
on the above structures exhibit a number of properties. For example, as
disclosed in
the examples below, the R substituents on the above structures are typically
encompassed by the structures of the side chain substituents of amino acids or
derivatives thereof. In particular, in most cases, R1 (and R4 in the center
structure)
is a proton. Independently, each of R2 and R3 (and R5 and R6) are typically
selected from the side chain substituents of amino acids. Typically, one of
the
substituents (such as R1) is a proton (H), while the others can be different
side
chain group of a specific amino acid. The placement of the proton (as either
R2 or
R3) is determined by the amino acid being of the L or D configuration. The
side
chain will be one of those from the family of naturally occurring L- or D-
amino acids,
or synthetic amino acids or derivatives thereof. Naturally occurring L- or D-
amino
acids (e.g. alanine, arginine, asparagine, aspartic acid, g-carboxyglutamate,
cysteine, glutamic acid, glutamine, glycine, histidine, hydroxyproline,
isoleucine,
leucine, lysine, methionine, phenylalanine, proline, serine, threonine,
tryptophan,
tyrosine and valine) and synthetic amino acids or derivatives thereof are well
known
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34
in the art. The side-chains of amino acids bearing polar functional groups
(e.g. NH2,
COOH, SH, imidazole) can be blocked with standard peptide protecting groups.
In a preferred embodiment, the metal is a group VIII transition metal and the
donor ligand(s) can be any of those given in Table 7. In another preferred
embodiment, the metal is nickel and the donor ligand is a 2,2'-bipyridyl
(bipy) moiety.
In another preferred embodiment, the R2 or R3 group comprises an amino acid
side
chain selected from the group consisting of side-chain protected NCA formed
from
arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine,
histidine,
lysine, methionine, serine, threonine, tryptophan, and tyrosine or an amino
acid side
chain selected from the group consisting of side-chain NCA formed from
alanine,
glycine, isoleucine, leucine, phenylalanine, proline and valine.
Adding NCA monomers
A related embodiment of the invention consists of a method of adding an
aminoacid-N-carboxyanhydride (NCA) to a polyaminoacid chain having an amido
containing metallacycle end group comprising combining the NCA with the
polyaminoacid chain so that the NCA is added to the polyaminoacid chain. In a
preferred embodiment of this method, the amido containing metallacycle end
group
is of a formula as follows:
R1
R2
N ~ R3
~~)nM~
N O
R4
wherein M is the low valent transition metal;
L is the Lewis Base ligand;
R1 comprises a constituent found in a side chain of an amino acid (e.g. a
hydrogen for glycine or a methyl group for alanine etc.) selected from the
group
consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic
acid,
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glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine,
phenylalanine,
proline, serine, threonine, tryptophan, tyrosine and valine;
R2 comprises a constituent found in a side chain of an amino acid selected
from the group consisting of alanine, arginine, asparagine, aspartic acid,
cysteine,
glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine,
methionine,
phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine;
R3 comprises a constituent found in a side chain of an amino acid selected
from the group consisting of alanine, arginine, asparagine, aspartic acid,
cysteine,
glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine,
methionine,
phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine;
and
R4 is the polyaminoacid chain.
In a preferred embodiment of the method of adding an aminoacid-N-
carboxyanhydride (NCA) to a polyaminoacid chain having an amido containing
metallacycle end group, the metal group of the amido containing metallacycle
is a
transition metal selected from the group consisting of nickel, palladium,
platinum,
cobalt, rhodium, iridium and iron and the Lewis Base ligand is selected from
the
group consisting of pyridyl ligands, diimine ligands, bisoxazoline ligands,
alkyl
phosphine ligands, aryl phosphine ligands, tertiary amine ligands, isocyanide
ligands, and cyanide ligands. In specific embodiments of the invention, the
NCA is
an a-aminoacid-N-carboxyanhydride selected from the group consisting of
alanine,
arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine,
glycine,
histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline,
serine,
threonine, tryptophan, tyrosine and valine.
Polymerization reactions
Another embodiment of the invention disclosed herein entails a method of
polymerizing aminoacid-N-carboxyanhydride monomers by combining a NCA
monomer with an initiator molecule complex comprised of a low valent
transition
metal-Lewis Base ligand. A specific embodiment of the invention disclosed
herein
entails a method of polymerizing aminoacid-N-carboxyanhydride monomers having
a
ring with a O-C5 and a O-C2 anhydride bond. The method consists of combining a
first NCA monomer with an initiator molecule complex. The complex is comprised
of
a low valent metal capable of undergoing an oxidative addition reaction,
wherein the
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36
oxidative addition reaction formally increases the oxidation state by two
electrons,
and an electron donor comprising a Lewis base. The initiator molecule opens
the
ring of the first NCA through oxidative addition across either the O-C5 or O-
C2
anhydride bond and combines with a second NCA monomer to form an amido-
containing metallacycle. A third NCA monomer then combines with the amido
containing metallacyle so that the amido nitrogen of the amido containing
metallacyle
attacks the carbonyl carbon of the NCA. The [WHICH ONE?} NCA is then added to
the polyaminoacid chain, and the amido containing metallacyle is regenerated
for
further polymerization.
In a preferred embodiment of the invention, the efficiency of the initiator is
controlled by allowing the reaction to proceed in a solvent selected for its
ability to
influence the reaction. In a specific embodiment of the invention, the solvent
is
selected from the group consisting of ethyl acetate, toluene, dioxane,
acetonitrile,
THF and DMF.
As illustrated in Example 5 below, the efficiency of various initiators can be
analyzed through polymerization experiments with Glu-NCA. The resulting
polymer,
PBLG, is a,-helical in many solvents, has been extensively studied, and is
readily
characterized. H. Block, Poly(g-benzyl-L-glutamate) and Other Glutamic Acid
Containing Polymers, Gordon and Breach, New York, (1983). Number-average
molecular weight of PBLG samples formed using bipyNi(COD) in DMF was found to
increase linearly as a function of the initial monomer to initiator ratios,
indicating the
absence of chain-breaking reactions. L.J. Fetters, "Monodisperse Polymers" in
Encyclopedia of Polymer Science and Engineering 2nd Ed., Wiley-Interscience,
New
York, 10:19-25 (1987); O. Webster, "Living Polymerization Methods" Science,
251:887-893 (1991). Such control over polypeptide molecular weight is a
substantial
improvement over conventional NCA polymerization systems (see Figure 1 ). The
polymers possessed narrow molecular weight distributions (Mw/Mn = 1.05 - 1.15)
and were obtained in excellent yields (95 - 99% isolated). Kinetic analysis
also
showed that the polymerizations were well behaved. The polymerizations were
first
order in monomer concentration over 4 half-lives in DMF (kobs = 2.7(1 ) x 10~
s ~ at
298K; [bipyNi(COD)] = 0.67 mM) showing none of the complexities of traditional
NCA
polymerizations. Our initiating system displays all of the characteristics of
a living
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37
chain growth process for Glu-NCA. Analysis of other NCA monomers (e.g. e-
carbobenzyloxy-L-lysine N-carboxyanhydride, Lys-NCA) also yielded controlled
polymerizations, illustrating the general utility of our initiating system for
preparation
of well-defined block copolypeptides with a variety of architectures.
Making block copo~ipeptides
Block copolymers have played an important part in materials science and
technology because they allow the effective combination of disparate
properties in a
single material. Block copolymers of styrene and dienes, for example, are
rubbers at
room temperature (a characteristic of the polydiene phase) but can be moulded
at
temperatures above the glass transition of the polystyrene phase. This
distinguishes
the block copolymers from most conventional rubbers, which must be chemically
cross-linked (vulcanized) in order to withstand the stresses that they
encounter in
use. Because chemical cross-linking is irreversible, it must be done while
making
the final part; and cross-linked rubber is difficult to reprocess. The 'cross-
linking' step
for styrene-diene block copolymers is instead a physical association of chains
in the
glassy polystyrene domains: the tendency of the two different chain sections
to
clump together, like with like. This association is robust enough to bear
loads at
room temperature, but is readily reversible upon heating.
Well-defined block copolymers assemble spontaneously into a variety of
intriguing nanostructures, and other, aligned nano-structure arrays can be
made
using fluid flow or other fields. Z.R. Chen, et al., Science, 277:1248-1253
(1997).
Because of this, block copolymers have enjoyed great commercial success, as
well
as the ardent attentions of polymer physicists. But block copolymers of amino
acids
have been little studied, largely because our synthetic methods do not have
fine
enough control to produce well-defined structures. F. Cardinauz, et al.,
Biopolymers,
16:2005-2028 (1977). The same is true of the synthesis of block copolypeptides
for
use as biomaterials or as selective membranes - the potential advantages of
the
protein-like architectures have remained unrealized for want of adequate
synthetic
tools. The disclosed methods and compositions promise to change that. By
treating
the monomer of interest with an initiator such as the zero-valent nickel
complex
bipyNi (COD), where bipy is 2,2' - bipyridyl and COD is 1,5-cyclooctadiene and
adding NCAs yields an intermediate molecule that can be isolated and that
remains
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active towards further ring-opening polymerization, and the target polypeptide
can be
prepared with essentially 100 percent yield.
One embodiment of the invention provides a method of making a block
copolypeptide consisting of combining an amount of a first aminoacid-N-
carboxyanhydride monomer with an initiator molecule comprising a low valent
transition metal-Lewis Base ligand complex so that a polyaminoacid chain is
generated and then combining an amount of a second aminoacid-N-
carboxyanhydride monomer with the polyaminoacid chain so that the second
aminoacid-N-carboxyanhydride monomer is added to the polyaminoacid chain. In a
preferred embodiment of this method, the initiator molecule combines with the
first
aminoacid-N-carboxyanhydride monomer to form an amido containing metallacycle
intermediate of the general formula:
R1
R2
N R3
N O
R4
wherein M is the low valent transition metal;
L is the Lewis Base ligand;
and each of R1 and R2 and R3 independently consist of a side chain of an
amino acid selected from the group consisting of alanine, arginine,
asparagine,
aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine,
isoleucine,
leucine, lysine, methionine, phenylalanine, proline, serine, threonine,
tryptophan,
tyrosine and valine; and
R4 is the polyaminoacid chain.
In a highly preferred embodiment of this method of making a block
copolypeptide, the low valent transition metal is selected from the group
consisting of
nickel, palladium, platinum, cobalt, rhodium, iridium and iron. In another
preferred
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embodiment of this method, the Lewis Base ligand is selected from the group
consisting of pyridyl ligands, diimine ligands, bisoxazoline ligands, alkyl
phosphine
ligands, aryl phosphine ligands, tertiary amine ligands, isocyanide ligands
and
cyanide ligands.
In yet another preferred embodiment of this method, the first a-aminoacid-N-
carboxyanhydride monomer is an NCA is an a-aminoacid-N-carboxyanhydride
selected from the group consisting of side-chain protected NCA formed from
arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine,
histidine,
lysine, methionine, serine, threonine, tryptophan, and tyrosine or an amino
acid side
chain selected from the group consisting of side-chain NCA formed from
alanine,
glycine, isoleucine, leucine, phenylalanine, proline and valine.
Block copolypeptides
Another embodiment of the invention entails a block copolypeptide wherein
the number of overall monomer units (residues) in the block copolypeptide is
greater
than about 100; and the distribution of chain-lengths in the block copolymer
composition is at least about 1.01 < Mw/Mn < 1.25, where Mw/Mn = weight
average
molecular weight divided by number average molecular weight. In one
embodiment,
the block copolypeptide has 10 consecutive identical amino acids per block. In
a
preferred embodiment, the block copolypeptide is composed of amino acid
components g-benzyl-L-glutamate and e-carbobenzyloxy-L-lysine. In another
preferred embodiment, the copolypeptide is a poly(e-benzyloxycarbonyl-L-Lysine-
block-g-benzyl-L-glutamate), PZLL-b-PBLG, diblock copolymer. In yet another
preferred embodiment the copolypeptide is a poly(g-benzyl-L-glutamate-block-e-
benzyloxycarbonyl-L-Lysine-block-g-benzyl-L-glutamate) triblock copolymer. In
related embodiments, the number of consecutive monomer units (residues) in the
block copolypeptide is greater than about 50 or 100 or 500 or 1000 (see e.g.
the
examples disclosed in Tables 1 and 4). In another related embodiment, the
total
number of overall monomer units (residues) in the block copolypeptide is
greater
than about 200, or greater than about 500, or greater than about 1000 (see
e.g. the
examples disclosed in Tables 1 and 4).
Illustrative embodiments of the invention that are disclosed in the examples
below include diblock copolymers composed of amino acid components g-benzyl-L-
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glutamate and e-carbobenzyloxy-L-lysine. The polymers were prepared by
addition
of Lys-NCA to bipyNi(COD) in DMF to afford living poly(e-carbobenzyloxy-L-
lysine),
PZLL, chains with organometallic end-groups capable of further chain growth.
Glu-
NCA was added to these polymers to yield the PBLG-PILL block copolypeptides.
The evolution of molecular weight through each stage of monomer addition was
analyzed using gel permeation chromatography (GPC) and data are given in Table
1
in Example 3 below. Molecular weight was found to increase as expected upon
growth of each block of copolymer while polydispersity remained low,
indicative of
successful copolymer formation. A. Noshay, et al., Block Copolymers, Academic
Press, New York, (1977).
The chromatograms of the block copolypeptides showed single sharp peaks
illustrating the narrow distribution of chain lengths (See Figure 2).
Copolypeptide
compositions were easily adjusted by variation of monomer feed compositions,
both
being equivalent. Successful preparation of copolypeptides of reverse sequence
(i.e. PZLL-PBLG) and of triblock structure (e.g. PBLGo,39-b-PZLLo.22-b-
PBLGo.39; M~ _
256,000, MW/Mn = 1.15) illustrate the sequence control using the nickel
initiator.
Block copolymerizations were not restricted to the highly soluble polypeptides
PBLG and PZLL. Copolypeptides containing L-leucine and L-proline, both of
which
form homopolymers which are insoluble in most organic solvents (e.g. DMF) have
also been prepared. Data for these copolymerizations are given in Table 1 in
Example 2 below. Because of the solubilizing effect of the PBLG and PZLL
blocks,
all of the products were soluble in the reaction media indicating the absence
of any
homopolymer contaminants. The block copolymers containing L-leucine were found
to be strongly associating in 0.1 M Liar in DMF, a good solvent for PBLG and
PZLL.
Once deprotected, the assembly properties of these materials are expected make
them useful as tissue engineering scaffolds, drug carriers, and morphology-
directing
components in biomimetic composite formation.
As discussed above, embodiments of the present invention provide a number
of novel methods and compositions for the generation of polypeptides having
varied
material properties. The description and illustrative examples disclosed
herein
provide a number of exemplary embodiments of the invention. Specific
embodiments of the invention include methods of adding aminoacid-N-
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carboxyanhydrides (NCAs) to polyaminoacid chains by exposing the NCA to
solutions containing polyaminoacid chains having an amido amidate metallacyle
end
groups and reacting the NCA with the amido amidate metallacyle end group so
that
the NCA is added to the polyaminoacid chain. Addition embodiments include
methods of controlling the polymerization of aminoacid-N-carboxyanhydrides by
reacting NCAs with initiator molecules and allowing initiator complexes to
regioselectively open the ring of the NCAs through oxidative addition across
the O-
C5 or O-C2 anhydride bond resulting a controlled polypeptide polymerization.
Other
embodiments include methods for making amido-containing metallacycles are
disclosed herein. Additional embodiments of the invention include compositions
for
use in peptide synthesis and design including five and six membered amido-
containing metallacycles and block copolypeptides.
In addition to block copolypeptides, a variety of other related of
polypeptides
can be generated utilizing the methods disclosed herein wherein an initiator
molecule combines with a first aminoacid-N-carboxyanhydride monomer to form an
amido containing metallacycle intermediate of the five and six ring formulae
disclosed herein. For example polypeptides can be generated where the domains
can either be repeats (2 or greater) of identical amino acids, or can be
repeats (2 or
greater) of mixtures of distinct amino acids, or a combination of the two. The
number, length, order, and composition of these domains can vary to include
all
possible amino acids in any number of repeats. Preferably the total number of
overall monomer units (residues) in these polypeptides having segregated
domains
of mixed monomers is greater than 100 and the distribution of chain-lengths in
the
polypeptide is about 1.01 < Mw/Mn < 1.25, where Mw/Mn = weight average
molecular weight divided by number average molecular weight.
An illustrative example of such a polypeptide could contain a sequence of, for
example, 50 residues of leucine in one domain, followed by a statistical
mixture of 20
valines and 20 glycines as the second domain, followed finally by a third
domain of
40 phenylalanines. Such polypeptides are substantially different than
"statistically
random" copolymers where the entire polypeptide is composed of statistical
mixtures
of amino acids in the chains, and there are no strict block domains. One
difference
is that these polypeptides have segregated domains where one statistical
mixture
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42
will be separated from the others. For example in a statistical copolymer, the
amino
acids will be distributed statistically (basically at random) along the entire
polypeptide
chain. In contrast, using the methods disclosed herein a polypeptide chain can
be
constructed such that in one domain there will be a statistical mixture of
leucine and
glycine, followed by a second domain consisting of a statistical mixture of
glycine
and valine. Although both copolymers have statistical mixtures of residues
along the
chain, these polypeptide differ in that the valine and leucine residues are
segregated
into separate domains.
