U.S. patent application number 15/243866 was filed with the patent office on 2017-02-16 for carbamate, thiocarbamate or carbamide comprising a biomolecular moiety.
The applicant listed for this patent is DSM IP ASSETS B.V.. Invention is credited to Aylvin Jorge Angelo Athanasius DIAS, Bartholomeus Johannes Margretha PLUM, Peter Jan Leonard Mario QUAEDFLIEG, Roel Wim WIERTZ.
Application Number | 20170044288 15/243866 |
Document ID | / |
Family ID | 39030846 |
Filed Date | 2017-02-16 |
United States Patent
Application |
20170044288 |
Kind Code |
A1 |
DIAS; Aylvin Jorge Angelo
Athanasius ; et al. |
February 16, 2017 |
CARBAMATE, THIOCARBAMATE OR CARBAMIDE COMPRISING A BIOMOLECULAR
MOIETY
Abstract
Compounds are provided which comprise (a) at least two
polymerisable moieties, (b) at least one amino acid residue of an
amino acid comprising at least two amine groups of which at least
two amine groups have formed a carbamate, a thiocarbamate or a
carbamide group, and (c) a biomolecular moiety linked directly or
via a spacer to the carboxylic acid moiety of the diamino acid
residue or a carboxylic acid to which such moiety can be linked. A
polymer obtained from such compounds is also disclosed.
Inventors: |
DIAS; Aylvin Jorge Angelo
Athanasius; (Maastricht, NL) ; PLUM; Bartholomeus
Johannes Margretha; (Ulestraten, NL) ; QUAEDFLIEG;
Peter Jan Leonard Mario; (Elsloo, NL) ; WIERTZ; Roel
Wim; (Brunssum, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DSM IP ASSETS B.V. |
Heerlen |
|
NL |
|
|
Family ID: |
39030846 |
Appl. No.: |
15/243866 |
Filed: |
August 22, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12513576 |
Nov 10, 2009 |
9458256 |
|
|
PCT/EP2007/009637 |
Nov 7, 2007 |
|
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15243866 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 27/16 20130101;
A61L 2420/02 20130101; C08F 220/365 20200201; A61L 2300/606
20130101; C08F 8/30 20130101; A61K 47/593 20170801; C09D 133/14
20130101; C07C 275/16 20130101; A61L 27/34 20130101; C08F 220/346
20200201; C08F 220/36 20130101; A61L 2400/18 20130101 |
International
Class: |
C08F 220/36 20060101
C08F220/36; A61L 27/16 20060101 A61L027/16; C09D 133/14 20060101
C09D133/14; A61L 27/34 20060101 A61L027/34; A61K 47/48 20060101
A61K047/48; C07C 275/16 20060101 C07C275/16 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 7, 2006 |
EP |
06023114.9 |
Claims
1. A formulation comprising: (a) a polymerizable compound
comprising (a) at least two radically polymerizable moieties
comprising an acrylate, an alkylacrylate, a methacrylate, an
alkylmethacrylate, a vinylether, a fumarate, a vinylsulphone, or an
itaconate, (b) at least one amino acid residue of an amino acid,
the amino acid comprising at least two amine groups, and (c) a
biomolecular moiety Z linked directly or via a spacer to the amino
acid residue via a carboxylic acid moiety of the amino acid,
wherein the polymerizable compound comprises at least two carbamate
groups, at least two carbamide groups, or at least one carbamate
group and at least one carbamide group, wherein the at least two
carbamate groups, at least two carbamide groups, or at least one
carbamate group and at least one carbamide group result from the
reaction of an amine group of the amino acid with one or more other
compounds; and (b) a photo-initiator.
2. The formulation according to claim 1, wherein the polymerizable
compound further comprises a polyester, a polythioester, a
polyorthoester, a polyamide, a polythioether, a polyanhydride, a
polydioxanone, or a polycarbonate moiety.
3. The formulation according to claim 1, further comprising (c) a
further polymerizable compound comprising a polymerizable moiety
comprising an acrylate, an alkylacrylate, a methacrylate, an
alkylmethacrylate, a vinylether, a fumarate, a vinylsulphone, or an
itaconate, wherein the further polymerizable compound is able to
copolymerize with the polymerizable compound.
4. The formulation according to claim 2, further comprising (c) a
further polymerizable compound comprising a polymerizable moiety
comprising an acrylate, an alkylacrylate, a methacrylate, an
alkylmethacrylate, a vinylether, a fumarate, a vinylsulphone, or an
itaconate, wherein the further polymerizable compound is able to
copolymerize with the polymerizable compound.
5. The formulation according to claim 3, wherein the polymerizable
compound and the further polymerizable compound each comprise an
acrylate or methacrylate group.
6. The formulation according to claim 1, wherein the at least two
radically polymerizable moieties are endgroups of the polymerizable
compound.
7. The formulation according to claim 1, wherein the amino acid
residue is a residue of lysine, ornithine, hydroxylysine,
N-alpha-methyl lysine, or diaminobutanoic acid.
8. The formulation according to claim 1, wherein the amino acid
residue is a residue of L-lysine, L-hydroxylysine, or
N-alpha-methyl-lysine.
9. The formulation according to claim 1, wherein the biomolecular
moiety Z is a peptide residue.
10. The formulation according to claim 1, wherein the biomolecular
moiety Z comprises a peptide residue comprising the sequence RGD,
GRGDS, RGDS, YIGSR, REDV, GTPGPQGIAGQRGW, PDGEA, KRSR, or
NSPVNSKIPKACCVPTELSAI.
11. The formulation according to claim 1, wherein the biomolecular
moiety Z comprises heparin.
12. The formulation according to claim 1, wherein the biomolecular
moiety Z comprises a signaling moiety for a cell.
13. The formulation according to claim 2, wherein the biomolecular
moiety Z comprises a peptide residue comprising the sequence RGD,
GRGDS, RGDS, YIGSR, REDV, GTPGPQGIAGQRGW, PDGEA, KRSR, or
NSPVNSKIPKACCVPTELSAI.
14. The formulation according to claim 8, wherein the biomolecular
moiety Z YIGSR, REDV, GTPGPQGIAGQRGW, PDGEA, KRSR, or
NSPVNSKIPKACCVPTELSAI, or the biomolecular moiety Z comprises
heparin.
15. A method of forming an article comprising the step of
polymerizing the formulation according to claim 3.
16. An article formed by polymerizing the formulation according to
claim 3.
17. An article formed by polymerizing the formulation according to
claim 8.
18. A formulation comprising: (a) A compound according to Formula
I: ##STR00013## wherein G is a residue of a --OH, --NH.sub.2, or
--RNH multifunctional polymer or oligomer comprising a polyester, a
polythioester, a polyorthoester, a polyamide, a polythioether, a
polyanhydride, a polydioxanone, or a polycarbonate, wherein R is as
defined below, or G is an X as defined below; each X independently
represents an acrylate or a methacrylate; each Y independently
represents, O or NR; each R independently represents hydrogen or a
group selected from substituted and unsubstituted hydrocarbons
which optionally contain one or more heteroatoms; L represents a
substituted or unsubstituted hydrocarbon that optionally contains
one or more heteroatoms; n is an integer having a value of 1 if G
is an X, and n is at least 2 if G represents a residue of the
multifunctional polymer or oligomer; and Z is a biomolecular moiety
linked directly or via a spacer to the remainder of the compound;
and (b) a photo-initiator.
19. A compound according to claim 18, wherein the NR-L(C.dbd.O)--NR
moiety of Formula I represents the residue of a lysine moiety, a
hydroxylysine moiety, or an N-alpha-methylated lysine moiety.
20. An article formed by polymerizing the formulation according to
claim 19.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of copending U.S.
application Ser. No. 12/513,576, filed Nov. 10, 2009 (now U.S. Pat.
No. ______), which is the national phase application of
international application PCT/EP2007/009637, filed Nov. 7, 2007,
which designated the US and claims priority to European Application
No. 06023114.9, filed Nov. 7, 2006, the entire contents of each of
which are hereby incorporated by reference.
FIELD
[0002] The invention relates to a polymerisable compound comprising
one or more carbamate, thiocarbamate and/or carbamide groups and a
biomolecular moiety The invention further relates to a method for
preparing such compound, a method for formulating said compound
with one or more other compounds, a method of polymerising the
compound and an article comprising the compound and a method of
preparing such an article.
BACKGROUND AND SUMMARY
[0003] The use of synthetic polymers in medical applications, such
as in the repair or regeneration of tissues, in particular
cartilage, bone or vasculature, has recently attracted significant
interest. However, synthetic polymers, for instance polyethylene
glycol (PEG) and polyacrylic polymers are generally not capable of
selectively facilitating adhesion of cells or facilitate another
biospecific function.
[0004] Further, several biomolecules, such as peptides, proteins
and glycopolymers are readily denatured by heat, proteases,
solvents, material processing conditions and/or the manner in which
implants are introduced into the body. It is a challenge to provide
compositions of synthetic polymers and such biomolecules, whilst
retaining the biomolecules in an active form.