[The living polymerization methods for NCAs that are disclosed herein will
lead to various polypeptides and block copolypeptides having a variety of new
and
useful properties. In this context, the disclosure provided herein
demonstrates the
successful synthesis of such materials, and creates a new family of
polypeptides that
link combination of acidic, basic and hydrophobic domains, all with excellent
control
of molecular architecture. The prospects for application in biomedical
engineering,
drug delivery and selective separations are excellent. In particular these
features
allow the preparation of complex polypeptide biomaterials which have potential
applications in biology, chemistry, physics, and materials engineering.
Potential
applications include medicine (drug delivery, tissue engineering), Osmart~ 2
hydrogels
(environmentally responsive organic materials), and in organic/inorganic
biomimetic
composites (artificial bone, high performance coatings).
Self-assembling amphiphilic block copolypeptides for biomedical applications
Yet another embodiment of the present invention entails the synthesis of
amphiphilic block copolypeptides, which contain at least one water soluble
block
polypeptide ("soluble block") conjugated to a water-insoluble polypeptide
domain
("insoluble block"). The soluble block includes oligo (ethyleneglycol)
terminated
amino acid (EG-aa) residues, whereas the insoluble block is primarily composed
of
nonionic amino acid residues. The amphiphilic nature of the polymers gives
them
the ability to self-assemble into a variety of well-defined structures in
aqueous
solutions, not unlike the assembly of small molecule lipids and surfactants.
The amphiphilic block copolypeptides of the present invention contain one or
more "soluble blocks." The amino acid composition of these soluble blocks
includes
one or more oligo(ethyleneglycol) functionalized amino acid residues.
Preferred
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oligo(ethyleneglycol) functionalized amino acid residues include EG-Lys, EG-
Ser,
EG-Cys, and EG-Tyr. A most preferred soluble block consists of
oligo(ethyleneglycol) terminated poly(lysine).
The amphiphilic block copolypeptides of the present invention also contain at
least one "insoluble block,"which is covalently linked to the soluble block.
The
insoluble block can contain a variety of amino acids residues or mixtures
thereof,
including the naturally occurring amino acids, ornithine, or blocks consisting
entirely
of one or more D-isomers of the amino acids. However, the insoluble block will
typically be composed primarily of nonionic amino acid residues, which
generally
form insoluble high molecular weight homopolypeptides. Such nonionic amino
acids
include, but are not limited to phenylalanine, leucine, valine, isoleucine,
alanine,
serine, threonine and glutamine. In a preferred embodiment, any given
insoluble
block will usually contain 2 - 3 different kinds of amino acid components in a
statistically random sequences.
Vesicle formation
The amphiphilic block copolypeptides of the present invention are included in
methods and compositions of matter, which form vesicular structures in aqueous
solutions. The method consists of suspending the amphiphilic block
copolypeptides
in an aqueous solution so that the copolypeptides spontaneously self assemble
into
vesicles. Accordingly, the vesicle-containing compositions are comprised of
the
amphiphilic block copolypeptides of the present invention and water.
The vesicles range in controllable size from microns to less than a hundred
nanometers in diameter, similar to liposomes yet more robust, which make them
potentially valuable for biomedical/biotechnological applications (i.e. drug
delivery).
In one specific embodiment, smaller vesicles having a diameter of about 50 nm
to
about 500 nm can be formed by sonicating the suspension of larger vesicles. In
addition, the diameter of the vesicles can be controlled by adjusting the
length and
amino acid composition of the amphiphilic block copolypeptide (see, e.g., the
examples below).
EXAMPLES
The following examples are offered by way of illustration and not by way of
limitation. The disclosures of all citations in the specification are
expressly
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incorporated herein by reference into this application in order to more fully
describe
the state of the art to which this invention pertains.
EXAMPLE 1 - Methods Using Amino Acid Derived Metallacycles: Intermediates in
Metal Mediated Polypeptide Synthesis
General Experimental Protocols and Reagents
Infrared spectra were recorded on a Perkin Elmer 1605 FTIR
Spectrophotometer calibrated using polystyrene film. Tandem gel permeation
chromatography/light scattering (GPC/LS) was performed on a Spectra Physics
Isochrom liquid chromatograph pump equipped with a Wyatt DAWN DSP light
scattering detector and Wyatt Optilab DSP. Separations were effected by 105,4
and
103A Phenomenex 5p columns using 0.1 M Liar in DMF eluent at 60 °C.
Optical
rotations were measured on a Perkin Elmer Model 141 Polarimeter using a 1 mL
volume cell (1 dm length). NMR spectra and bulk magnetic susceptibility
measurements (Evans method) were measured on a Bruker AMX 500MHz
spectrometer. [D.F. Evans, J. Chem. Soc., 2003-2009 (1959); J.K. Becconsal, J.
Mol. Phys., 15:129-135 (1968)]. C, H, N elemental analyses were performed by
the
Microanalytical Laboratory of the University of California, Berkeley Chemistry
Department. Chemicals were obtained from commercial suppliers and used without
purification unless otherwise stated. (COD)ZNi was obtained from Strem
Chemical
Co., and'3C~-L-leucine and '3C-phosgene were obtained from Cambridge Isotope
Labs. L-leucine isoamylamide hydrochloride, g-benzyl-L-glutamate NCA and L-
leucine NCA were prepared according to literature procedures. M. Bodanszky, et
al.,
The practice of Peptide Synthesis, 2"d Ed., Springer, BerIiniHeidelberg,
(1994); E.R.
Blout, et al., J. Am. Chem Soc., 78:941-950 (1956); H. Kanazawa, et al., Bull.
Chem
Soc. Jpn., 51:2205-2208 (1978). Hexanes, THF, and THF-d8 were purified by
distillation from sodium benzophenone ketyl. DMF and DMF-d~ were purified by
drying over 4A molecular sieves followed by vacuum distillation.
(S)-f NiNHC(H)RC(O)NCH2R]X, R = -CH2CHZC O OCH2C6H5; 1
In the dry box, Glu NCA (15 mg, 0.058 mmol) was dissolved in THF (0.5 mL)
and added to a stirred homogeneous mixture of PPh3 (31 mg, 0.12 mmol) and
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(COD)2Ni (16 mg, 0.058 mmol) in THF (1.5 mL). The red/brown solution was
stirred
for 24 hours, after which the solvent was removed in vacuo to leave a dark red
oily
solid. This was extracted with hexanes (3 x 5 mL) to yield a red/brown hexanes
solution and a yellow solid. Evaporation of the hexanes solution gave a red
oil
containing (PPh3)2Ni(CO)2 [1R (THF): 2000, 1939 cm-1 (_nCO, vs); 18 mg;
Literature:
IR (CH2CICH2C1): 1994, 1933 cm-1)], and drying of the solid gave the product
as a
yellow powder (10 mg, 75 % yield). J. Chatt, et al., J. Chem. Soc., 1378-1389
(1960). An'H NMR spectrum could not be obtained in THF-d8, most likely because
of paramagnetism of the complex (only broad lines for the benzyl ester groups
were
observed). meff(THF, 293 K) = 1.08 me. Osmotic molecular weight in THF (vs.
ferrocene; ca. 7 mg/mL): 910 g/mol; this corresponds to a degree of
aggregation of
1.94. 1R (THF): 3281 cm-1 (nNH, s br), 1734 cm-1 (nCO, ester, vs), 1577 cm-1
(nCO, amidate, vs). Anal. calcd. for NiC23H2sN205: 58.87%C, 5.59%H, 5.96%N;
found: 59.07%C, 5.67%H, 5.56%N. [a]p2° (THF, c = 0.0034) _ -71.
(S)- CI-+H3NC(H)RC(O)NHCHZR, R = -CH2CH(CH3~2
In the dry box, L-leucine NCA (9.2 mg, 0.058 mmol) was dissolved in THF
(0.5 mL) and added to a stirred homogeneous mixture of PPh3 (31 mg, 0.12 mmol)
and (COD)2Ni (16 mg, 0.058 mmol) in THF (1.5 mL). The red/brown solution was
stirred for 24 hours, after which the solvent was removed in vacuo to leave a
dark
red oily solid. This was extracted with cold hexanes (0 °C, 3 x 2 mL)
to yield a
red/brown hexanes solution and a pale orange solid. Evaporation of the hexanes
solution gave a red oil containing (PPh3)2Ni(CO)2 [1R (THF): 2000, 1939 cm-1
(nCO,
vs); 17 mg], and drying of the solid gave an orange powder which could be
purified
by precipitation from THF/hexanes to give (S)-[NiNHC(H)RC(O)NCH2R]X, R = -
CH2CH(CH3)2 as a yellow powder (6 mg, 80 % yield). An'H NMR spectrum could
not be obtained in THF-d8, most likely because of paramagnetism of the
complex. 1R
(THF): 3290 cm-1 (nNH, s br), 1580 cm-1 (nCO, amidate, vs). [a]p2°
(THF, c =
0.001 ) _ -185.
This product was dissolved in THF (5 mL) in a round bottom Schlenk flask in
the dry box. The flask was placed under N2 atmosphere on a Schlenk line and
HCI
(90 mL of a 1.0M solution in EtzO) was then added. The yellow solution turned
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orange and then became hazy as it slowly turned green. After 2h, the solvent
was
removed in vacuo to leave a green gummy solid. This solid was extracted with
D20
to isolate the amino acid containing products. The single isolated compound (4
mg,
73 %) was found to be identical to an authentic sample of L-leucine
isoamylamide
hydrochloride (Figure 1). 'H NMR (D20): d 3.94 (t, NH3CH(CH2CH(CH3)2)C(O)-,
1H,
J = 7.5 Hz), 3.33, 3.14 (dm, -C(O)NHCH2CH2CH(CH3)2, 2H, Jgem = 107 Hz, Jmutt =
6
Hz, 13 Hz), 1.72 (dd, NH3CH(CHZCH(CH3)2)C(O)-, 2H, J = 6 Hz, 7 Hz), 1.68 (m,
NH3CH(CH2CH(CH3)2)C(O)-, 1 H, J = 7 Hz~, 1.63 (m, -C(O)NHCH2CH2CH(CH3)2, 1 H,
J = 7 Hz), 1.43 (ddd, -C(O)NHCH2CH2CH(CH3)2, 2H, J = 7 Hz), 0.98, 0.96 (dd,
NH3CH(CH2CH(CH3)2)C(O)-, 6H, J = 6 Hz), 0.92, 0.90 (dd, -
C(O)NHCH2CH2CH(CH3)2, 6H, J = 6 Hz). (a]p2° (THF, c = 0.0033) _
+10.3.
Authentic Sample: [a]p2° (THF, c = 0.0033) _ +10.5.
Reaction of (PPh3~2Ni(COD) with'3C2-L-Leucine NCA
The procedure given above for the reaction using unlabeled L-leucine NCA
was followed exactly, except for the substitution of'3C2-L-Leucine NCA
[prepared
from L-leucine and O'3CC12; IR (CHC13): 3299 cm-1 (nNH, s br), 1836, 1745 cm-1
(nCO, anhydride, vs);'3C f'H} NMR (THF-d8): d 152 (s, -NC(O)O-]. The product
was
extracted with cold hexanes (0 -°C, 3 x 2 mL) to yield a red/brown
hexanes solution
and a pale orange solid. Evaporation of the hexanes solution gave a red oil
containing (PPh3)2Ni(CO)2 [1R (THF): 2000, 1939 cm-1 (nCO, vs)], and drying of
the
solid gave an orange powder which could be purified by precipitation from
THF/hexanes to give (S)-[NiNHC(H)RC(O)NCH2R]X, R = -CH2CH(CH3)2 (5 mg, 66
yield). 1R (THF): 3288 cm-1 (nNH, s br), 1580 cm-1 (nCO, amidate, vs).
Reaction of (PPh3~2Ni(COD) with '3C5-L-Leucine NCA
The procedure given above for the reaction using unlabeled L-leucine NCA
was followed exactly, except for the substitution of'3C5-L-Leucine NCA
[prepared
from'3C,-L-leucine and OCC12; IR (KBr): 3308 cm-1 (nNH, s br), 1818, 1763 cm-1
(nCO, anhydride, vs);'3C {'H~ NMR (THF-d$): d 171 (s, -CHRC(O)O-]. The product
was extracted with cold hexanes (0 -°C, 3 x 2 mL) to yield a red/brown
hexanes
solution and a pale orange solid. Evaporation of the hexanes solution gave a
red oil
containing (PPh3)2Ni('3C0)2 ['3C{'H~NMR (THF-d$): d 202 (t, Ni('3C0)2, JP_c =
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15Hz); IR (THF): 1954, 1895 cm-1 (n'3C0, vs)], and drying of the solid gave an
orange powder which could be purified by precipitation from THF/hexanes to
give
(S)-[NiNHC(H)R'3C(O)NCH2R]X, R = -CH2CH(CH3)2 (6 mg, 80 % yield). 1R (THF):
3290 cm-1 (nNH, s br), 1536 cm-1 (n'3C0, amidate, vs). '3C ~'H} NMR (THF-d8):
d
182 (s, [NiNHC(H)R'3C(O)NCH2R]X).
(S)-(2,2'-bipyridyl)NiNHC(H)RC(O)NCH2R, R = -CH2CH2C O OCH2C6H5 2
In the dry box, a yellow solution of (S)-[NiNHC(H)RC(O)NCH2R]X, R = -
CH2CH2C(O)OCH2C6H5 (40 mg, 0.085 mmol) in DMF (0.5 mL) was added to a
solution of 2,2'-bipyridyl (54 mg, 0.35 mmol) in DMF (0.5 mL). The homogeneous
mixture was stirred for 2d at 50 -°C, during which the color changed
from yellow to
blood red. THF (1 mL) and toluene (5 mL) were layered onto this solution
resulting
in precipitation of a red powder. This powder was reprecipitated from
DMF/THF/toluene (1:2:10) two additional times to give (S)-(2,2'-
bipyridyl)NiNHC(H)RC(O)NCH2R, R = -CH2CH2C(O)OCH2C6H5 as a red powder (49
mg, 92 % yield). An'H NMR spectrum could not be obtained in THF-d8, most
likely
because of paramagnetism of the complex (only broad lines for the benzyl ester
groups were observed). 1R (THF): 3281 cm-1 (nNH, s br), 1732 cm-1 (nCO, ester,
vs), 1597 cm-1 (nCO, amidate, vs). Anal. calcd. for NIC33H34N4O5: 63.37%C,
5.49%H, 8.95%N; found: 63.72%C, 5.49%H, 8.86%N. [a]o2° (THF, c = 0.001)
_ -
135.
Polymerization of Glu-NCA using (S)-(2,2'-bipyridyl)NiNHC(H)RC(O)NCH2R
R=-CH2CH2C(O)OCH2C6H5
In the dry box, Glu NCA (50 mg, 0.2 mmol) was dissolved in DMF (0.5 mL)
and placed in a 25 mL reaction tube which could be sealed with a Teflon
stopcock.
An aliquot of (S)-(2,2'-bipyridyl)NiNHC(H)RC(O)NCH2R, R =
-CH2CH2C(O)OCH2C6H5 (50 ml of a 40 mM solution in DMF) was then added via
syringe to the flask. A stirbar was added and the flask was sealed, removed
from
the dry box, and stirred in a thermostated 25 °C bath for 16 hours.
Polymer was
isolated by addition of the reaction mixture to methanol containing HCI (1 mM)
causing precipitation of the polymer. The polymer was then dissolved in THF
and
reprecipitated by addition to methanol. The polymer was dried in vacuo to give
a
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48
white solid, PBLG (39 mg, 93 % yield). '3C {'H} NMR,'H NMR, and FTIR spectra
of
this material were identical to data found for authentic samples of PBLG. H.
Block,
Poly(g-benzyl-L-glutamate) and Other Glutamic Acid Containing Polymers, Gordon
and Breach, New York, (1983). GPC of the polymer in 0.1 M Liar in DMF at 60
°C:
M~ = 21,600; M,N/M" = 1.09.
Reaction of (2,2'-bipyridyl)Ni(COD) with '3C2-L-Leucine NCA
In the dry box, five equivalents of'3C2-L-Leucine NCA (14.5 mg, 0.091 mmol)
was added to a solution of bipyNi(COD) (5.9 mg, 0.018 mmol) in THF (1 mL). The
mixture slowly turned from purple to red and was stirred for 16 hours. The
crude
product was isolated by evaporation of the solvent to yield a red oily solid.
FTIR
analysis of the crude reaction mixture confirmed the presence of (2,2'-
bipyridyl)Ni(CO)2 [1R (THF): 1978, 1904 cm-1 (nCO, vs); Literature: IR
(diethyl ether):
1983, 1914 cm-1)], polyleucine [1R (THF): 1653 cm-1 (nAmide I, vs); 1546 cm-1
(nAmide II, vs)] as well as the '2C-amidate endgroup [IR(THF): _n(CO) = 1577
cm-'].
R.S. Nyholm, et al., J. Chem Soc., 2670 (1953). The reaction was also run in
DMF-
d~ (0.5 mL) under otherwise identical conditions. '3C {'H} NMR (DMF-d~): d 126
(s,
'3C02).
Reaction of (2,2'-bipyridyl)Ni(COD) with'3C5-L-Leucine NCA
In the dry box, five equivalents of'3C5-L-Leucine NCA (14.5 mg, 0.091 mmol)
was added to a solution of bipyNi(COD) (5.9 mg, 0.018 mmol) in THF (1 mL). The
mixture slowly turned from purple to red and was stirred for 16 hours. The
crude
product was isolated by evaporation of the solvent to yield a red oily solid.