[0005] There would be great value in attaching biologically active
molecules to polymers. Free radical polymerisation is a common
method to create protein-polymer hybrid materials as shown in Van
Hest et. al. Advances in Polymer Science 2006, 202, 19-52. This
free radical polymerisation can also be performed at lower
temperatures than condensation polymerisation thereby reducing the
risk of denaturing the proteins or biomolecules which are expensive
and synthetically intensive to generate. For example, free radical
photopolymerisation is often used to prepare hydrogels.
[0006] To prepare such protein polymer hybrids by free radical
polymerization the protein or peptide is normally furnished with
one polymerisable group as shown by Hubbel et. al. J. Biomed.
Mater. Res. 1998, 39, 266. The technique described in this
publication may lead to polymers with network defects since the
monofunctional peptide functions as a dangling chain end. The
consequences are that there is a risk that the peptide is not
effectively incorporated into the network and that the resulting
polymer be plasticised, to the extent that mechanical properties of
the resulting biomaterial are adversely affected. This adverse
effect is particularly pronounced with hydrogels.
[0007] In Macromolecules, Volume 39, Number 4 (2006), page
1305-1307, Junmin Zhu et al. describe the synthesis of a
polyethylene glycol diacrylate macromer with a cell-adhesive
peptide ligand. The macromer is prepared by reacting a hexapeptide
attached to the carboxylic acid of diaminopropionic acid with
acryloyl-PEG-anhydrous succinamide (Acr-PEG-NHS), thereby forming
amide bonds. In particular, if diamino propionic acid is used in a
polymer prone to enzymatic or hydrolytic attack then this non
natural amino acid could give rise to an undesired side effect.
[0008] In the synthetic approach by Zhu, the polymerisable entities
are attached to the peptide to make a crosslinking peptide
prepolymer. However the present inventors propose a route where the
prepolymer can be furnished with one or more reactive groups that
will be able to react with a peptide or activated peptide. This
allows better control of peptide density along the polymer chain
and furthermore the prepolymer can be polymerised to generate a
network that can be subsequently furnished with the biomolecules,
in particular peptides. This is in particular advantageous, when
the polymer processing conditions are aggressive and the peptides
are preferably attached at the end of a process, e.g. in the
manufacture of a sensor.
[0009] It is an object of the invention to provide a novel polymer
or article that may serve as an alternative to known polymers
respectively articles, in particular for use in a medical
application, a sensor, a diagnostic application and/or a drug
delivery application.
[0010] It is an object of the invention to provide a polymer or
article that shows satisfactory biocompatibility in vivo (such a
low tendency or no tendency to cause an immune response) and/or
that is biodegradable, in particular in vivo. In particular, it is
an object to provide a polymer of which the biodegradation rate
under in vivo conditions is well controlled.
[0011] It is a further object of the invention to provide a method
for efficiently introducing one or more biologically active
molecules, such as one or more functional peptides, into a polymer
having a polymerisable functionality larger than 1.
[0012] It is a further object of the invention to provide a polymer
with good degradation behaviour, in particular reduced acidity
during degradation.
[0013] It is a further object of the invention to provide a novel
polymer with good mechanical properties such as a good
elasticity.
[0014] It is a further object to provide a polymer or article that
shows selective interaction with a cell tissue or biological fluid
to promote, suppress or balance a specific biological response.
[0015] It is a further object of the invention to provide a polymer
matrix that is suitable for sensing purposes and/or for targeted
drug delivery.
[0016] It is a further object to provide a novel compound that can
be used to prepare a polymer or article.
[0017] It is a further object to provide a novel compound, polymer
or article, that can be used in vivo, which comprises a
biomolecular moiety that is capable of interacting with
(autologous) cells or with a specific biochemical component, or
that comprises a functional group that can be covalently attach
with such biomolecular moiety.
[0018] It is a further object of the invention to provide an
article comprising a coating based on the polymer which can be used
to coat implanted articles
[0019] One or more other objects that may be solved in accordance
with the present invention will become apparent from the
description, below.