FTIR
analysis of the crude reaction mixture confirmed the presence of (2,2'-
bipyridyl)Ni('3C0)2 [1R (THF): 1933, 1862 cm-1 (nCO, vs)J as well as '3C-
labeled
polyleucine [1R (THF): 1613 cm-1 (nAmide I, vs); 1537 cm-1 (nAmide II, vs)].
The
reaction was also run in DMF-d7 (0.5 mL) under otherwise identical conditions.
'3C
{'H} NMR (DMF-d~): d 198 (s, bipyNi('3CO)2); 177 (s,
bipyNiN(H)C(H)R'3C(O)N(CH(R)'3C(O)NH]~CH2R), 174 (s, bipyNiN(H)C(H)-
R'3C(O)N[CH(R)'3C(O)NH]"CHzR).
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Polymerization of Glu-NCA with (2,2'-bipyridyl)Ni(COD)
In the dry box, Glu NCA (50 mg, 0.2 mmol) was dissolved in DMF (0.5 mL)
and placed in a 25 mL reaction tube which could be sealed with a Teflon
stopcock.
An aliquot of bipyNi(COD) (50 ml of a 40 mM solution in DMF) was then added
via
syringe to the flask. A stirbar was added and the flask was sealed, removed
from
the dry box, and stirred in a thermostated 25 °C bath for 16 hours.
Polymer was
isolated by addition of the reaction mixture to methanol containing HCI (1 mM)
causing precipitation of the polymer. The polymer was then dissolved in THF
and
reprecipitated by addition to methanol. The polymer was dried in vacuo to give
a
white solid, PBLG (41 mg, 98 % yield). '3C {'H} NMR,'H NMR, and FTIR spectra
of
this material were identical to data found for authentic samples of PBLG. H.
Block,
Poly(g-benzyl-L-glutamate) and Other Glutamic Acid Containing Polymers ,
Gordon
and Breach, New York, (1983). GPC of the polymer in 0.1 M Liar in DMF at 60
°C:
Mn = 22,100; M,N/M~ = 1.15.
As discussed above, a-Amino acid-N-carboxyanhydrides (NCAs) were
reacted with zerovalent nickel complexes of the type L2Ni(COD) to yield
metallacyclic
oxidative addition products. These oxidative addition reactions were found to
result
in the addition across either the O-C5 or the O-C2 bond of the NCAs,
ultimately
giving, after addition of a second equivalent of NCA, chiral amido-amidate
nickelacycles. The origins and structures of these complexes were elucidated
by
use of selectively'3C labeled NCA reagents. Stable metallacycles were obtained
when L = PPh3. When other donor ligands were used, the metallacycle
intermediates were found to quickly react with additional NCA molecules to
form
polypeptides in quantitative yield and with narrow molecular weight
distributions.
These reactions provide a facile route to unusually stable metallacyclic amido-
containing nickel intermediates.
When two equivalents of PPh3 and one Ni(COD)2 were reacted with one
equivalent of g-benzyl-L-glutamate-N-carboxyanydride (Glu-NCA) in THF at room
temperature, rapid consumption of the NCA was observed. From the golden brown
solution, an alkane soluble brown oil and a THF soluble yellow powder were
isolated.
Analysis of the oil confirmed the presence of (PPh3)2Ni(CO)2 [IR(THF): -n(CO)
_
2000, 1939 cm-'] which was produced by the decarbonylation of an intermediate
six-
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membered metallacycle followed by trapping of the carbon monoxide with
(PPh3)2Ni(COD). H. Kanazawa, et al., Bull. Chem Soc. Jpn., 51:2205-2208
(1978).
Infrared analysis of the yellow powder showed carbonyl stretches at 1734 and
1577
cm-' which were assigned, respectively, to the side-chain benzyl esters and
amidate
group of the chiral nickelacycle (see scheme 2 of Figure 3).
The structures and origins of these products were elucidated when'3C5-L-
leucine-N-carboxyanhydride was reacted with (PPh3)2Ni(COD) in THF. An infrared
spectrum of the crude reaction mixture showed a stretch at 1536 cm-' for
the'3C-
amidate group ['3C {'H} NMR (THF-d8): 182 ppm] as well as (PPh3)2Ni('3C0)2
stretches at 1954 and 1895 cm-' ['3C {'H~ NMR (THF-d$): 202 ppm] which were
isotopically shifted from the unlabeled compounds (see equation 1 of Figure
4).
When'3C2-L-leucine-N-carboxyanhydride was reacted with (PPh3)2Ni(COD) in THF,
analysis of the products showed exclusive formation of (PPh3)2Ni('2C0)2
[IR(THF):
_n(CO) = 2000, 1939 cm-'] and the'2C amidate [IR(THF): _n(CO) = 1580 cm-']
(see
equation 2 of Figure 4). Since no mixed'3C/'2C products were observed, it was
concluded that oxidative addition was occurring either at the C5-O or the C2-O
bond
followed by decarbonylation and addition of a second NCA molecule to yield an
amido-containing nickelacycle (see scheme 2 of Figure 3).
While not being bound to a specific mechanism or theory, the transformation
of the initially formed 6-membered amido-alkyl nickelacycle to a 5-membered
amido-
amidate nickelacycle might occur through a proton-transfer mediated ring
contraction
induced by addition of an additional NCA monomer (see equation Figure 4). Such
a
transformation has been observed for related nickel complexes.
Furthermore, quenching of polymerization reactions with DCI and HCI has
shown conclusively that the nickel-alkyl bond of the 6-membered metallacycle
is not
present after addition of NCA molecules to the initial metallacycle, providing
additional supporting evidence for the ring-contraction to the amido-amidate
structure.
The structure of these metallacyclic products was further confirmed by
elemental analysis and acidolysis of the complexes. The product metallacycles
contain no phosphine by elemental analysis and were found to consist of the
empirical formula (NiNHC(H)RC(O)NCH2CHR]X. Osmotic molecular weight
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measurements in THF (ca. 7 mg/mL ) showed that the complexes aggregate as
dimers. Treatment of the metallacyclic complex derived from L-leucine NCA with
HCI in THF gave only a single organic product. Analysis of this product by'H
NMR
spectroscopy and polarimetry, and comparison of the data with an authentic
sample,
showed it to be optically pure L-leucine isoamylamide~HCl (see scheme 2 of
Figure
3).
When the donor ligands bound to the nickel (0) precursor were varied (e.g.
alkyl phosphines, a,a'-diimines), the only products isolable from
stoichiometric
reactions with Glu-NCA in THF were some starting nickel(0) compound and poly(g-
benzyl-L-glutamate), PBLG. When 100 equivalents of Glu-NCA was added to
bipyNi(COD) in DMF, all of the nickel precursor was consumed and PBLG was
isolated in excellent yield (>95%) with narrow molecular weight distribution
(M~ _
22,100, MW/Mn = 1.15). L2 = donor ligand(s); COD = 1,5-cyclooctadiene; bipy =
2,2'-
bipyridyl. It has been shown that bipyNi(COD) initiates the living
polymerization of
NCAs. T.J. Deming, Nature, 390:386-389 (1997). It was suspected that
bipyNi(COD) oxidatively adds Glu-NCA to form the active polymerization
initiator in
situ which then rapidly consumes the remainder of the monomer. To identify
this
active initiator, a series of experiments were performed where bipyNi(COD) was
reacted with selectively'3C labeled NCA monomers.
In order to completely consume all the bipyNi(COD) in reactions with NCAs, at
least a fivefold excess of NCA monomer was used. BipyNi(COD) was reacted with
five equivalents of 3C5-L-leucine-N-carboxyanhydride in THF. 1R and '3C {'H}
NMR
analysis of the crude products verified the presence of bipyNi('3C0)2
[IR(THF):
n('3C0) = 1933, 1862 cm-'; '3C {' H} NMR (DMF-d~): 198 ppm], '3C-labeled poly-
L-
leucine [1R (THF): 1613 cm-1 (nAmide I, vs); 1537 cm-1 (nAmide II, vs);'3C
{'H}
NMR (DMF-d7): 177 ppm (bipyNiN(H)C(H)R'3C(O)N[CH(R)'3C(O)-NH]~CH2R)], and
the labeled nickel-amidate endgroup ['3C {'H} NMR (DMF-d~): 174 ppm
(bipyNiN(H)C(H)R'3C(O)N-[CH(R)'3C(O)NH]~CH2R)]. The reaction with '3C2-L-
leucine-N-carboxyanhydride gave similar products, except for location of
the'3C
label. The presence of bipyNi('2C0)2 [IR(THF): n(CO) = 1978, 1904 cm-'],'2
poly-L-
leucine [1R (THF): 1653 cm-1 (nAmide I, vs); 1546 cm-1 (nAmide II, vs)],'3 as
well as
the'zC-amidate endgroup [IR(THF): n(CO) = 1577 cm-'] was identified. When the
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52
reaction was run in DMF-d~, the presence of liberated '3C02 was also confirmed
using'3C ~'H~ NMR [126 ppm (s,'3C02)].
All of these experiments were consistent with initial addition of the NCA to
bipyNi(COD) across the C5-O bond, analogous to the reactions using
(PPh3)2Ni(COD). The primary influence of the ligands manifests itself in the
reactivity of the resulting products. The ligand free complex from the PPh3
reaction
was inert toward further reactivity with NCAs, while the bipy complex and
complexes
formed with other a,a'-diimines and alkyl phosphines were efficient NCA
polymerization initiators. This phenomenon was directly verified by synthesis
of the
reactive metallacycle intermediate formed in the bipyNi(COD)/NCA reactions.
The
stable metallacycle (S)-[NiNHC(H)RC(O)NCH2R]X, R = -CH2CH2C(O)OCH2C6H5)
was reacted with an excess of bipy in DMF to form the ligand adduct (S)-(2,2'-
bipyridyl)NiNHC(H)RC(O)NCHZR, R = -CH2CH2C(O)OCH2C6H5 (see scheme 2 of
Figure 3). Reaction of (S)-(2,2'-bipyridyl)NiNHC(H)RC(O)NCH2R, R = -
CH2CH2C(O)OCH2C6H5 with 100 equivalents of Glu-NCA in DMF resulted in rapid
polymer formation. The PBLG formed in this reaction was identical to that
formed
using bipyNi(COD) under otherwise identical conditions (Mn = 21,600, MW/M~ =
1.09).
The bipyNi(COD) mediated polymerizations of NCAs are therefore thought to
proceed via amido-amidate nickelacycle active endgroups (see equation 4 of
Figure
4).
EXAMPLE 2 - Initiators for Chain-End Functionalized PolVpeptides and Block
Copolypeptides
Na allyloxycarbonyl-amino acid amides were reacted with zerovalent nickel
complexes LNi(1,5-cyclooctadiene) (L = 2,2'-bipyridine (bpy), 1,10-
phenanthroline
(phen), 1,2-bis (dimethylphosphino)ethane (dmpe), and 1,2-
bis(diethylphosphino)ethane (depe)), to yield amido-amidate metallacycles of
the
general formula: LNiNHC(R')HC(O)NR". These complexes were found to initiate
polymerization of a-amino acid-N-carboxyanhydrides (NCAs) yielding
polypeptides
with defined molecular weights, narrow molecular weight distributions, and
with
quantitative incorporation of the initiating ligand as an end-group. A napthyl
substituent was incorporated as a fluorescent end-group to demonstrate the
feasibility of this methodology.
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General Experimental Protocols and Reagents
Infrared spectra were recorded on a Perkin Elmer 1605 FTIR
Spectrophotometer calibrated using polystyrene film. Tandem gel permeation
chromatography/light scattering (GPC/LS) was performed on an SSI Accuflow
Series
III liquid chromatograph pump equipped with a Wyatt DAWN DSP light scattering
detector and Wyatt Optilab DSP. Separations were effected by 105 A, 103 A, and
500 R Phenomenex 5p columns using 0.1 M Liar in DMF eluent at 60 °C.
NMR
spectra were measured on Bruker AVANCE 200MHz spectrometer. FAB Mass
Spectrometry was performed at the facility in the Chemistry Department at the
University of California, Santa Barbara. MALDI mass spectra were collected
using a
Thermo BioAnalysis DYNAMO mass spectrometer running in positive ion mode with
samples prepared by mixing solutions of analyte in TFA with solutions of 2,5-
dihydroxybenzoic acid in TFA and allowing the mixtures to air dry.
Fluorescence
measurements were conducted on a SPEX FluoroMax.-2. Chemicals were obtained
from commercial suppliers and used without purification unless otherwise
stated.
Alloc-L-amino amides, E-CBZ-L-lysine NCA, (S)-phenylglycine NCA, and ~y-benzyl-
L-
glutamate NCA were prepared according to literature procedures (Tirrell, J.
G.;
Fournier, M. J.; Mason, T. L.; Tirrell, D. A. Chem. & Engr. News 1994, 72, 40-
51;
Viney, C.; Case, S. T.; Waite, J. H.. Biomolecular Materials, Mater. Res. Soc.
Proc.
292, 1992.; and Deming, T. J. J. Am. Chem. Soc., 1998,120, 4240-4241:
incorporated herein by reference). Hexanes, THF, and THF-d$ were purified by
first
purging with dry nitrogen, followed by passage through columns of activated
alumina
3 DMF and DMF-d7 were purified by drying over 4 A molecular sieves followed by
vacuum distillation.
Sample Procedure for Synthesis of Alloc-L-Amino Acid Amides: Alloc-L
Leucine-Isoamylamide
Isoamylamine (1.4 mL, 12 mmol) was added to a solution of Alloc-L-leucine-
N-hydroxysuccinimidyl ester (2.5 g, 8.0 mmol) in THF (5 mL). The reaction was
stirred for 1 hr after which the resulting precipitate was removed by
filtration and the
solution was diluted with ethyl acetate (100 mL). This solution was
sequentially
washed with dilute aqueous HC1 (2 x 30 mL), saturated aqueous NaHC03 (2 x 30
mL), and then saturated aqueous NaCI (2 x 30 mL) followed by drying over
MgS04.
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54
The solvent was then evaporated in vacuo to leave the product (1.7 g, 77 %).
IR(THF): 1724 cm-' (vCO, Alloc, s), 1674 cm-' (vCO, amide, s).'H NMR(CDC13): S
6.00 (br s, (CH3)2_CHCH2CH(NHC(O)OCH2CH=CH2)C(O)NHCH2CH2CH(CH3)2~2H),
5.85 (m, (CH3)2-CHCH2CH(NHC(O)OCH2CH=CH2)C(O)NHCH2CH2CH(CH3)2~1 H),
5.25 (t, (CH3)2-CHCH2CH(NHC(O)OCH2CH=CHZ)C(O)NHCH2CH2CH(CH3)z~2H),
4.57 (d, CH3CHCH2CH(NHC(O)OCH2CH=CH2)C(O)NHCH2CH2CH(CH3)2, 2H), 4.12
(m, (CH3)2CHCH2CH(NHC(O)OCH2CH=CH2)C(O)NHCH2CH2CH(CH3)2, 1 H), 3.24
(q, (CH3)2CHCH2CH(NHC(O)OCH2CH=CHZ)C(O)NHCH2CH2CH(CH3)2, 2H), 1.57
(m, (CH3)2CHCH 2CH(NHC(O)OCH 2CH=CH 2)C(O)NHCH2CH 2CH(CH3)2, 2H), 1.40
(m, (CH3)2CHCH2CH(NHC(O)OCH2CH=CH2)C(O)NHCH2CH 2CH(CH3)2, 4H), 0.92
(d, (CH3)2CHCH2CH(NHC(O)OCH2CH=CH2)C(O)NHCH2CH2CH(CH3)2, 12H).
(S)-phenNiNHC(H)RC(O)O. R = -CHZCH(CH3~2
1,10-Phenanthroline (phen) (13 mg, 0.073 mmol) was added to a suspension
of Ni(COD)2 (20 mg, 0.073 mmol) in DMF (2 mL and let stand at room temperature
for 30 min after which a solution of Phen-Ni(COD) had formed. Alloc-L-leucine
allyl
ester (20 mg, 0.073 mmol) was added to the purple solution, which subsequently
became brown in color. After standing at room temperature for 5 h the solution
was
green, indicative of formation of the single oxidative-addition product. The
green
solution was heated at 80 °C for 20 h to yield a purple solution. The
product was
isolated from this solution by precipitation into diethyl ether (10 mL)
followed by
washing with THF (2 x 10 mL) and drying-, in vacuo to give a purple powder (16
mg,
68%). IR(THF): 1620 cm-' (vCO, carboxylate, s br). An'H NMR spectrum could not
be obtained in DMF-d, most likely because of paramagnetism of the complex
(only
broad lines for the methyl groups were observed). ~e~(296K) = 2.05,x,8.
~S)-phenNiNHC(H)RC(O)NCH2R, R = -CHZCH(CH3~2 2
Phen (13 mg, 0.073 mmol) was added to a suspension of Ni(COD)2 (20 mg,
0.073 mmol) in DMF (2 mL) and let stand at room temperature for 30 min after
which
a solution of phenNi(COD) had formed. Alloc-L-leucine isoamyl amide (20 mg,
0.073
mmol) was then added to the purple solution, which subsequently became brown
in
color. After standing at room temperature for 5 h the solution was green,
indicative of
formation of the single oxidative-addition product. The green solution was
heated at
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80 °C for 20 h to yield a purple solution. The product was isolated
from this solution
by precipitation into diethyl ether (10 mL) followed by washing with THF (2 x
10 mL)
and drying in vacuo to give a purple powder (23 mg, 75%). IR(THF): 1578 cm-1
(vCO, amidate, s br). An'H NMR spectrum could not be obtained in DMF-d" most
likely because of paramagnetism of the complex (only broad lines for the
methyl
groups were observed).p.eff (296K) = 2.34 ~.~8.