[0020] It has been found possible to meet one or more objects of
the present invention by providing a compound comprising (a) at
least two polymerisable moieties, (b) at least one amino acid
residue of an amino acid comprising at least two amine groups of
which at least two amine groups have formed a carbamate, a
thiocarbamate or a carbamide group, and (c) a biomolecular moiety
linked directly or via a spacer to the carboxylic acid moiety of
the diamino acid residue.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 shows confocal microscopy pictures of the gel
according to Example 2 below;
[0022] FIGS. 2-4 are weight loss graphs of the cured films
according to Example 3 below;
[0023] FIGS. 5-10 are weight loss graphs of the cured films
according to Example 4 below;
[0024] FIG. 11 is a graphical representation of the tensile test
according to Example 5 below;
[0025] FIGS. 12-13 are graphical representations of the extractable
measurement concluded according to Example 6 below;
[0026] FIG. 14 are photomicrographs showing the morphology of cells
grown on PTGL1000-(TDI-HEA).sub.2/RGD-LDI-(HEA).sub.2 coating in
comparison to cells grown on PTGL1000-(TDI-HEA).sub.2 coating
according to Example 7 below; and
[0027] FIG. 15 are photomicrographs showing cell attachment to the
PEG600-(m-LDI-HEA).sub.2/GRGDS-(LDI-HEA).sub.2 coating under serum
free and serum containing conditions as compared to control polymer
PEG600-(m-LDI-HEA).sub.2 according to Example 8 below.
DETAILED DESCRIPTION
[0028] In particular the invention relates to a compound, which is
polymerisable, represented by formula I
##STR00001##
wherein [0029] G is a residue of a polyfunctional compound having
at least n functional groups or a moiety X [0030] each X
independently represents a moiety comprising a polymerisable group;
[0031] each Y independently represents, O, S or NR; [0032] each R
independently represents hydrogen or a group selected from
substituted and unsubstituted hydrocarbons which optionally contain
one or more heteroatoms, preferably hydrogen or a C1-C20
hydrocarbon, more preferably hydrogen or a C1-C8 alkyl; [0033] L
represents a substituted or unsubstituted hydrocarbon which
optionally contains one or more heteroatoms. [0034] n is an integer
having a value of 1 in case G represents an X and n is at least 2,
preferably 2-8, in case G represents a residue of a polyfunctional
compound having at least n functional groups; [0035] Z is a
biomolecular moiety linked directly or via a spacer to the
remainder of the compound.
[0036] Within the context of the present invention the term
"hydrocarbon" is meant to include substituted and unsubstituted
hydrocarbons, hydrocarbons with one or more heteroatoms (such as S,
N, O, P) or hydrocarbons without heteroatoms, unless specifically
mentioned otherwise. Substituents may in particular be selected
from OH and halogen atoms (Br, Cl, F, I).
[0037] The term "alkyl" and "alkylene" is meant to include
unsubstituted and substituted alkyl respectively alkylene, unless
specified otherwise. Substituents may in particular be selected
from OH and halogen atoms (Br, Cl, F, I).
[0038] In principle, the polymerisable moiety ("X") in the
polymerisable compound according to the present invention can be
any moiety that allows the formation of a polymer. In particular it
may be chosen from moieties that are polymerisable by an addition
or radical reaction. The addition reaction has been found easy and
well-controllable. Further, it may be carried out without formation
of undesired side products, such as products formed from leaving
groups.
[0039] Preferably, the moiety allows radical polymerisation. This
has been found advantageous as it allows initiating a
polymerisation, in the presence of a photo-initiator, by
electromagnetic radiation, such as UV, visible, microwave, Near IR,
gamma radiation, or by electron beam instead of thermally
initiating the polymerisation reaction. This allows rapid
polymerisation, with no or at least a reduced risk of thermal
denaturation or degradation of (parts of) the compound/the
polymer.
[0040] Thermal polymerisation may be employed, in particular in
case no biological moiety or moieties are present that would be
affected by heat. E.g. heat-polymerisation may be employed when one
or more short chain peptides and/or proteins are present of which
the bio-active sites are not affected by the high temperature,
required for polymerization.
[0041] Preferred examples of polymerisable groups X include groups
comprising an unsaturated carbon carbon bond such as a C.dbd.C bond
(in particular a vinyl group) or a C.ident.C group (in particular
an acetylene group), thiol groups, epoxides, oxetanes, hydroxyl
groups, ethers, thioethers, HS--, H.sub.2N--, --COOH,
HS--(C.dbd.O)-- or a combination thereof, in particular a
combination of thiol and C.dbd.C groups.
[0042] In particular preferred is a polymerisable moiety X selected
from the group consisting of acrylates methacrylates,
alkyl(meth)acrylates, hydroxyl alkyl (meth)acrylates; vinylethers;
alkylethers; itaconates, unsaturated diesters and unsaturated
diacids or salts thereof (such as fumarates); vinylsulphones,
vinylphosphates, alkenes, unsaturated esters, fumarates, maleates
and combinations thereof. Such moieties X can be introduced in the
polymer of the present invention starting from readily available
starting materials and show good biocompatibility, which makes them
particularly useful for an in vivo or other medical
application.