(S)-phenNiNHC(H)R'3C(O)NCH2R, R = -CHZCH(CH3~2, 2-'3C
Phen (13 mg, 0.073 mmol) was added to a suspension of Ni(COD)2 (20 mg,
0.073 mmol) in DMF (2 mL) and let stand at room temperature for 30 min after
which
a solution of phenNi(COD) had formed. '3C(amide)-alloc-L-leucine isoamyl amide
(20 mg, 0.073 mmol) was then added to the purple solution, which subsequently
became brown in color. After standing at room temperature for 5 h the solution
was
green, indicative of formation of the single oxidative- addition product. The
green
solution was heated at 80°C for 20 h to yield a purple solution. The
product was
isolated from this solution by precipitation into diethyl ether (10 mL)
followed by
washing with THF (2 x 10 mL) and drying in vacuo to give a purple powder (22
mg,
70%) IR(THF): 1541 cm-' (v'3C0, amidate, s br).
(S)-depeNiNHC(H)R'C(O)NCH2R2-, R' _ -CH2~3~2, R2= -CH2CH3, 4
1,2-Bis(diethylphosphino)ethane, depe (17 ~.L, 0.073 mmol) was added to a
solution of Ni(COD)2 (20 mg, 0.073 mmol) in THF (1 mL) and let stand at room
temperature for 5 min after which a solution of depeNi(COD) had formed. Alloc-
L-
valine n-propyl amide (18.5 mg, 0.073 mmol) in DMF (1 mL) was then added to
the
yellow solution, which subsequently became orange-yellow in color. The
solution
was heated at 80 °C for 20 h to yield an orange solution. The solvent
was removed
in vacuo and the residue was redissolved in THF and isolated from this
solution by
precipitation into hexanes (10 mL). Drying of the solid in vacuo gave 4 as a
yellow
powder (16 mg, 53%). IR(THF): 1 578 cm-' (vCO, amidate, s br). An'H NMR
spectrum could not be obtained in DMF-d" most likely because of paramagnetism
of
the complex (only broad lines for the alkyl groups were observed).~eff (296K)
= 2.08
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Isolation of L-Leucine-HCI from (S)-phenNiNHC(H)RC(O)O, R =
CH2CH(CH322
Anhydrous 4M HC1 in dioxane solution (1.0 mL) was added to a solution of
(S)-PhenNiNHC(H)RC(O)O, R = -CH2CH(CH3)2 (10 mg, 0.027 mmol) in CH2C12. The
solution immediately changed color from purple to orange. It was stirred for 2
hours
and then the solvents were removed in vacuo. The remaining solid was extracted
with water and the insoluble nickel -containing residue was removed by
filtration. The
water was then removed by freeze-drying to yield the desired product. FAB-MS:
M-
CI-: 132.19 calcd, 132 found.
Isolation of L-Leucine Allylamide-HCI from (S)-bpyNiNHC(H)R~C(O)NR2 R2 =
'CH2C~3~2~ R2 = _CHzCH=CH2, 1
Anhydrous 4M HC1 in dioxane solution (1.0 mL) was added to a solution of 1
(10 mg, 0.025 mmol) in CH2CIz. The solution immediately changed color from
purple
to orange. It was stirred for 2 hours and then the solvents were removed in
vacuo.
The remaining solid was extracted with water and the insoluble nickel-
containing
residue was removed by filtration. The water was then removed by freeze-drying
to
yield the desired product. 'H NMR (D20): 8 5.91 (m, (CH3)2CHCH2CH(NH2)C(O)-
NHCH2CH=CH2, 1 H), 5.73 (t, (CH3)2CHCH2CH(NH2)-C(O)NHCH2CH=CH, 2H), 3.95
(m, (CH3)2CHCH2CH(NH2)C(O)NHCH2CH=CH2, 1 H), 3.81 (br s,
(CH3)2CHCH2CH(NH2)-C(O)NHCH2CH=CH2, 2H), 1.74 (d,
(CH3)2CHCH2CH(NH2)C(O)NHCH2CH=CH2 3H), 0.96 (d,
(CH3)2CHCH2CH(NH2)C(O)NHCH2CH=CH2, 6H). FAB-MS: M-CI-: 171.28 calcd, 171
found.
Isolation of L-Leucine Isoamylamide-HCI from (S)-phenNiNHC(H)RC(O)-
NCH2R, R = -CH2CH(CH3~2, 2
Anhydrous 4M HCI in dioxane solution (1.0 m-L) was added to a solution of 2
(10 mg, 0.024 mmol) in CH2C12. The solution immediately changed color from
purple
to orange. It was stirred for 2 hours and then the solvents were removed in
vacuo.
The remaining solid was extracted with water and the insoluble nickel-
containing
residue was removed by filtration. The water was then removed by freeze-drying
to
yield the desired product.'H NMR (D20): s 3.94 (t, NH3CH(CH2CH(CH3)2)C(O)-,
1H,
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57
J = 7.5 Hz), 3.3 3, 3.14 (dm, -C(O)NHCH2CHzCH(CH3)2, 2H, Jgen, = 10.7 Hz,
Jm~~t = 6
Hz, 13 Hz), 1.72 (dd, NH3CH(CH2CH(CH3)2C(O)-, 2H, J = 6 Hz, 7 Hz), 1.68 (m,
NH3CH-(CH2CH(CH3)z)C(O)-, 1 H, J = 7 Hz), 1.63 (m, -C(O)NHCH2CH2CH(CH3)2,
1 H, J = 7 Hz), 1.43 (ddd, -C(O)NHCH2CH2CH(CH3)2, 2H, J = 7 Hz), 0.98 (d,
NH3CH(CH2CH(CH3)2)-C(O)-, 3H, J = 6 Hz), 0.96 (d, NH3CH(CH2CH(CH3)2)C(O)-,
3H, J = 6 Hz), 0.92 (d, C(O)-NHCH2CH2CH(CH3)2, 3H, J = 6 Hz), 0.90 (d, -
C(O)NHCH2CH2CH-(CH3)2, 3H, J = 6 Hz). FAB-MS: M-CI-: 201.36 calcd, 201 found.
Polymerization of Glu NCA Using (S)-depeNiNHC(H)R'C(O)NR2 R' _ -
CH2CH(CH3~2s R2 = -1-Naphthyl, 3
In the dry box, y-benzyl-L-glutamate-N-carboxyanhydride, Glu NCA (50 mg,
0.2 mmol) was dissolved in DMF (1.0 mL) and placed in a 25 mL reaction tube
which
could be sealed with a Teflon stopcock. An aliquot of 3 (140 ~L of a 14 mM
solution
in DMF) was added via syringe to the flask. A stir bar was added and the flask
was
sealed, removed from the dry box and stirred at 25 °C in a thermostated
bath for 24
h. Polymer was isolated by addition of the reaction mixture to methanol
containing
HC1 (1 mM) causing precipitation of the polymer. The polymer was dried in
vacuo to
give a white solid, PBLG (19 mg, 90% yield)'3C~'H} NMR,'H NMR, and FTIR
spectra of this material were identical to data found for authentic samples of
PBLG.4
GPC of the polymer in 0.1 M Liar in DMF at 60 °C: M~= 26,100;
M,N/M~= 1.15.
Isolation of Mixed Hexenes From Oxidative Addition of N~-trans 2-
Hexenyloxycarbonyl-L-Leucine Isoamyl Amide to Nickel
A solution of trans 2hexenyloxycarbonyl-L-leucine isoamyl amide (231 mg,
0.015 mmol) in THF (0.5 mL) was added to a solution of (PPh3)4Ni (741 mg,
0.015
mmol) in THF (0.5 mL) resulting in a yellow-orange solution. This mixture was
heated at 80 °C for 2 days during which the color changed to dark
brown. The
volatiles of the reaction were then vacuum distilled into an NMR tube to
remove the
paramagnetic nickel products. The presence of a mixture of 1-hexene, 2-hexenes
and 3-hexenes in the distillate was verified by'3C NMR. The mixture of hexenes
was likely formed by facile isomerization of the intermediate ~3hexenyl-nickel
species
formed in the reaction. '3C NMR(THF): s 139.05 (CH3CH2CH2CH2CH=CH2), 132.15
(trans-CH3CHzCH=CHCH2CH3), 131.44 (CH3CH2CH2CH=CHCH3), 130.53 (trans-
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58
CH3CH2CH2CH=CHCH3), 128.56 (cis-CH3CH2CH=CHCHzCH3), 125.92 (cis-
CH3CH2CH2CH=CHCH3), 124.79 (trans-CH3CH2CH2CH=CHCH3), 114.10
(CH3CH2CH2CH2CH=CH2), 34.95 (trans-CH3CH2CH2CH=CHCH3), 33.73
(CH3CH2CH2CH2CH=CH2), 31.44 (CH3CH2CH2CH2CH=CH2), 29.71 (cis-
CH3CH2CH2CH=CHCH3), 22.89 (CH3CH2CH2CH2CH=CH2), 22.38 (trans-
CH3CH2CH2CH=CHCH3), 18.05 (trans-CH3CH2CH2CH=CHCH3), 15.04 (cis-
CH3CH2CH2CH=CHCH3), 14.44 (trans-CH3CH2CH=CHCH2CH3), 13.79 (cis-
CH3CH2CH2CH=CHCH3), 13.60 (CH3CH2CH2CH2CH=CHZ), 13.32 (trans-
CH3CH2CH2CH=CHCH3), 13.20 (cis-CH3CH2CH2CH=CHCH3).
Polymerization of Glu NCA with (S)-PhenNiNHC(H)RC(O)O, R = -
CHzCH(CH3~?
In the dry box, Glu NCA (50 mg, 0.2 mmol) was dissolved in DMF (1.0 mL)
and placed in a 25 mL reaction tube which could be sealed with a Teflon
stopcock.
An aliquot of the initiator (100 ~L of a 36 mM solution in DMF) was added via
syringe
to the flask. A stir bar was added and the flask was sealed, removed from the
dry
box and stirred at 25 °C in a thermostated bath for 24 h. Polymer was
isolated by
addition of the reaction mixture to methanol containing HCI (1 mM) causing
precipitation of the polymer. The polymer was dried in vacuo to give a white
solid,
PBLG (18.1 mg, 87% yield) ~3C~'H} NMR,'H NMR, and FTIR spectra of this
material
were identical to data found for authentic samples of PBLG (Stevens, C.;
Watanabe,
R. J. Am. Chem. Soc., 1950, 72, 725-727). GPC of the polymer in 0.1 M Liar in
DMF at 60 °C: M~ = 45,500; M,N/M~ = 1.24.
Fluorescence Measurements of 1-Napthyl Functionalized PBLG
A solution of 1-napthyl funtionalized PBLG (26.5 mg) in THF (2 ml) was
placed into a cuvette. The sample was excited at a frequency of 324 nm which
yielded an emission with maximum intensity at 390 nm. This emission was
characteristic of the 1-napthyl end-group. When the molecular weights of the
polymers were varied, the corresponding, emission intensities were found to
vary
inversely with chain length, indicating that the number of end-groups was
proportional to the number of chains. Control experiments showed that the
emission
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59
from labeled polymers was an order of magnitude greater than that from
unlabeled
PBLG (see Table 3 below).
Table 3
Entry Polymer Mass(mg) M" Intensity(cps)
I PBLG-Nap 26.5 14400 7593550
2 PBLG-Nap 26.5 26100 4238040
3 PBLG 26.5 36100 800000
Isolation of cis-5-Norbornene-endo-2-Carboxylic acid-3-Carboxyl L-Valine n-
Propyl Amide from Reaction of 4
A solution of cis-5-norbornene-endo-2,3-dicarboxylic anhydride (6.0 mg, 0.037
mmol) in THF (1 mL) was added to a solution of 4 (16 mg, 0.037 mmol) in THF (1
ml). The yellow solution was heated at 40 °C for 2 d until the
anhydride stretch at
1780 cm' was no longer detectable by FTIR. A dilute solution of HCI in water
(0.5
mL) was added to the reaction which then immediately changed color from yellow
to
orange. The mixture was stirred for 2 h and then the volatiles were removed in
vacuo. The remaining- solid was extracted with THF, filtered, and then added
to Et20
to precipitate the nickel-containing byproducts. The solubles were then
condensed in
vacuo to yield the product (11 mg, 92%). FAB-MS: MH+: 323.8 calc, 323 found.
Polymerization of (S)-phenylglycine NCA.Using.(S)-depeNiNHC(H)R'-
C(O)NR2, R' _ -CHzCH(CH3~z~ R2 = -CH2CHZCH(CH3~2 7
In the dry box, (S)-phenylglycine NCA (50 mg, 0.28 mmol) was dissolved in
THF (1.0 mL) and placed in a 25 mL reaction tube which could be sealed with a
Teflon stopcock. An aliquot of 7 (560 ~L of a 50 mM solution in THF) was added
via
syringe to the flask. A stir bar was added and the flask was sealed, removed
from
the dry box and stirred at 25 °C in a thermostated bath for 24 h.
Polymer was
observed to precipitate from solution during this time period. Polymer was
isolated by
addition of the reaction mixture to methanol containing HCI (1 mM) followed by
centrifugation. The polymer was washed with excess water, methanol, and then
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diethyt ether and then dried in vacuo to give the product as a white solid (35
mg,
93% yield). 'H NMR(TFA-~ and FTIR spectra of this material were identical to
data
found for authentic samples of poly (S)-phenylglycine. MALDI mass spectroscopy
of
the polymer showed a distribution of masses ranging from ca. 1000 - 4500 Da,
with
the separation between the peaks equal to the mass of the phenylglycine
repeats
(133.15 Da). Below 1000 Da, the spectra were complicated by the presence of
large
amounts of matrix peaks. Analysis of the absolute masses of the peaks revealed
that
nearly all chains were end-functionalized with the leucine residue of the
initiator
(Figure 7). Some of the chains contained the intact leucine isoamylamide end-
group
(b-series), while the remainder contained a leucine end-group where the C-
terminal
amide had been cleaved by hydrolysis after dissolution in wet TFA (a-series).
As an
example peak, 9a: expected MH+: 1331.44 Da; found MH+: 1330.13 Da. Only very
small peaks were observed where non-functionalized oligo(phenylglycines)
should
appear (c-series), and these peaks may also contain adducts formed with
functionalized chains. For example, 10c (1350.43 Da) has a mass nearly equal
to
9a+O (1347.44 Da) [peak observed at 1347.53 Da]. From comparison of the peak
intensities for the a- and b-series of peaks (and adducts) to the c-series of
peaks, it
was determined that the degree of chain functionalization was greater than
98%.
The initiators described above were generated using bis-1,5-cyclooctadiene
nickel (Ni(COD)2) as the nickel source in conjunction with a variety of donor
ligand
components. We have also successfully used other sources of zerovalent nickel
(e.g. Ni(CO)4) as well as other donor ligands (e.g., PR3 [R = Me, Et, Bu,
cyclohexyl,
phenyl], R2PCH2CH2PR2 [R = Me, phenyl], (a,,a'-diimine ligands [1,10-
phenanthroline, neocuproine], diamine ligands [tetramethylethylene diamine],
and
isocyanide ligands [tert-butyl isocyanide]) to generate these initiators. The
use of
other sources of zerovalent nickel (e.g. nickel-olefin complexes, nickel-
carbonyl
complexes, nickel-isocyanide or cyanide complexes, and nickel nitrogen or
phosphorus donor ligand complexes) are possible modifications which should not
be
interpreted as going beyond the concept of this invention. Likewise, the use
of other
donor ligands (nitrogen or phosphorus based in particular) or reaction
solvents are
logical extensions of this work. Finally, we have found that other transition
metals,
specifically palladium, platinum, cobalt, rhodium and iridium, might also be
used in
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61
the reaction with alloc-amino acid amides to form amido-amidate metallacycle
initiators, and are thus additional potential modifications to this invention.
EXAMPLE 3 - Facile Synthesis of Block Copolypeptides of Defined Architecture.
General Experimental Protocols and Reagients.
Infrared spectra were recorded on a Perkin Elmer 1605 FTIR
Spectrophotometer calibrated using polystyrene film. Tandem gel permeation
chromatography/light scattering (GPC/LS) was performed on a Spectra Physics
Isochrom liquid chromatograph pump equipped with a Wyatt DAWN DSP light
scattering detector and Wyatt Optilab DSP. Separations were effected by 105A
and
103A Phenomenex 5N columns using 0.1 M Liar in DMF at 60 °C as eluent.
Optical
rotations were measured on a Perkin Elmer Model 141 Polarimeter using a 1 mL
volume cell (1 dm length). NMR spectra were measured on a Bruker AMX 500MHz
spectrometer. Chemicals were obtained from commercial suppliers and used
without purification unless otherwise stated. (COD)2Ni was obtained from Strem
Chemical Co., and'3C~-L-leucine and '3C-phosgene were obtained from Cambridge
Isotope Labs. g-Benzyl-L-glutamate NCA were prepared according to literature
procedures. Hexanes, THF, and THF-d$ were purified by distillation from sodium
benzophenone ketyl. DMF and DMF-d; were purified by drying over 4A molecular
sieves followed by vacuum distillation.