[0043] Good results have in particular been achieved with moieties
X being hydroxyethylacrylate and hydroxyethylmethacrylate
[0044] In an advantageous embodiment, the polymerisable moiety X is
represented by the formula --R.sub.1R.sub.2C.dbd.CH.sub.2, wherein
[0045] R.sub.1 is chosen from the group consisting of substituted
and unsubstituted, aliphatic, cycloaliphatic and aromatic
hydrocarbon groups that optionally contain one or more moieties
selected from the group consisting of ester moieties, ether
moieties, thioester moieties, thioether moieties, carbamate
moieties, thiocarbamate moieties, amide moieties and other moieties
comprising one or more heteroatoms, in particular one or more
heteroatoms selected from S, O, P and N. R.sub.1 may be linear or
branched. In particular R.sub.1 may comprise 2-20 carbon atoms,
more in particular it may be a substituted or unsubstituted C.sub.1
to C.sub.20 alkylene; more in particular a substituted or
unsubstituted C.sub.2 to C.sub.14 alkylene; and [0046] R.sub.2 is
chosen from the group consisting of hydrogen and substituted and
unsubstituted alkyl groups, which alkyl groups optionally contain
one or more heteroatoms, in particular one or more heteroatoms
selected from P, S, O and N. R.sub.2 may be linear or branched. In
particular, R.sub.2 may be hydrogen or a substituted or
unsubstituted C.sub.1 to C.sub.6 alkyl, in particular a substituted
or unsubstituted C.sub.1 to C.sub.3 alkyl.
[0047] The amino acid residue ("L") is a substituted or
unsubstituted hydrocarbon, which may contain heteroatoms, such as
N, S, P and/or O. In case of a substituted hydrocarbon the
substituent may be a hydroxyl.
[0048] The amino acid residue may be based on a D-isomer or an
L-isomer. Preferably, L is C1-C20 hydrocarbon, more preferably, L
is a linear or branched C1-C20 alkylene, even more preferably
C1-C12 alkylene, most preferably C3-C8 alkylene, wherein the
alkylene may be unsubstituted or substituted, in particular with a
hydroxyl, and/or optionally contains one or more heteroatoms. In
view of desirable hydrophilic properties, the amount of carbons is
preferably relatively low, such as 8 or less.
[0049] Particularly preferred are amino acid residues selected from
lysine, ornithine, hydroxyllysine, N-alpha-methyl lysine or
diaminobutanoic acid residues.
[0050] In particular in case the compound/polymer/article of the
present invention is intended to be used in a medical application,
more in particular in case it is intended to be used in vivo, it is
preferred that the amino acid residue is based upon a naturally
amino acid, usually an L-isomer. This is in particular desired in
case the compound/polymer/article is biodegradable. In view
thereof, preferred amino acid residues are residues of L-lysine,
L-hydroxylysine or N-alpha-methyl-lysine. Good results have in
particular been achieved with L-lysine.
[0051] In case the compound/polymer of the invention degrades (for
instance in vivo), an amino acid (corresponding to the residue "L")
may be one of the degradation products. As the compound/polymer
degrades, acid (protons, H.sub.3O.sup.+) may be released. This may
cause an inflammation or similar reaction, under in vivo
conditions. The inventors consider that the amino acid may
contribute to avoiding an inflammation of tissue in the vicinity of
a biodegrading implanted article of the invention. Without being
bound by theory, it is contemplated that the amino acid may
scavenge the acid, thereby contributing to avoiding an inflammation
of the tissue. For this purpose lysine is found particularly
suitable.
[0052] L-Hydroxylysine may be useful in that it allows the
attachment of a peptide via the C terminus of the peptide to be
attached. It may also be used for providing a polymer with a higher
hydrophilicity than a comparable polymer based on L-lysine.
[0053] The amino acid which may be formed upon degradation of a
compound/polymer of the invention may serve a physiological
function, such as contribute to the healing of a wound (L-arginine,
L-glutamine) or affect the nervous system (L-asparagine), e.g. in
case of a nerve guide comprising the polymer.
[0054] As indicated above, Z is a biomolecular moiety linked
directly or via a spacer to the remainder of the compound. The
spacer may be present to provide selective surface or bulk
patterning of the biomolecular moiety. The biomolecular moiety Z
can in principle be any biologically active molecule bound
(directly or via a spacer) to the carboxylic acid group of the
amino acid residue. Such molecule can be a naturally occurring
molecule or a synthetic molecule.
[0055] Preferably, the biomolecular moiety Z is selected from cell
signalling moieties, moieties capable of improving cell adhesion to
the compound/polymer/article, moieties capable of controlling cell
growth (such as stimulation or suppression of proliferation),
antithrombotic moieties, moieties capable of improving wound
healing, moieties capable of influencing the nervous system,
moieties having selective affinity for specific tissue or cell
types, epitopes and antimicrobial moieties. The moiety may exert an
activity when bound to the remainder of the
compound/polymer/article and/or upon release from the compound.
Preferably, it is active when bound.
[0056] Preferably, the biomolecular moiety Z is selected from amino
acids, peptides, including cyclic peptides, oligopeptides,
polypeptides, glycopeptides and proteins, including glycoproteins;
nucleotides, including mononucleotides, oligonucleotides and
polynucleotides and carbohydrates.
[0057] For instance, an amino acid may be linked for stimulating
wound healing (L-arginine, L-glutamine) or to modulate the
functioning of the nervous system (L-asparagine).
[0058] In a preferred embodiment, the bioactive moiety is a peptide
residue, more preferably an oligopeptide residue. Peptides with
specific functions are known in the art and may be chosen based
upon a known function. For instance, the peptide may be selected
from growth factors and other hormonally active peptides. In
particular, Z may be selected from a peptide residue comprising the
sequences as given below, which are composed of amino acids known
by a man skilled in the art.
TABLE-US-00001 Peptide residue suggested function RGD, GRGDS, RGDS
Enhance bone and/or cartilage tissue formation; Regulate neurite
outgrowth; Promote myoblast adhesion, proliferation and/or
differentiation; Enhance endothelial cell adhesion and/or
proliferation KQAGDV Smooth muscle cell adhesion YIGSR Cell
adhesion REDV Endothelial cell adhesion GTPGPQGIAGQRGVV (P-15) Cell
adhesion (osteoblasts) PDGEA Cell adhesion (osteoblasts) IKVAV
Neurite extension RNIAEIIKDI Neurite extension KHIFSDDSSE Astrocyte
adhesion VPGIG Enhance elastic modulus of artificial extra cellular
matrix (ECM) FHRRIKA Improve osteoblastic mineralization KRSR
Osteoblast adhesion KFAKLAARLYRKA Enhance neurite extension
KHKGRDVILKKDVR Enhance neurite extension YKKIIKKL Enhance neurite
extension NSPVNSKIPKACCVPTELSAI Osteoinduction APGL Collagenase
mediated degradation VRN Plasmin mediated degradation AAAAAAAAA
Elastase mediated degradation Acetyl-GCRDGPQ-GIWGQDRCG Encourage
cell-mediated proteolytic degradation, remodeling and/or bone
regeneration (with RGD and BMP presentation in vivo)
[0059] In an embodiment, Z is angiotensin. Angiotensin may be used
to impart vasoconstriction, increased blood pressure, and/or
release of aldosterone from the adrenal cortex.
[0060] A preferred example of a cyclic peptide is gramicidin S,
which is an antimicrobial.
[0061] Further examples of suitable peptides in particular include:
vascular endothelial growth factor (VEGF), transforming growth
factor B (TGF-B), basic fibroblast growth factor (bFGF), epidermal
growth factor (EGF), osteogenic protein (OP), monocyte
chemoattractant protein (MCP 1), tumour necrosis factor (TNF).
[0062] Examples of proteins which may in particular form part of
the compound of the present invention include growth factors,
chemokines, cytokines, extracellular matrix proteins,
glycosaminoglycans, angiopoetins, ephrins and antibodies.
[0063] A preferred carbohydrate is heparin, which is
antithrombotic.
[0064] A nucleotide may in particular selected from therapeutic
oligo-nucleotides, such as a oligo-nucleotide for gene therapy and
oligo-nucleotide that are capable of binding to cellular or viral
proteins, preferably with a high selectivity and/or affinity.
[0065] Preferred oligo-nucleotides include aptamers. Examples of
both DNA and RNA based aptamers are mentioned in Nimjee at. Al.
Annu. Rev. Med. 2005, 56, 555-583. The RNA ligand TAR (Trans
activation response), which binds to viral TAT proteins or cellular
protein cyclin Ti to inhibit HIV replication, is an example of an
aptamer. Further, preferred nucleotides include VA-RNA and
transcription factor E2F, which regulates cellular
proliferation.
[0066] As indicated above, the biomolecular moiety may be linked
via spacer. In principle any spacer may be used that can be coupled
with both the carboxylic acid of the amino acid residue of the
compound/polymer of the invention and the biomolecule to be
covalently attached. Suitable spacers include polyalkylene glycols,
such as PEG, oligomeric esters or peptide segments that have no
signalling functions e.g. oligopeptides or polypeptides based on
one amino acid, such as glycine.