Reaction of (2,2'-bipyridyl)Ni(COD) with'3C2-L-Leucine NCA
In the dry box, five equivalents of'3C2-L-Leucine NCA (14.5 mg, 0.091 mmol)
was added to a solution of bipyNi(COD) (5.9 mg, 0.018 mmol) in THF (1 ml). The
mixture slowly turned from purple to red and was let stir for 16 hours. The
crude
product was isolated by evaporation of the solvent to yield a red oily solid.
FTIR
analysis of the crude reaction mixture confirmed the presence of (2,2'-
bipyridyl)Ni(CO)2 [1R (THF): 1978, 1904 cm-1 (nCO, vs), polyleucine [1R (THF):
1653
cm-1 (nAmide I, vs); 1546 cm-1 (nAmide II, vs)] as well as the '2C-amidate
endgroup
[IR(THF): _n(CO) = 1577 cm-']. The reaction was also run in DMF-d7 (0.5 mL)
under
otherwise identical conditions. '3C {'H} NMR (DMF-d~): d 126 (s,'3C0z).
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62
Reaction of (2,2'-bipyridyl)Ni(COD) with '3C5-L-Leucine NCA
In the dry box, five equivalents of C5-L-Leucine NCA (14.5 mg, 0.091 mmol)
was added to a solution of bipyNi(COD) (5.9 mg, 0.018 mmol) in THF (1 ml). The
mixture slowly turned from purple to red and was let stir for 16 hours. The
crude
product was isolated by evaporation of the solvent to yield a red oily solid.
FTIR
analysis of the crude reaction mixture confirmed the presence of (2,2'-
bipyridyl)Ni('3C0)2 [1R (THF): 1933, 1862 cm-1 (nCO, vs)] as well as '3C-
labeled
polyleucine [1R (THF): 1613 cm-1 (nAmide I, vs); 1537 cm-1 (nAmide II, vs)].
The
reaction was also run in DMF-d7 (0.5 mL) under otherwise identical conditions.
'3C
{'H} NMR (DMF-d7): d 198 (s, bipyNi('3C0)2); 177 (s,
bipyNiN(H)C(H)R'3C(O)N[CH(R)'3C(O)-NH]~CH2R)), 174 (s,
bipyNiN(H)C(H)R'3C(O)N[CH(R)'3C(O)NH]~CH2R).
Polymerization of Glu-NCA with (2,2'-bipyridyl)Ni(COD)
In the dry box, Glu NCA (50 mg, 0.2 mmol) was dissolved in DMF (0.5 mL)
and placed in a 25 mL reaction tube which could be sealed with a Teflon
stopcock.
An aliquot of bipyNi(COD) (50 ml of a 40 mM solution in DMF) was then added
via
syringe to the flask. A stirbar was added and the flask was sealed, removed
from
the dry box, and placed in a thermostated 25 °C bath for 16 hours.
Polymer was
isolated by addition of the reaction mixture to methanol containing HCI (1 mM)
causing precipitation of the polymer. The polymer was then dissolved in THF
and
reprecipitated by addition to methanol. The polymer was dried in vacuo to give
a
white stringy solid, PBLG (41 mg, 98 % yield). '3C {'H} NMR,'H NMR, and FTIR
spectra of this material were identical to data found for authentic samples of
PBLG.
GPC of the polymer in 0.1 M Liar in DMF at 60 °C: M~ = 22,000; MW/M~
= 1.05.
As an illustrative embodiment of the invention, diblock copolymers composed
of amino acid components g-benzyl-L-glutamate and e-carbobenzyloxy-L-lysine
were synthesized. The polymers were prepared by addition of Lys-NCA to
bipyNi(COD) in DMF to afford living poly(e-carbobenzyloxy-L-lysine), PZLL,
chains
with organometallic end-groups capable of further chain growth. Glu-NCA was
added to these polymers to yield the PBLG-PZLL block copolypeptides. The
evolution of molecular weight through each stage of monomer addition was
analyzed
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63
using gel permeation chromatography (GPC) and data are given in Table 1 below.
Molecular weight was found to increase as expected upon growth of each block
of
copolymer while polydispersity remained low, indicative of successful
copolymer
formation. A. Noshay, et al., Block Copolymers, Academic Press, New York,
(1977).
The chromatograms of the block copolypeptides showed single sharp peaks
illustrating the narrow distribution of chain lengths (See Figure 2).
Copolypeptide
compositions were easily adjusted by variation of monomer feed compositions,
both
being equivalent. Successful preparation of copolypeptides of reverse sequence
(i.e. PZLL-PBLG) and of triblock structure (e.g. PBLGo.3s-b-PZLLo.22-b-
PBLGo.39; M~ _
256,000, MW/M~ = 1.15) illustrate the potential for sequence control using the
nickel
initiator.
Block copolymerizations were not restricted to the highly soluble polypeptides
PBLG and PZLL. Copolypeptides containing L-leucine and L-proline, both of
which
form homopolymers which are insoluble in most organic solvents (e.g. DMF) were
prepared. Data for these copolymerizations are given in Table 4 below. Because
of
the solubilizing effect of the PBLG and PZLL blocks, all of the products were
soluble
in the reaction media indicating the absence of any homopolymer contaminants.
The block copolymers containing L-leucine were found to be strongly
associating in
0.1 M Liar in DMF, a good solvent for PBLG and PZLL. Once deprotected, the
assembly properties of these materials are expected to make them useful as
tissue
engineering scaffolds, drug carriers, and morphology-directing components in
biomimetic composite formation.
First segmentt Diblock Copolymer$
First Monomer* Second Monomer* Mn MW/M~ M~ MW/Mn Yield (%)~
52 Lys-NCA 181 Glu-NCA 15,000 1.12 66,000 1.21 95
~~
90 Glu-NCA 78 Lys-NCA 28,500# 1.12 52,700** 1.13 93
104 Lys-NCA 40 Leu-NCA 29500 1.13 34,000 1.20 93
~~ ~~
182 Glu-NCA 90 Pro-NCA 57,600# 1.07 86,000# 1.14 92
120 Glu-NCA 40 Leu-NCA 38,000# 1.08 79,000# 1.13 96
Table 4 Preparation and analysis of block copolypeptides. Polymerization
initiator
was bipyNi(COD) in DMF in all cases. Molecular weight (Mn) and polydispersity
(Mw
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64
/Mn) were determined by tandem GPC/light scattering in 0.1 M Liar in DMF at 60
°C
using dn/dc values measured in this solvent at IO = 633 nm. * First and second
monomers added stepwise to the initiator; number indicates equivalents of
monomer
per bipyNi(COD). Leu-NCA = L-leucine-N-carboxyanhydride. Pro-NCA = L-proline-
N-carboxyanhydride. t Molecular weight and polydispersity after polymerization
of
the first monomer. $ Molecular weight and polydispersity of the complete block
copolymer. ~ Total isolated yield of block copolymer. ~~ dn/dc = 0.123 mL/g. ~
dn/dc
= 0.108 mL/g. # dn/dc = 0.104 mL/g. ** dn/dc = 0.115 mL/g.
The initiators described above were generated using bis-1,5-cyclooctadiene
nickel (Ni(COD)2) as the nickel source and 2,2'-bipyridyl (bipy) as the donor
ligand
component in tetrahydrofuran (THF) solvent. Other sources of zerovalent nickel
(e.g. Ni(CO)4) as well as other donor ligands (e.g. PR3 [R = Me, Et, Bu,
cyclohexyl,
phenyl], RZPCH2CH2PR2 [R = Me, phenyl], a, a'-diimine ligands [1,10-
phenanthroline, neocuproine], diamine ligands [tetramethylethylene diamine],
and
isocyanide ligands [tert-butyl isocyanide]) can be used to initiate these
polymerizations. The use of other sources of zerovalent nickel (e.g. nickel-
olefin
complexes, nickel-carbonyl complexes, nickel-isocyanide or cyanide complexes,
and
nickel nitrogen or phosphorous donor ligand complexes) are possible
embodiments
which should not be interpreted as going beyond the concept of this invention.
Likewise, the use of other donor ligands (nitrogen or phosphorous based in
particular) or polymerization solvents are logical extensions of this work.
Finally,
other transition metals, specifically palladium, platinum, cobalt, rhodium,
iridium and
iron are also able to polymerize NCA monomers. The use of metals in "Group
VIII"
(i.e. Co, Rh, Ir, Ni, Pd, Pt, Fe, Ru, Os) are thus additional potential
embodiments of
this invention.
Illustrative Diblock and Triblock Copolypeptides and their Synthesis
1. Poly(e-benzyloxycarbonyl-L-Lysine-block-g-benzyl-L-glutamate). PILL-b-
PBLG, Diblock Copolymer
In the dry box, Glu NCA (50 mg, 0.19 mmol) was dissolved in
dimethylformamide (DMF) (0.5 mL) and placed in a 25 mL reaction tube which
could
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be sealed with a Teflon stopcock. An aliquot of (2,2'-bipyridyl)Ni(COD) (50 ml
of a
40 mM solution in DMF, prepared by mixing equimolar amounts of 2,2'-bipyridyl
and
Ni(COD)2) was then added via syringe to the flask. A stirbar was added, the
flask
was sealed and then stirred for 16 hours. An aliquot (50 mL) was removed from
the
polymerization for GPC analysis (M~ = 28,500; MW/M~ = 1.12). e-
benzyloxycarbonyl-
L-Lysine-N-carboxyanhydride, Lys-NCA, (50 mg, 0.16 mmol) dissolved in
dimethylformamide (DMF) (0.5 mL) was then added to the reaction mixture. After
stirring for an additional 16h, polymer was isolated by addition of the
reaction mixture
to methanol containing HCI (1 mM) causing precipitation of the polymer. The
polymer was then dissolved in THF and reprecipitated by addition to methanol.
The
polymer was dried in vacuo to give a white solid, PZLL-b-PBLG (79 mg, 93 %
yield).
'3C ~'H} NMR, 'H NMR, and FTIR spectra of this material were identical to a
combination of data found for authentic individual samples of PBLG and PZLL.
GPC
of the block copolymer in 0.1 M Liar in DMF at 60 °C: M~ = 52,700;
MW/M~ = 1.13.
2. Poly (M-benzyloxycarbonyl-L-Lysine-block-K-benzyl-L-alutamate) PZLL-b-
PBLG. Diblock Copolymer usin (q PMe3~4Co
In the dry box, Glu NCA (50 mg, 0.19 mmol) was dissolved in DMF (0.5 mL)
and placed in a 15 mL reaction tube which could be sealed with a TEFLONT"~
stopper. An aliquot of (PMe3)4)Co (50TL of a 40 mM solution in DMF:THF (1:1))
was
then added via syringe to the flask. A stirbar was added, the flask was scaled
and
then stirred for 16 h. An aliquot (50 TL) was removed from the polymerization
for
GPC analysis (M~=21,500; MW/Mn = 1.12). Lys-NCA, (50 mg, 0.16 mmol) dissolved
in DMF (0.5 mL) was then added to the reaction mixture. After stirring for an
additional 16 h, polymer was isolated by addition of the reaction mixture to
methanol
containing HCI (1 mM) causing precipitation of the polymer. The polymer was
then
dissolved in THF and reprecipitated by addition to methanol. The polymer was
dried
in vacuo to give a white solid, PZLL-b-PBLG (82 mg, 97% yield). '3C {'H~
NMR,'H
NMR, and FTIR spectra of this material were identical to a combination of data
found
for authentic individual samples of PBLG and PZLL. GPC of the block copolymer
in
0.1 M Liar in DMF at 60°-C; Mn-44,700; MW/Mn = 1.13.
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3. Poly(a-benzyl-L-glutamate-block-e-benzyloxycarbonyl-L-Lysine-block-c~-
benzyl-L-Qlutamate) Triblock Copolymer.
In the dry box, Glu NCA (250 mg, 0.95 mmol) was dissolved in
dimethylformamide (DMF) (1.5 mL) and placed in a 25 mL reaction tube which
could
be sealed with a Teflon stopcock. An aliquot of (2,2'-bipyridyl)Ni(COD) (50 ml
of a
40 mM solution in DMF, prepared by mixing equimolar amounts of 2,2'-bipyridyl
and
Ni(COD)2) was then added via syringe to the flask. A stirbar was added, the
flask
was sealed and then stirred for 16 hours. An aliquot (50 mL) was removed from
the
polymerization for GPC analysis (M~ = 100,100; MW/M~ = 1.11). Lys-NCA, (125
mg,
0.42 mmol) dissolved in dimethylformamide (DMF) (0.5 mL) was then added to the
reaction mixture, which was stirred for 16h. A second aliquot (50 mL) was
removed
from the polymerization for GPC analysis (M~ = 156,200; MW/M~ = 1.12).
Finally,
Glu-NCA, (250 mg, 0.95 mmol) dissolved in dimethylformamide (DMF) (1.5 mL) was
then added to the reaction mixture. After stirring for an additional 16h,
polymer was
isolated by addition of the reaction mixture to methanol containing HCI (1 mM)
causing precipitation of the polymer. The polymer was then dissolved in THF
and
reprecipitated by addition to methanol. The polymer was dried in vacuo to give
a
white solid, PBLG-b-PZLL-b-PBLG (505 mg, 96 % yield). '3C ~'H~ NMR,'H NMR,
and FTIR spectra of this material were identical to a combination of data
found for
authentic individual samples of PBLG and PILL. GPC of the block copolymer in
0.1 M Liar in DMF at 60 °C: M~ = 256,300; MW/M~ = 1.15.
4. General Preparation of Block Copolypeotides with Metal Initiators
Other diblock and triblock copolymers were prepared by a procedure identical
to that described above for either PZLL-b-PBLG and PBLG-b-PZLL-b-PBLG, except
that either different monomers, or different amounts of monomers, were used
for the
individual polymerization reactions. Examples are given in Tables 4 (abovee)
and
Table 5(below). The nature of the amino acid monomer was found to be
unimportant
in limiting the effectiveness of these polymerizations. All amino acid NCAs
tried
were incorporated into block copolypeptides in any sequential order, as
determined
by the order of addition to the initiator. Representative monomers include,
but are
not limited to: the naturally occurring L-amino acids, naturally occurring D-
amino
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67
acids, a-disubstituted a-amino acids, racemic a-amino acids, and synthetic a-
amino
acids. Block copolypeptides could be prepared using initiators other than
(2,2'-
bipyridyl)Ni(COD). The initiators given in Tables 7 and 8 below (except those
that
gave no yield of polymer) all were able to prepare block copolypeptides.
Monomers Diblock Triblock
and 1St segment$ copolymer~l
order segments
of
addition*
~ st 2n 3rd i
monomer monomer monomer Mn MW/Mn Mn MW/Mn Mn MW/Mn Yield
450 Glu 200 Lys 450 Glu 100 1.11 156 1.12 256 1.15 96
~
130 Glu 120 Ala 130 Glu 34 1.07 47 1.14 82 1.20 94
250 Glu 130 Leu 250 Glu 68 1.12 83 1.18 152 1.20 97
~ ~
250 Glu 300 Pro 250 Glu 67 1.10 98 1.18 96
' ~ 1.17 j
167
Table 5 Preparation and analysis of triblock copolypeptides. Polymerization
initiator
was bipyNi(COD) in DMF in all cases. Molecular weight (Mn) and polydispersity
(Mw
/Mn) were determined by tandem GPC/light scattering in 0.1 M Liar in DMF at 60
°C
using dn/dc values measured in this solvent at Ip = 633 nm. * First, second
and third
monomers added stepwise to the initiator; number indicates equivalents of
monomer
per bipyNi(COD). Lys = Lys-NCA. Glu = Glu-NCA. Ala = L-alanine-N-
carboxyanhydride. Leu = L-leucine-N-carboxyanhydride. Pro = L-proline-N-
carboxyanhydride. t Molecular weight (x 10-3) and polydispersity after
polymerization of the first monomer. $ Molecular weight (x 103) and
polydispersity
after polymerization of the second monomer. ~~ Molecular weight (x 10-3) and
polydispersity of the complete triblock copolymer. ~ Total isolated yield (%)
of
triblock copolymer.
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EXAMPLE 4 - General Initiator Features; Assessment of Multiple Initiators and
Effect of Chemical Structure of Efficiency; Effects of Reaction Conditions on
Polymerization; and Initiator Mediated Block Copolypeptide Synthesis
General Protocols and Reagents.
Infrared spectra were recorded on a Perkin Elmer 1605 FTIR
Spectrophotometer calibrated using polystyrene film. Optical rotations were
measured on a Perkin Elmer Model 141 Polarimeter using a 1 mL volume cell (1
dm
length). NMR spectra and bulk magnetic susceptibility measurements (Evans
method) were measured on a Bruker AMX 500MHz spectrometer. D.F. Evans, J.
Chem. Soc., 2003-2009 (1959); J.K. Becconsal, J. Mol. Phys., 15:129-135
(1968).