[0067] The moiety G which may be present in the compound/polymer of
the present invention may be the residue of any molecule comprising
at least n functionalities that can be linked with the moiety L via
a --Y--(C.dbd.O)--NR-- bond. In particular such residue may be
selected from the group consisting of multifunctional polymers and
oligomers comprising one or more of the following functionalities:
--OH, --NH.sub.2, --RNH, --SH, wherein R is as defined above.
[0068] In particular, the polyfunctional molecule respectively G
may be selected from poly (lactic acid) (PLA); polyglycolide (PGA);
poly(anhydrides); poly(trimethylenecarbonates); poly(orthoesters);
poly(dioxanones); poly(.epsilon.-caprolactones) (PCL);
poly(urethanes); poly (vinyl alcohols) (PVA); poly alkylene
glycols, preferably PEG; polyalkylene oxides, preferably selected
from poly (ethylene oxides) and poly(propylene oxides); poloxamers;
meroxapols; poloxamines; poly (hydroxy acids); polycarbonates;
polyaminocarbonates; poly (vinyl pyrrolidones); poly (ethyl
oxazolines); carboxymethyl celluloses; hydroxyalkylated celluloses,
such as hydroxyethyl cellulose and methylhydroxypropyl cellulose;
and natural polymers, such as polypeptides, polysaccharides and
carbohydrates, such as polysucrose, hyaluranic acid, dextran and
similar derivatives thereof, heparan sulfate, chondroitin sulfate,
heparin, alginate, and proteins such as gelatin, collagen, albumin,
or ovalbumin; and co-oligomers, copolymers, and blends of any of
these moieties.
[0069] The moiety G may be chosen based upon its
biostability/biodegradability properties. For providing a
compound/polymer/article with a high biostability, polyethers,
polythioethers, aromatic polyesters, aromatic thioesters are
generally particularly suitable. Preferred examples of oligomers
and polymers that impart biodegradability include aliphatic
polyesters, aliphatic polythioesters, aliphatic polyamides and
aliphatic polypeptides.
[0070] Preferably G is selected from polyesters, polythioesters,
polyorthoesters, polyamides, polythioethers, polyethers,
polyanhydride or polydioxanone. Good results have in particular
been achieved with a polyalkylene glycol, more in particular with a
PEG.
[0071] For a hydrophobic polymer, G may suitably be selected from
hydrophobic polyethers such as polybutylene oxide or
poly(-methyl-1,4-butanediol)co(tetramethyleneglycol) (PTGL).
[0072] A polyalkylene glycol, such as PEG is advantageous in an
application wherein the compound or polymer of the present
invention may be in contact with a protein containing body fluid
for instance blood, plasma, serum or the extracellular matrix. It
may in particular show a low tendency to foul (low non-specific
protein absorption) and/or have an advantageous effect on the
adhesion of biological tissue. A low fouling is desirable, in order
to avoid shielding of group Z by fouling proteins and the like.
[0073] The number average molecular weight (Mn) of the moiety G is
usually at least 200 g/mol, in particular at least 500 g/mol. For
an improved mechanical property, Mn preferably is at least 2000
g/mol. The number average molecular weight of the moiety G is
usually up to 100 000 g/mol. The number average molecular weight is
as determinable by size exclusion chromatography (GPC).
[0074] The invention further relates to a method for preparing the
compound according to the present invention comprising first
reacting a compound with formula III
##STR00002##
wherein R is hydrogen or a protecting group with a compound of the
formula X--Y--H and--if G is different from X--a compound of the
formula G-Y--H wherein the hydrogen or protecting group is
selectively removed to covalently attach the biomolecular moiety
directly or via a spacer to the carboxylic acid moiety attached to
L.
[0075] It is an advantage of the method of the invention that it
can be carried out without the formation of undesired by-products
(molecules formed from as leaving groups).
[0076] Suitable and preferred reaction conditions may be based on
conditions known in the art for reacting an isocyanate with an
amine, alcohol or thiol. If desired, the protective group may be
removed in a manner known in the art. For instance it may be
removed by exposure to light in case of a photocleavable group. An
alkyl may chemically be removed (for instance methyl), by exposure
to a base (for instance methyl) or by acidic hydrolysis, e.g. in
trifluoro acetic acid (for instance t-butyl).
[0077] The present invention also relates to a polymer comprising
the polymerisable compound and to an article, in particular an
article for medical use, comprising the polymerisable compound.
[0078] The present invention also relates to a polymer comprising a
certain proportion of the polymerisable compounds and a radically
or addition polymerisable compound such that an optimal biologic
effect is observed. The radically or addition polymerisable
compound may be chosen from the above described polymerisable
moieties X.