C, H, N elemental analyses were performed by the Microanalytical Laboratory of
the
University of California, Berkeley Chemistry Department. Metal analyses were
conducted using a Thermo Jarrell Ash IRIS HR ICP analyzer. Chemicals were
obtained from commercial suppliers and used without purification unless
otherwise
stated. (COD)2Ni was obtained from Strem Chemical Co., and '3C~-L-leucine and
'3C-phosgene were obtained from Cambridge Isotope Labs. L-leucine isoamylamide
hydrochloride, g-benzyl-L-glutamate NCA and L-leucine NCA were prepared
according to literature procedures. M. Bodanszky, et al., The practice of
Peptide
Synthesis, 2"d Ed., Springer, BerIin/Heidelberg, (1994); E.R. Blout, et al.,
J. Am.
Chem Soc., 78:941-950 (1956); H. Kanazawa, et al., Bull. Chem Soc. Jpn.,
51:2205-
2208 (1978). Hexanes, THF, and THF-d8 were purified by distillation from
sodium
benzophenone ketyl. DMF and DMF-d7 were purified by drying over 4A molecular
sieves followed by vacuum distillation.
General Features for Formation of Active Metal Initiators
The efficient, controlled polymerization of NCAs using transition metal
compounds requires the general formation of an amido-containing 5- or 6-
membered
metallacycle (Figure 6), which is the active intermediate in the
polymerizations. With
regard to results described in this disclosure, these metallacycles are formed
by
reaction of 1 or 2 equivalents of an NCA with a metal complex, which is
capable of
undergoing an oxidative-addition reaction where its valence formally increases
by
two. A variety of NCAs can be used for this reaction (i.e. L-leucine NCA, Glu-
NCA,
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and L-phenylalanine NCA) and there is no reason why any NCA of general
structure
shown in Figure 6 would not work for this reaction. The metals which most
commonly undergo two-electron oxidative-addition reactions are those in Group
VIII
(i.e. Fe, Ru, Os, Co, Rh, Ir, Ni, Pd, Pt) and hence these are the metals
studied most
extensively. Collman, J. P.; Roper, W. R. Adv. Orgmet. Chem., 1968, 7, 53-94.
Amido-containing metallacycles can be formed with Fe, Co, Rh, Ir and Ni, and
that
these complexes give controlled polymerization of NCAs. Pd and Pt complexes
are
also able to promote polymerization of NCAs. Virtually any low-valent
transition
metal (i.e. a metal in a low oxidation state) with the proper combination of
electron
donor ligand(s) can react with NCAs to yield amido-containing metallacyclic
intermediates which could act as active polymerization initiators. Other
metals which
clearly fall into this category are Au, Mn, Cr, Nlo, W, and V.
The range of substituents (R) which can be placed on the amido-containing
metallacycles was investigated. These include the side chain functions found
in
amino acids themselves (e.g. R = CHZC6H5 from phenylalanine, R = CH2CH(CH3)2,
or R = CH2CH2COZCH2C6H5 from g-benzylglutamate), and should thus include any
organic moiety attached to an a-amino acid.
Determination of Initiator Efficiency.
Efficiencies were quantified by measurement of product polymer molecular
weights and molecular weight distributions, and measurement of polymerization
reaction rates. Polymer molecular weights and molecular weight distributions
were
measured using tandem gel permeation chromatographyllight scattering (GPC/LS)
which was performed on a Spectra Physics Isochrom liquid chromatograph pump
equipped with a Wyatt DAWN DSP light scattering detector and Wyatt Optilab DSP
interoferometric refractometer. Separations were effected by 105A and 103A
Phenomenex 5N columns using 0.1 M Liar in DMF eluent at 60 °C.
Polymerization
reaction rates were obtained from kinetic data which were measured by
periodically
removing aliquots from a thermostated polymerization of Glu-NCA, diluting
these
(10-fold) with anhydrous chloroform to a known volume, and recording the
intensity
of the unreacted anhydride stretch at 1790 cm-~ in the solution by FTIR
spectroscopy. NCA concentrations were determined by use of an empirical
calibration curve (transmittance vs. concentration) of Glu-NCA in chloroform.
Plots
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of log (concentration) versus time gave pseudo first order polymerization
rates for
the different initiators.
(S)-[NiNHC(H)RC(OlNCH2RlX, R = -CH~CHZC O)OCHZCSHS; NiGlu2
In the dry box, Glu NCA (15 mg, 0.058 mmol) was dissolved in THF (0.5 mL)
and added to a stirred homogeneous mixture of PPh3 (31 mg, 0.12 mmol) and
(COD)2Ni (16 mg, 0.058 mmol) in THF (1.5 mL). The red/brown solution was
stirred
for 24 hours, after which the solvent was removed in vacuo to leave a dark red
oily
solid. This was extracted with hexanes (3 x 5 mL) to yield a red/brown hexanes
solution and a yellow solid. Evaporation of the hexanes solution gave a red
oil
containing (PPh3)ZNi(CO)2 [1R (THF): 2000, 1939 cm-~ (nCO, vs); 18 mg. J.
Chaff, et
al., J. Chem. Soc., 1378-1389 (1960). 1R (CH2CICH2C1): 1994, 1933 cm-~)], and
drying of the solid gave the product as a yellow powder (10 mg, 75 % yield).
An'H
NMR spectrum could not be obtained in THF-d8, most likely because of
paramagnetism of the complex (only broad lines for the benzyl ester groups
were
observed). meff(THF, 293 K) = 1.08 mg. Osmotic molecular weight in THF (vs.
ferrocene; ca. 7 mg/mL): 910 g/mol; this corresponds to a degree of
aggregation of
1.94. 1R (THF): 3281 cm-~ (nNH, s br), 1734 cm-~ (nCO, ester, vs), 1577 cm-~
(nCO,
amidate, vs). Anal. calcd. for NiC23HzsN20s: 58.87%C, 5.59%H, 5.96%N; found:
59.07%C, 5.67%H, 5.56%N. [a]o2° (THF, c = 0.0034) _ -71.
(Sl-fNiNHClH)RC(O)NCH2RIx. R = -CHZCsHS: NiPhez
In the dry box, L-phenylalanine NCA (45 mg, 0.24 mmol) was dissolved in
THF (0.5 mL) and added to a stirred homogeneous mixture of PPh3 (124 mg, 0.48
mmol) and (COD)2Ni (64 mg, 0.24 mmol) in THF (1.5 mL). The red/brown solution
was stirred for 24 hours, after which the solvent was removed in vacuo to
leave a
dark red oily solid. This was extracted with cold hexanes (0 °C, 3 x 2
mL) to yield a
red/brown hexanes solution and a pale orange solid. Evaporation of the hexanes
solution gave a red oil containing (PPh3)2Ni(CO)2 [1R (THF): 2000, 1939 cm-~
(nCO,
vs)], and drying of the solid gave an orange powder which could be purified by
precipitation from THF/hexanes to give (S)-[NiNHC(H)RC(O)NCHzR]X, R = -CHzC6H5
as a yellow powder (31 mg, 80 % yield). An'H NMR spectrum could not be
obtained
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71
in THF-d8, most likely because of paramagnetism of the complex. 1R (THF): 3290
cm-~ (nNH, s br), 1574 cm-~ (nCO, amidate, vs). [a]p'° (THF, c = 0.001)
_ 170.
(S)-(2,2'-bipyridyl)NiNHC(H)RC(O)NCHZR. R = -CH2CHZC O OCHZC6Hsi
(2.2'-bioyridyll N iGluz
In the dry box, a yellow solution of NiGlu2 (40 mg, 0.085 mmol) in DMF (0.5
mL) was added to a solution of 2,2'-bipyridyl (54 mg, 0.35 mmol) in DMF (0.5
mL).
The homogeneous mixture was stirred for 2d at 50 °C, during which
the color
changed from yellow to blood red. THF (1 mL) and toluene (5 mL) were layered
onto
this solution resulting in precipitation of a red powder. This powder was
reprecipitated from DMF/THF/toluene (1:2:10) two additional times to give
(2,2'-
bipyridyl)NiGluz as a red powder (49 mg, 92 % yield). An'H NMR spectrum could
not be obtained in THF-d8, most likely because of paramagnetism of the complex
(only broad lines for the benzyl ester groups were observed). 1R (THF): 3281
cm-~
(nNH, s br), 1732 cm-~ (nCO, ester, vs), 1597 cm-~ (nCO, amidate, vs). Anal.
calcd.
for NIC33H34N4~5~ 63.37%C, 5.49%H, 8.95%N; found: 63.72%C, 5.49%H, 8.86%N.
[a]p2° (THF, c = 0.001) _ -135.
Preparation of Other LZNiGlu2 and LzNiPhe~ Initiators
The procedures for synthesis of these compounds were identical to that
described for preparation of (2,2'-bipyridyl)NiGIu2 except for substitution
or' different
ligands (L2) for 2,2'-bipyridyl or the use of NiPhez instead of NiGlu2. The
range of
ligands included phen, LiCN, and tmeda. All of the complexes gave satisfactory
analysis.
1. (PMe322FePhez
In the dry box, L-phenylalanine NCA (32 mg, 0.16 mmol) was dissolved in
THF (0.5 mL) and added to a stirred homogeneous solution of (PMe3)4Fe (30 mg,
0.083 mmol) in EtzO (4 mL). The pale orange solution was stirred for 24 hours,
after
which the resulting off-white precipitate was isolated by centrifugation. This
solid
was washed with EtzO (3 x 5 mL) and then dried to give an off-white powder.
The
powder was purified by dissolving in THF and precipitating with hexanes (36
mg,
91 %). An ' H NMR spectrum could not be obtained in THF-d8, most likely
because of
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paramagnetism of the complex (only broad lines for the phenyl groups were
observed). 1R (THF): 3296 cm-~ (nNH, s br), 1603 cm-~ (nCO, amidate, vs).
2. tBuNC zFePhe2
In the dry box, (PMe3)ZFePhe2 (20 mg, 0.042 mmol) was dissolved in THF (2
mL) and mixed with tBuNC (24 mL, 0.252 mmol) in THF (2 mL). The solution was
stirred overnight during which it slowly turned from brown to yellow. The
product
was isolated by repeated precipitation of a yellow powder from THF by addition
to
hexanes. Drying gave a yellow solid (19 mg, 94%). 1R (THF): 3289 cm-~ (nNH, s
br), 2150 cm-~ (nNC, tBuNC, vs), 1626 cm-~ (nCO, amidate, vs).
3. X2.2'-bipyridyl)FePhe2
In the dry box, (PMe3)2FePhe2 (20 mg, 0.042 mmol) was dissolved in THF (2
mL) and mixed with tBuNC (33 mg, 0.168 mmol) in THF (2 mL). The solution was
stirred overnight during which it slowly turned from brown to deep red. The
product
was isolated by repeated precipitation of a red powder from DMF:THF (1:1 ) by
addition to hexanes. Drying gave a red solid (18 mg, 89%). 1R (THF): 3291 cm-~
(nNH, s br), 1600 cm-~ (nCO, amidate, vs).
Polymerization of Glu-NCA with (2.2'-bipyridyl)Ni(COD)
In the dry box, Glu NCA (50 mg, 0.2 mmol) was dissolved in tetrahydrofuran
(THF) (0.5 mL) and placed in a 25 mL reaction tube which could be sealed with
a
Teflon stopcock. An aliquot of (2,2'-bipyridyl)Ni(COD) (50 ml of a 40 mM
solution in
THF, prepared by mixing equimolar amounts of 2,2'-bipyridyl and Ni(COD)2) was
then added via syringe to the flask. A stirbar was added and the flask was
sealed,
removed from the dry box, and stirred in a thermostated 25 °C bath for
16 hours.
Polymer was isolated by addition of the reaction mixture to methanol
containing HCI
(1 mM) causing precipitation of the polymer. The polymer was then dissolved in
THF
and reprecipitated by addition to methanol. The polymer was dried in vacuo to
give
a white solid, PBLG (41 mg, 98 % yield). '3C {'H} NMR,'H.NMR, and FTIR spectra
of this material were identical to data found for authentic samples of PBLG.
H.
Block, Poly(g-benzyl-L-glutamate) and Other Glutamic Acid Containing Polymers
,
Gordon and Breach, New York, (1983). GPC of the polymer in 0.1 M Liar in DMF
at
60 °C: M" = 98,100; M,N/M~ = 1.15.
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General Polymerization of Glu-NCA with (2,2'-bipyridyl)Ni(CODI in Different
Solvents
The procedure followed was identical to that used for the (2,2'-
bipyridyl)Ni(COD) in THF except for substitution of different solvents for
THF. The
range of other solvents included: toluene, dioxane, acetonitrile, ethyl
acetate, and
DMF. The results of these polymerizations are given in Table 6 below. The
initiator
efficiencies were determined by analysis of polymer yields, proximity of the
found
molecular weights to the theoretical values, and the narrowness of the
molecular
weight distributions.
Notebook # Solvent Yield (%) M" MW/M
3-23 Ethyl acetate99 ' 109,000 1.12
2-148 I Toluene 97 146,000 1.11
I
3-23 Dioxane 96 126,000 1.20
3-23 Acetonitrile 62 75,000 1.45
2-144 THF I 96 142,000 1.05
2-151 DMF 97 40,000 1.19
Table 6: Effect of solvent on polymerizations of Glu-NCA using 2,2'-
bipyridylNi(COD)
initiator. w9oles monomer:moles initiator = 180:1. All polymerizations were
run at
20°C for 16 hours under nitrogen atmosphere.
General Polymerization of Glu-NCA with (LZINi(COD) Initiators
The procedure followed was identical to that used for the (2,2'-
bipyridyl)Ni(COD) initiator except for substitution of different ligand
molecules (L2) for
2,2'-bipyridyl. The range of ligands (L2) included: tricyclohexylphosphine
(PCy3, 1
and 2 equivalents per metal), tent-butyl isocyanide (tBuNC, 2 and 4
equivalents),
lithium cyanide (2 equivalents), trimethylphosphine (PMe3, 2 equivalents),
triethylphosphine (PEt3, 2 equivalents), tributylphosphine (PBu3, 2
equivalents),
triphenylphosphine (PPh3, 1 and 2 equivalents), 1,2-
bis(diphenylphosphino)ethane
(DIPHOS), 1,2-bis(dimethylphosphino)ethane (dmpe), tetramethylethylenediamine
(tmeda), (-)-sparteine, 1,10-phenanthroline (phen), neocuproine (ncp), as well
as the
compounds shown in Figure 5. The results of these polymerizations are given in
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Table 7 below. The initiator efficiencies were determined by analysis of
polymer
yields, proximity of the found molecular weights to the theoretical values,
and the
narrowness of the molecular weight distributions.
Notebook Ligand (L2) [M]:[I]Solvent Yield M~ x 10- ~ MWIM
# (%)
2-144 2,2=-bipyridyl180 THF 96 142 1.05
2-148 DIPRIM " " 60 165 i 1.21
2-148 dmpe " " 90 275 ~ 1.04
2-148 COD " " 0 - -
2-151 DMIM " " i 0 ' -
2-151 2 PPh2 " " 0 - -
2-151 1 PPh2 " " 78 126 i 1.26
2-151 phen 90 " 94 151 1.15
2-151 ncp " " 94 293 i 1.17
3-2 DPIM " " 0 -
3-2 DPOX " ~ " 96 189 1.06
3-2 2 PMe3 " " 94 244 1.16
3-10 tmeda " " 96 305 i 1.09
3-11 DIPHOS " " 0 -
I 3-21 ~ 2- PCy3 " 0 _
i
I
3-21 1 PCy3 " " 0
3-34 2 t-BuNC " " 76 218 1.09
3-34 4 t-BuNC " ~ " 74 190 1.15
3-37 2 PEt3 " " 92 251 1.08
3-37 2 PBu3 " " 88 196 1.14
i ~
JJ-Rep (-)sparteine 200 " 92 174 ~ 1.05
JJ-Rep ' TMOX " " 98 I 170 i 1.14
JJ-Rep DMOX-py " " 90 ' 157 ' 1.03
2-151 2,2=-bipyridyl152 DMF 97 35 1.14
3-11 tmeda 90 " 96 87 i 1.36
3-11 dmpe " " 96 60 1.33
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3-11 phen " " ~ 98 41 1.21
3-11 ncp " " ~ 99 48 1.45
3-11 DIPHOS " " 94 100 1.50
I
'i 3-21 1 PCy3 " " 35 46 1.18
Table 7: Effect of Ligands on LzNi(COD) initiators for polymerizations of Glu-
NCA.
[M):[I] = moles monomer:moles initiator. All polymerizations were run at 20
°C for
16 hours under nitrogen atmosphere. L2:Ni(COD)2 = 1:1 unless specified
otherwise.
General Polymerization of Glu-NCA with Other Transition Metal Initiators
The procedure followed was identical to that used for the (2,2'-
bipyridyl)Ni(COD) initiator except for substitution of different metal
complexes for
(2,2'-bipyridyl)Ni(COD). The range of metal complexes included: (2,2'-
bipyridyl)Ni(CO)2; (S)-[NiNHC(H)RC(O)NCHZR]x, R = -CHzCHzC(O)OCH2C6H5
(NiGlu2); (S)-(2,2'-bipyridyl)NiNHC(H)RC(O)NCHzR, R = - CH2CH2C(O)OCHzC6H5
(2,2'-bipyridyINiGlu2); (S)-LiZ(CN)zNiNHC(H)RC(O)NCHzR, R = -
CHzCH2C(O)OCH2C6H5 (LiZ(CN)2NiGlu2); (S)-(phen)NiNHC(H)RC(O)NCH2R, R = -
CHZCH2C(O)OCH2C6H5 (phenNiGlu2); (S)-(phen)NiNHC(H)RC(O)NCH2R, R = -
CH2C6H; (phenNiPhe2); (S)-(tmeda)NiNHC(H1RC(O)NCH2R, R = -CH2C6H5
(tmedaNiPhe2); dmpeCoPhe2; (PMe3)2CoPhe2; (PMe3)4Co; dmpeRhCl; dmpelrCl; h5-
CSHSCo(CO)2 (CpCo(CO)z); (2,2'-bipyridylCo(CO)2)2; ((PPh3)ZCo(CO)2)2;
(PMe3)4Fe;
(2,2'-bipyridyl)2Fe; (S)-(PMe3)2FeNHC(H)RC(O)NCHZR, R = -CHZC6H5
((PMe3)2FePhe2); (S)-(tBuNC)zFeNHC(H)RC(O)NCHZR, R = -CH2C6H5
((tBuNC)2FePhe2); (S)-(2,2'-bipyridyl)FeNHC(H)RC(O)NCHZR, R = -CH2C6H5 ((2,2'-
bipyridyl)FePhe2); (PPh3)4Pd; tris(dibenzylideneacetone)dipalladium
(Pd2(DBA)3)
plus 4 equivalents of PEt3; (PEt3)2Pt(COD); and (dmpe)2Co. The results of
these
polymerizations are given in Table 8 below. The initiator efficiencies were
determined by analysis of polymer yields, proximity of the found molecular
weights. to
the theoretical values, and the narrowness of the molecular weight
distributions.
Polymerization of Glu-NCA with (PMe3~aCo
In the dry box, Glu NCA (50 mg, 0.2 mmol) was dissolved in DMF (0.5 mL)
and placed in a 15 mL reaction tube which could be sealed with a TEFLONT"~
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stopper. An aliquot of (PMe3)a)Co (50TL of a 40 mM solution in DMF:THF (1:1))
was
then added via syringe to the flask. A stirbar was added and the flask was
sealed,
removed from the dry box, and stirred in a thermostated 25°-C bath for
16 h. Polymer
was isolated by addition of the reaction mixture to methanol containing HCI (1
mM)
causing precipitation of the polymer. The polymer was then dissolved in THF
and
reprecipitated by addition to methanol. The polymer was dried in vacuo to give
a
white solid, PBLG (42 mg, 99% yield). '3C f'H} NMR,'H NMR, and FTIR spectra of
this material were identical to data found for authentic samples of PBLG. GPC
of the
polymer in 0.1M Liar in DMF at 60°-C: M~=21,600; MW/M~ = 1.11.
(S)-~CoNHC(H)RC(0)NCH2~~5 R=CHZC6H6: CoPhe2
In the dry box, Phe NCA (9.0 mg, 0.046 mmol) was dissolved in THF (0.5 mL)
and added to a stirred homogeneous solution of (PPh3)3Co(N~) {40 mg, 0.046
mmol)
in THF (1.5 mL). The red/brown solution was stirred for 24 h, after which the
solvent
was removed in vacuo to leave a red/orange oily solid. This was extracted with
hexanes (3 x 5 mL) to yield an orange hexanes solution and a tan solid.
Evaporation
of the hexanes solution gave a brown oil containing [(PPh3)3Co(CO)]2 [1R
(THF):
1909, 1875 cm-' (nCO, vs); 15 mg; Literature: IR (KBr): 1904, 1877 cm-')], and
drying of the solid gave the product as a tan powder (11 mg, 74% yield). An'H
NMR
spectrum could not be obtained in THF-d$ most likely because of paramagnetism
of
the complex (only broad lines for the phenyl rings were observed). 1R (THF):
3310
cm-' (nNH, s br), 1600 cm-' (nCO, amidate, vs).
(S)-(dmpe)CoNHC(H)RC(O)NCHZR~R = -CH2C5H5 dmr~eCoPhez
In the dry box, a light brown solution of 1 (40 mg, 0.12 mmol) in DMF (0.5 mL)
was added to a solution of bis(dimethylphosphino)ethane, dmpe, (35TL, 0.21
mmol)
in DMF (0.5 mL). The homogeneous mixture was stirred for 2d at 50°-C,
during
which the color changed from yellow to orange/red. THF (1 mL) and toluene (5
mL)
were layered onto this solution resulting in separation of a brown oil. This
oil was
isolated from DMF/THF/toluene (1:2:10) two additional times to give the
product (49
mg, 86% yield). An'H NMR spectrum could not be obtained in THF-d8, most likely
because of paramagnetism of the complex (only broad lines for the phenyl and
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methyl groups were observed). 1R (THF): 3295 cm-' (nN11, s br), 1603 cm-'
(nCO,
amidate, vs).
Polymerization of Glu-NCA using dmpeCoPhez
In the dry box, Glu NCA (50 mg, 0.2 mmol) was dissolved in DMF (0.5 mL)
and placed in a 15 mL reaction tube which could be sealed with a TEFLONTM
stopper. An aliquot of dmpeCoPhez (50TL of a 40 mM solution in DMF) was then
added via syringe to the flask. A stirbar was added and the flask was sealed,
removed from the dry box, and stirred in a thermostated 25°C bath for
16 h. Polymer
was isolated by addition of the reaction mixture to methanol containing HCI (1
mM)
causing precipitation of the polymer. The polymer was then dissolved in THF
and
reprecipitated by addition to methanol. The polymer Was dried in vacuo to give
a
white solid, PBLG (41 mg, 98% yield). '3C {'H} NMR,'H NMR, and FTIR spectra of
this material were identical to data found for authentic samples of PBLG. GPC
of the
polymer in 0.1 M Liar in DMF at 60°-C: M~ = 20,900: MW/M~ = 1.07.
Notebook Metal complex [M]:[l]Solvent Yield M~ MWIM
# (%) x1
p-s
3-40 2,2'-bipyridylNi(CO)z50 THF 54 38 1.38
3-45 NiGluz '' DMF 84 35 1.26
~ 3-34 2,2'-bipyridyINiGluzj " THF 97 142 1.12
i 3-58 ~ Liz(CN)zNiGluz i " " i 96 1.27
~ i
100
3-64 ~ phenNiGluz ' 30 " i 97 i 83 1.15
" phenNiPhez " " 98 98 1.11
i
3-62 tmedaNiPhez " " 96 100 1.10
3-61 2,2'-bipyridyINiGluz90 DMF 94 60 1.18
3-64 phenNiGluz " " 97 36 1.15
i 3-68 i phenNiPhez " CH2CIz 93 86 1.09
~ I ~
3-58 CpCo(CO ' " ~ THF 0 i - -
3-36 2,2'-bipyridylCo(CO)z)z" " 0 -
~ 1-
3-36 ~ ((PPhs)zCo(CO)z)z " " 0 ~ -
_
3-73 ~ (dmpe)zCo i 505 97 80 1.09
AG-Rep (PPh3)4Pd ~ 150 " 82 220 1.05
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AG-Rep Pd2(DBA)3 + 4 150 " 81 254 1.06
PEt3
AG-Rep (PEt3)2Pt(COD) 150 " 84 236 1.04
~ 3-61 (2,2'-bipyridyl)2Fe90 " 80 50 1.10
3-68 (PMe3)4Fe 50 " 0 - _
3-69 (t-BuNC)2FePhez 90 " 0 - _
3-75 (PMe3)2FePhe2 ~ 50 " 96 84 1.11
-
3-75 (PMe3)zFePhe2 90 DM 50 25 1.21
F
3-75 (2,2'-bipyridyl)FePheZ" " 97 36 1.18
3-75 (t-BuNC)2FePhe2 " I " 96 38 1.15
3-127 ~ (PMe3)4Co ~ 100 DMF 97 22 1.11
3-124 (PMe3)4Co 50 THF 98 47 1.17
3-117 dmpelrCl 25 THF 97 67 1.28
3-117 (PMe3)IrCI ~ 25 THF 96 89 1.14
i 3-112 (PEt3)ZIrCI 50 THF 25 50 1.40
3-113 (CH2(PEt2)z)zIrC150 THF 97 122 1.21
3-114 (CHZ(PCy2)z)ZIrCI50 THF ~ 94 188 1.17
~ ~ ~
3-118 ~ dmpeRhCl 50 THF 98 99 i 1.16
3-118 ~ (PMe3)2RhCl 50 THF ' 39 238 1.22
i
3-103 dmpeCoPhe2 100 DMF 98 21 1.07
i
Table 8: Efficiency of different transition metal initiators far
polymerization of Glu-
NCA. [M]:[I] = moles monomer:moles initiator. All polymerizations were run at
20 °C
for 16 hours under nitrogen atmosphere.
EXAMPLE 5 - Method of Preparing Oliqo(Ethylenealycol) Functionalized Amino
Acids and Their Polymers: New Water Solulble Biocompatible Polypeotides
The following experiments describe the synthesis of of oligo(ethyieneglycol)
functionalized lysine, serine, cysteine, and tyrosine NCA monomers, and their
subsequent polymerization into oligo(ethyleneglycol) functionalized
polypeptides.
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General
Tetrahydrofuran (THF), hexane, N,N-dimethylformamide, and diethyl ether
were dried by passage through an alumina column under nitrogen prior to use.
All
reactions were conducted under an anhydrous nitrogen atmosphere, unless
otherwise noted. The chemicals were purchased from commercial suppliers and
used without purification. Co(PMe3)4 was prepared according to the procedure
of
Klein, et al. [Klein, et al., Methyltetrakis(trimethylphosphin)kobalt and
seine Derivate,
Chem Ber., 108:944-955 (1975);1 ncorporated herein by reference]. The infrared
spectra were recorded on a Perkin Elmer RX1 FTIR Spectrophotometer calibrated
using polystyrene film. 'H NMR spectra were recorded on a Bruker AVANCE 200
MHZ spectrometer and were referenced to internal solvent resonances. Tandem
gel
permeation chromatography/light scattering (GPC/LS) was performed on a SSI
pump equipped with a Wyatt DAWN DSP light scattering detector and Wyatt
Optilab
DSP. Separations were effected by 105 A, 104 P,, and 103 A Phenomenex 5 pm
columns using 0.1 M Liar in DMF as eluent at 60°C. Circular dichroism
measurements were carried out on a Olis Rapid Scanning Monochromator at room
temperature. The path length of the quartz cell was 1.0 mm and the
concentration of
peptide was 0.5 mg/mL. MALDITOF mass spectra were collected using a Thermo
BioAnalysis DYNAMO mass spectrometer r unning in positive ion mode with
samples
prepared by mixing solutions of analyte in THF with solutions of 2,5-
dihydroxybenzoic acid in THF and allowing the mixture to air dry.
N-Hydroxysuccinimidyl 2-(2-Methoxyethoxv)Ethoxy~ Acetate. 1
Preparation of Na-tert-butyloxvcarbonyl-O-(2-(2-methoxyethoxy)ethyl)-L-
serine. Compound 1. Scheme II
Na-tert-butyloxycarbonyl-L-serine (4.59 g, 22.3 mmol) was dissolved in N,N-
dimethylformamide (100 mL), the solution was then cooled to 0°C and
treated with
sodium hydride (1.97 g, 49.2 mmol). 1-Bromo-2-(2-methoxyethoxy)ethane (10.0 g,
49.2 mmol) was added to the solution and the reaction mixture was stirred at
ambient temperature for 3 h. The solvent was then removed under a reduced
pressure at 40 °C bath temperature. The residue was dissolved in water
(75 mL)
and washed twice with diethyl ether (30 mL each time). The aqueous layer was
then
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acidified to pH 3 with 1 M HCI and then extracted with ethyl acetate. The
organic
layer was dried over anhydrous MgS04 and the solvent was removed in vacuo to
yield the compound as a yellow oil (4.1 g, 63 %). The compound has the
following
characteristics: FTIR (CHC13): 1735 (~CO, s), 1712 (~CO, s). 'H NMR (CDC13): b
5.63 (d, (CH3)3COC(O)NHCH(CHzOCHZCH20CH2CH20CH3)C(O)OH, 1 H), 4.38 (s,
(CH3)3COC(O)NHCH(CHZOCHZCH20-CH2CHZOCH3)C(O)OH, 1 H), 4.14-3.53 (m,
(CH3)3COC(O)NHCH(CHzOCHzCH20-CH~CH20CH3)C(O)OH,10H), 3.47 (s,
(CH3)3COC(O)NHCH(CHZOCH2CH20CH2-CH20CH3)C(O)OH, 3H), 1.52 (s,
(CH3)3COC(O)NHCH(CHZO-CHZCHZOCH2CH2-OCH3)C(O)OH, 9H). MALDITOF-
MS: MH+: 307.34 calcd, 309.19 found.
Preparation of O-(2-(2-methoxyethoxy)ethy[)-L-serine. Compound 2. Scheme
The serine derivative N°-tert-butyloxycarbonyl-O-(2-(2-methoxy-
ethoxy)ethyl)-
L-serine was used without further purification. The serine derivative (4.13 g,
16.9
mmol) was dissolved in concentrated acetic acid (50 mL). After placing the
solution
in an ice bath, 1 M HCI (34 mL) was then added and the mixture stirred for 30
min.
The stirring was continued at ambient temperature for 2 h and the solution was
then
concentrated under reduced pressure to yield a yellow oil. The oil was then
neutralized with Et3N and the amine salt was removed by extraction with CH3CN.
The insoluble product was collected as a white solid (2.4 g, 58 %). ' H NMR
(D20):
~ 3.87 (m, NH2CH(CH20CHzCH20CHzCH20CH3)C(O)OH, 3H), 3.67-3.60 (m,
NH2CH(CHzO-CHZCHZOCHZCH20CH3)C(O)OH, 8H), 3.34 (s,
NH2CH(CH20CHzCHzOCHzCH20-CH3)C(O)OH, 3H). '3C f'H} NMR (Dz0): b
173.05 (NH2CH(CHZOCHZCHzOCH2-CHZOCH3)C(O)OH), 71.97, 70.87, 70.64,
70.48, 69.80 (NH2CH(CHZOCH2CHZOCH2-CH20CH3)C(O)OH), 59.10
(NHZCH(CHZOCHzCHzOCH2-CHZOCH3)C(O)OH)), 55.67
(NH2CH(CHZOCH2CH~OCH2-CHzOCH3)C(O)OH). MALDITOF-MS: MH+: 207.22
calcd, 208.48 found. ~a]p23 = -11.2 (c = 0.05, H20).
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Preparation of O-(2-(2-methoxyethoxy)ethyl)-L-serine NCA. Compound 3
Scheme II
To O-(2-(2-methoxyethoxy)ethyl)-L-serine (0.64 g, 2.6 mmol) was added THF
(100 mL) and COC12 (1.63 mL of a 1.93M toluene solution) and the mixture was
stirred for 5 h at room temperature. The resulting solution was concentrated
to give
a yellow oil as the crude product (0.49 g, 80 %). The oil was crystallized
from a
tetrahydrofuran, toluene and hexane mixture (1:4:4) at -30 °C to give
the product as
a white solid (0.27 g, 45 %). FTIR (THF): 1858 cm-' (vCO, s), 1792 cm-', (vCO,
s),.
'H NMR (CDC13): b 7.53 (s, RC(H)C(O)OC(O)NH, R = -
CHzOCH2CH20CH2CH20CH3, 1 H) 4.40 (t, RC(H)C(O)OC(O)NH, R = -
CH20CHzCH20CH2CHzOCH3 IH), 3.90 (d, RC(H)C(O)OC(O)NH, R = -
CH20CH2CH20CH2-CH20CH3 2H), 3.70-3.55 (m, RC(H)C(O)OC(O)NH, R = -
CH~OCH2CH~OCHzCHzOCH3, 8H), 3.41 (s, RC(H)C(O)OC(O)NH, R = -
CHzOCH2CH20CHz-CHZOCH3, 3H). '3C {'H} NMR (CDC13): b 169.82
(RC(H)C(O)OC(O)NH, R = -CH20CHZCH20CH2-CH20CH3), 153.63
(RC(H)C(O)OC(O)NH, R = -CHZOCH2CH20CH2-CHZOCH3), 72.75, 72.26, 71.76,
71.32, 71.07 (RC(H)C(O)OC(O)NH, R = -CH20CH2CHzOCH2-CH20CH3) 59.95
(RC(H)C(O)OC(O)NH, R = -CHZOCHZCHZOCHZ-CH20CH3), 59.78
(RC(H)C(O)OC(O)NH, R = -CH20CH2CH20CH2-CHZOCH3) ~a]o23 = -3 7.6 (c = 0.0
17, TH F).
Preparation of Poly(O-(2-(2-methoxyethoxy)ethyl)-L-serine) Compound 4
Scheme II
O-(2-(2-methoxyethoxy)ethyl)-L-serine NCA (120 mg, 0.52 mmol) in DMF (2
mL) was mixed with Co(PMe3)4 (3.8 mg, 0.010 mmol) in THF (0.5 mL) and stirred
for
18 h. The polymer was precipitated from this solution by addition to hexane
(20 mL).
The polymer dissolved in Hz0 (5 mL) and dialyzed to remove impurities and then
freeze-dried to give the product as a white solid (70 mg, 71 %). FTIR (KBr):
1631
cm-' (amide I, s br), 1523 cm~' (amide II, s br). 'H NMR: b 8.45 (d, -
(NHCH(CHZOCHzCH?OCHZCHZOCH3)-C(O))~_, IH), 4.61 (m,
-(NHCH(CHZOCHZCHZOCHzCH20CH3)C(O))~_, 1 H), 3.80 (br s,
-(NHCH(CHzOCH2CH20CH2CH20CH3)C(O))~_, 2H), 3.67-3.61 (br m,
-(NHCH(CH20CHzCH2OCH2CH2OCH3)C(O))~_, 8H), 3.36 (s,
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-(NHCH(CHZOCHzCH20CH2CH20CH3)C(O))~_. 3H).'3C {'H} NMR: b 174.23
(-(NHCH(CH20CH2CH20CHZCH20CH3)C(O))~_), 72.06, 71.15, 70.75, 70.59
(-(NHCH(CH~OCHZCHZOCHZCH20CH3)C(O))~_), 59.15 (-(NHCH(CHZOCHzCH20-
CHZCH20CH3)C(O))~_, 54.52 (-(NHCH(CH20CHZCH20CHZCHZOCH3)C(O))~_). [a]o
z3 = -28.3 (c = 0.012, H20).
Preparation of (2-(2-methoxyethoxy)ethy1l chloroformate, Compound 5
Scheme II
Di(ethyleneglycol) monomethyl ether (5.0 g, 42 mmol) in THF (30 ml) was
mixed with COC12 (32.3 ml of a 1.93M solution in toluene) at 0 °C and
stirred for one
hour. It was then stirred at 10°C for additional two hours. The solvent
and excess
phosgene were removed under reduced pressure to yield clear oil (7.0 g, 92 %).
FTIR(CHzCI~): 1780 cm''. This compound was used without further purification.
Preparation of O-(2-(2-methoxyethoxy)ethyl)carbonyl-NaCbz-L-tyrosine,
Compound 6, Scheme II
Na-Cbz-L-tyrosine (5.00 g, 15.9 mmol) was dissolved in NaOH (0.634 g, 15.9
mmol) in water (100 mL). (2-(2-methoxyethoxy)ethyl) chloroformate (4.34 g,
23.8
mmol) and 23.8 ml of 1 M NaOH were added simultaneously at 0 °C and the
resulting mixture was stirred for one hour at this temperature. The mixture
was then
stirred for an additional three hours at ambient temperature. The solution was
acidified with 1 N1 HCI and extracted with ethyl acetate. The organic layer
was dried
over MgSOa and solvent removed under reduced pressure to yield the product as
a
yellow oil (6.42 g, 91 %). This compound was used without further
purification. FTIR
(CH2C12): 1764, 1725 cm''. 'H NMR (CDC13): b 3.39 (s, 3H), 3,57-3.79 (in, 8H),
4.38
(m, 2H), 4.63 (s, 1 H), 5.05 (s, 2H), 6.93-7.57 (m, 9H).
Preparation of O-(2-(2-methoxyethoxy)ethyl)carbonyl-L-tyrosine NCA
Compound 7, Scheme II
O-(2-(2-methoxyethoxy)ethyl)carbonyl-Na-Cbz-L-tyrosine (6.43 g, 14.3 mmol)
was dissolved in CH2C12 (150 ml) and (a,a-dichloromethyl methyl ether (2.46 g,
21.5
mmol) was added to the solution. The mixture was then refluxed for 40 h, and
then
the solvent was removed to yield the product as a yellow oil (3.42 g, 70.1 %).
FTIR
(THF): 1790, 1855 cm''. 'H NMR (CDC13): b 7.04 (s, 4H), 4.53 (m, 1H), 4.31 (m,
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2H), 3.71 (m, 2H), 3.59 (m, 2H), 3.50 (m, 2H), 3.30 (s, 3H), 3.09-2.97 (m,
2H). '3C
NMR (CDC13): b 169.64, 153.73, 152.07, 150.55, 132.30, 130.74, 121.60, 71.84,
70.52, 68.89, 67.92, 59.00, 58.81, 36.94.
Preparation of Poly(O-(2-(2-methoxyethoxy)ethyllcarbonyl-L-tyrosine)
Compound 8, Scheme II
O-(2-(2-ethoxymethoxy)ethyl)carbonyl-L-tyrosine NCA (3.42 g, 10.0 mmol)
was dissolved in THF (10 mL). Sodium t-butoxide (9.6 mg, 0.10 mmol) was then
added. The resulting solution was stirred over night to give the polymer as an
off-
white precipitate that was isolated by washing with diethyl ether (50 mL)
(1.94 g,
65%). FT-IR (THF): 1660, 1547 cm-'.
Preparation of S-(2-(2-methoxvethoxy)ethyl)carbonyl-L-cysteine Compound
9, Scheme II
L-cysteine hydrochloride (2.00 g, 12.7 mmol) was dissolved in 1 M aqueous
sodium bicarbonate (25.4 ml) and the solution was diluted with water (75 mL).
The
solution was cooled in an ice bath and then covered with ether (50 ml). (2-(2-
methoxyethoxy)ethyl) chloroformate (2.31 g, 12.7 mmol) was added to the
solution in
one portion with vigorous stirring for one hour at 0 °C. The
temperature was allowed
to rise to 10 °C and stirred for an additionional two hours. The
solvents were then
removed under reduced pressure. The resulting solid was washed with methanol
and the methanol layer was evaporated to yield the product as a white oil
(2.21 g, 65
%). FTIR: 1709 cm-'. 'H NMR (D20): b 4.43 (m,1lH), 4.24 (m, 2H), 3.833.47 (m,
8H), 3.41 (s, 3H).
Preparation of S-(2-(2-methoxyethoxy)ethyl)carbonyl-L-cysteine NCA
Compound 10. Scheme II
S-(2-(2-ethoxymethoxy)ethoxy)carbonyl-L-cysteine (2.21 g, 8.26 mmol) was
mixed with THF (100 mL) and COC12 (5.13 ml of a 1.93 M solution in toluene).
The
mixture was stirred at ambient temperature for 5 hours and the solvent was
then
removed under reduced pressure to yield the product as a yellow oil (1.94 g,
80.1
%). FTIR: 1791, 1862 cm-'.
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Preparation of Poly(S-(2-(2-methoxyethoxy)ethvl)carbonyl-L-cysteine)
Compound 11. Scheme II
O-(2-(2-ethoxymethoxy)ethoxy)carbonyl-L-cysteine NCA (1.94 g, 6.61 mmol)
was dissolved in THF (10 mL) and sodium t-butoxide (6.4 mg, 0.067 mmol) was
then
added. The initially homogeneous solution was stirred over night to give the
polymer
as an off-white precipitate that was isolated by washing with diethyl ether
(50 mL)
(1.17 g, 70.9 %). FTIR: 1635, 1517 cm-'.
A mixture of 2-[2-(2-methoxyethoxy)ethoxy) acetic acid (10 g, 58 mmol) and
N-hydroxysuccinimide (7.5 g, 64 mmol) dissolved in THF (ca. 300 mL) in a round
bottom flask was cooled using an ice water bath. Dicyclohexylcarbodiimide (12
g, 58
mmol) was then added with stirring. A white precipitate was observed to form
after 5
min and the reaction mixture was then let stand in a refrigerator (4
°C) for 16 h. The
white precipitate, dicyclohexylurea, was removed by filtration and the
filtrate was
concentrated undre vacuum to give an oil. This crude product was then
dissolved in
a small amount of THF (ca. 10 mL) and the resulting suspension was filtered to
remove the precipitate. This procedure was repeated until a clear solution was
obtained upon dissolution in THF. Removal of the residual THF under vacuum
gave
the product an oil (9.0 g, 59%). 'H NMR(CDC13): ~ 4.49 (s, -OC(O)CHzO-, 2H),
3.77
(m, -OC(O)CHZOCH~CH~O-, 2H), 3.65 (m, -OCHzCH2CHz-, 4H), 3.52 (m, -
OCHZCH~OCH~, 2H), 3.34 (s, -CH~OCH~, 3H), 2.82 (s,-C(O)CH~CH~C(O)-,4H).
NE-2-(2-(2-Methoxyethoxy)Ethoxyl Acetyl-NC CBZ-L-Lysine. Compound 2.
Scheme III
To a mixture of Na-CBZ-L-Lysine (4.9 g, 17 mmol) and NaHC03 (2.0 g, 23
mmol) in THF:H~O (75 mL:75 mL) was added 1 (3.2 g, 12 mmol) in THF (10 mL).
After stirring for 1 h at 20 °C, the THF was removed under vacuum. The
product
was extracted with ethyl acetate (2 x 50 mL), the organic fractions were
combined,
and the solvent was removed under vacuum to leave a white solid. This crude
product was recrystallized from MeOH and diethyl ether to give 2 as white
crystals
(3.0 g, 59%). MP = 115-117 °C. 'H NMR(CDC13): c~ 7.25 (m, -CHzC6H5,
5H), 5.18
(s, -CH2C6H5, 2H), 4.65 (t, -NHCH(R)C(O)OH, 1 H), 3.72 (m, -
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NHCH((CHZ)3CHzC(O)R)C(O)- + - O(CH2CHz0)2CH3, 15H), 1.70 (m,-
NHCH((CH2)3CH2C(O)R)C(O)-, 6H).
Ne-2-f2-(2-Methoxyethoxy)Ethoxyl Acetyl-L-Lysine-N-Carboxyanhydride
Compound 3. Scheme 111
To a solution of 2 (4.9 g, 11 mmol) in anhydrous CH2C12 (125 mL) under
nitrogen was added 1,1-dichlorodimethylether (1.5 mL, 17 mmol). The solution
was
then heated to reflux for 20 h, after which the solvent was removed under
vacuum.
The crude oil was crystallized from THF and hexanes to give 3 as white
crystals (2.8
g, 75%). ' H NMR(CDC13): b 7.68 (br s, -NH, 1 H), 7.35 (br s, -NH, 1 H), 4.30
(t,
NHCH(R)C(O)O-, 1 H), 3.15 (m, -NHCH((CHz)3CH2C(O)R)C(O)- + -O(CHCH20)zCH3,
15H), 1.70 (m, -NHCH((CH2)3CH2C(O)R)C(O)-, 6H. FTIR(THF): 1856 cm'' (vCO,
anhydride s), 1789 cm-' (vCO, anhydride, vs), 1677 cm-' (vCO, amide, s).
Polv Ne-2-(2-Methoxyethoxy)Ethoxyl Acetyl-L-Lysine) Compund 4 Scheme III
In the dry box, 3 (730 mg, 2.2 mmol) was dissolved in THF (15 mL) and
placed in a 75 mL reaction tube which could be sealed with a Teflon stopper.
An
aliquot of 2,2'-bipyridyl)Ni(1,5-cyclooctadiene) (600 ~,L of a 36 mM solution
THF) was
then added via syringe to the flask. A stirbar was added and the flask was
sealed,
removed from the dry box, and stirred in a thermostated 25 °C bath for
24 h.
Polymer was isolated by addition of the reaction mixture to diethyl ether
causing
precipitation of the polymer. The polymer was then dissolved in THF and
reprecipitated by addition to diethyl ether. The polymer was dried in vacuo to
give 4
as a white fibrous solid (550 mg, 87 % yield). GPC of the polymer in 0.1 M
Liar in
DMF at 60 °C: M~= 101,000; MW/M~ = 1.21. FTIR(THF): 1672 cm-'
(vCO,amide,s),
1650 cm~' (vCO, Amide I, br vs), 1538 cm-' (vCO, Amide II, br s).
EXAMPLE 6 -Synthesis of Self-Assembling Amphiphilic Block Cooolypeptides for
Biomedical Applications
In the examples below, we prepared di- and tri-block copolypeptides of the
general architectures: (EG-Lys)X-(insoluble blocky and (EG-Lys)X-(insoluble
block)Y-
(EG-Lys)Z, where x, y, and z represent the number of amino acids in each
domain.
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Sample Synthesis of a Poly(NE-2-f2-(2-Methoxyethoxylethoxylacetyl-L-
Lysine)-block-(L-Leucine/L-Valine) diblock copolymer.
In the dry-box, 100 mg of NE-2[2-(2-Methoxyethoxy)ethoxyJacetyl-L-Lysine)-N-
carboxyanhydride, EG-Lys NCA, was dissolved in anhydrous THF (3mL) and placed
in a 15 mL reaction tube, which could be sealed with a Teflon stopper. Into
the
reaction tube, which contained a stirbar, an aliquot of (PMe3)4)Co (2.37 mg in
1 ml of
THF) was added. The flask was sealed and stirred overnight to form the poly(EG-
Lys) block. A mixture of L-leucine-N-carboxyanhydride, Leu NCA, (5mg) and L-
valine-N-carboxyanhydride, Val NCA, (1 mg) was dissolved in anhydrous THF (1
mL) and then added to the reaction flask. After stirring for an additional 16
h, the
block copolymer was isolated and purified by removing the solvent from the
reaction
mixture in vacuo followed by resuspension of the residue in double distilled
water
and dialysis of this solution against 4 liters of double distilled water for 8
h using a
Spectrapore dialysis membrane with a molecular weight cut-off of 1000. The
dialysis
was repeated twice and the copolymer was isolated by freeze-drying of the
solution
(yield: 76 mg). GPC analysis of the polymer: M~=39,000 and MW/M~=1.2.
FTIR(THF): 1650 cm-' (vCO, amide I, vs), 1540 cm-' (vCO, amide II, s).
Sample Synthesis of a Polv(N~-2-f 2-(2-fVlethoxyethoxylethoxylacetyl-L-
L~~sine)-block-(L-Leucine/L-Valine)-block-(N~-2- 2-(2-
Methoxvethoxv)ethoxylacetyl-L-Lysine triblock copolymer.
In the dry-box, 50 mg of NE-2[2-(2-Methoxyethoxy)ethoxy]acetyl-L-Lysine)-N-
carboxyanhydride, EG-Lys NCA, was dissolved in anhydrous THF (3mL) and placed
in a 15 mL reaction tube, which could be sealed with a Teflon stopper. Into
the
reaction tube, which contained a stirbar, an aliquot of (PMe3)4)Co (2.37 mg in
1 ml of
THF) was added. The flask was sealed and stirred overnight to form the poly(EG-
Lys) block. A mixture of L-ieucine-N-carboxyanhydride, Leu NCA, (5mg) and L-
valine-N-carboxyanhydride, Val NCA, (1 mg) was dissolved in anhydrous THF (1
mL) and then added to the reaction flask to form the central block. After
stirring for
an additional 16 h, 50 mg of EG-Lys NCA was dissolved in THF (1 mL) and added
to
the reaction tube, which was stirred for an additional 16 h to form the final
poly(EG-
Lys) block of the complete triblock copolymer. The copolymer was isolated and
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purified by removing the solvent from the reaction mixture in vacuo followed
by
resuspension of the residue in double distilled water and dialysis of this
solution
against 4 liters of double distilled water for 8 h using a Spectrapore
dialysis
membrane with a molecular weight cut-off of 1000. The dialysis was repeated
twice
and the copolymer was isolated by freeze-drying of the solution (yield: 78
mg). GPC
analysis of the polymer: M~=41,000 and MW/M~=1.1. FTIR(THF): 1650 cm-' (vCO,
amide I, vs), 1540 cm-' (vCO, amide II, s).
Vesicle Formation
Spontaneous vesicle formation from di- and tri-block copolypeptides was
observed under a light microscope after initial dissolution in water which
gave milky
suspensions. The size of the vesicles varied widely, with diameters up to
several
microns. Smaller vesicles were prepared by sonication of the solutions of
large
vesicles, similar to the way lipid vesicles are manipulated. Polymers were
typically
dissolved in doubly distilled water at concentrations of 5 mg/mL and then
sonicated
for 9 min at 13 watts of power using a Via Cell sonicator. The sonication was
repeated 3 times, after which the opalescent suspensions became clear
solutions.
Vesicle Characterization
Light Nticroscopy was performed on a Nikon Optiphot2-POL. Initial samples
were approximately 5 mg/ml in doubly distilled water and 50 Nof sample was
deposited on a glass slide and topped with a cover-slip for visualization.
Light scattering was performed on a Brookhaven Instruments Dynamic Light
Scattering (DLS) system, with a laser power of 30 mwatts and wavelength of 546
nm. Samples were sonicated as indicated above and added to DLS cuvettes. The
sonicated vesicles had average diameters ranging from 50 nm to 500 nm,
depending
on copolymer chain length and amino acid composition (e.g., see Table 9
below).
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Table 9
Name* ~ Composition i Molecular Weight
! Vesicle Diameter
PLV10 i 90% P, 10% LV 10 kDa 132 nm
PLV10 90% P, 10% LV i 22 kDa i 147
nm
PLV10 90% P, 10% i 40 kDa 197 nm
LV
i PLV10 I 90% P, 10% LV 90 kDa ' 230 nm
PLV20 ~ 80% P, 20% LV 25 kDa 175 nm
PLV30 70% P, 30% LV 30 kDa 207 nm
PLV40 60,i P, 40% LV 35 kDa 217 nm
*Key: P = poly (EGz-L lysine) domain; LV = hydophobic domain composed of a
random copolymer of L-leucine (L) and L-valine (V) with an internal
composition of
75% leucine and 25% valine. Thus, PLV10 is a diblock copolymer where the P
block
makes up 90 mole percent of the total polymer size and the LV block makes up
10%
of the total copolymer.
Although the present invention has been described in considerable detail with
reference to certain preferred versions thereof, other versions are possible.
For
example, the lengths of each domain, and composition of the insoluble block
domains. can be varied during the synthesis to modify the self-assembled
structures
that are formed. In addition, D- or L- or stereomixtures of amino acids can be
used
in these block copolymers to modify polypeptide secondary structure or to
modify
biological stability and interactions. Therefore, the spirit and scope of the
present
invention should not be limited to the description of the preferred versions
contained
herein.