[0079] The polymers according to the present invention preferably
further comprise compounds of formula II
##STR00003##
wherein R is selected from hydrogen, substituted or unsubstituted
alkyl, substituted or unsubstituted aryl or a metal salt.
[0080] As used herein, the term "polymer" denotes a structure that
essentially comprises a multiple repetition of units derived,
actually or conceptually, from molecules of low relative molecular
mass. Such polymers may include homopolymers, copolymers, block
copolymers, crosslinked networks, branched polymers and linear
polymers. Oligomers are considered a species of polymers, i.e.
polymers having a relatively low number of repetitions of units
derived, actually or conceptually, from molecules of low relative
molecular mass.
[0081] As used herein, the term "prepolymer" denotes a polymer
comprising one or more polymerisable functionalities, for instance
vinyl groups.
[0082] Polymers may have a molecular weight of 200 Da or more, 400
Da or more, 800 Da or more, 1000 Da or more, 2000 Da or more, 4000
Da or more, 8000 Da or more, 10 000 Da or more, 100 000 Da or more
or 1 000 000 Da or more. Polymers having a relatively low mass,
e.g. of 8000 Da or less, in particular 4000 Da or less, more in
particular 1000 Da or less may be referred to as oligomers.
[0083] It has in particular been found that the polymer or the
article of the present invention show one or more of the following
properties: being hypo- or non-allergenic, having a high
biocompatibility, having a good elasticity, elongation until
rupture and/or high toughness, showing a low tendency to fouling,
showing favorable cell adhesion, being capable of allowing cell
colonisation, being biodegradable or biostable, showing reduced
acidity upon degradation, more effective tying in of biologically
active moieties and low cytotoxicity.
[0084] More in particular, it has been found possible to provide a
polymer that shows low or no fouling by non specific protein
absorption, e.g., when contacted with a body fluid that contains a
protein and/or that allows adhesion of cells and/or colonisation by
cells, in vivo and/or in vitro.
[0085] It is further contemplated that the polymer according to the
present invention may protect a biomolecular moiety, at least to
some extent to a detrimental effect, such as loss of activity, as a
result of denaturation by heat, proteases, solvents, material
processing conditions and/or the manner the polymer may be
introduced into the body (e.g. as an implant).
[0086] The article of the present invention may be tubes,
microspheres, nanospheres, porous monolith wax, woven or non-woven
fibrous material, filaments, films, foams, implants, gels,
hydrogels, sponges, coatings and artificial body tissues.
[0087] The polymerisable compound or polymer according to the
invention may in particular be used to provide a medical device,
more in particular a prosthesis or another substitute for a tissue,
a drug delivery device, microspheres, an implantable device or an
extracorporeal medical device. The polymer is in particular
suitable to prepare a biostable or biodegradable polymer device for
engineering of tubular tissues. These tissues include intestine,
blood vessels, tracheas, ureters and nerve guides.
[0088] The polymers according to the present invention may also be
used to prepare coatings, films, sealants and adhesives for medical
applications. The polymer may also be given a 3 D shape by a 3D
modelling (also known as rapid manufacturing) process, such as a
layer by layer manufacturing process.
[0089] The invention further relates to a method for the
preparation of the polymer according to the present invention by
polymerizing the compounds of formula I.
[0090] The invention further relates to a method for preparing the
polymer by polymerizing a compound of formula II,
##STR00004##
wherein R is selected from hydrogen or a protecting group wherein
hydrogen or the protecting group is selectively removed and
subsequently the biomolecular moiety is covalently attached
directly or via a spacer to the carboxylic acid moiety attached to
L.
[0091] The biomolecular moiety may be covalently attached to the
carboxylic acid in a manner known in the art, in particular using
an amidation or esterification reaction.
[0092] The polymer of the invention may be obtained by polymerising
the polymerisable moieties of a compound according to the
invention. This may be done based upon a method known in the art
for the particular polymerisable moiety, e.g. step growth
polymerisation or radical polymerisation. The polymerisation may be
initiated using a low temperature thermal initiator or a
photo-initiator. Preferably the polymerisation is initiated using a
photo-initiator.
[0093] A single photo initiator or two or more photo initiators can
be included. In order to increase curing speeds a combination of
photo initiators may be advantageously used, especially if
colorants are present.
[0094] Suitable photo initiators are well known and within the
skill of the art, and include free-radical photo initiators.
Free-radical photo initiators are generally divided into two
classes according to the process by which the initiating radicals
are formed.
[0095] Compounds that undergo uni-molecular bond cleavage upon
irradiation are termed Type I photo initiators. If the excited
state photo initiator interacts with a second molecule (a
coinitiator COI) to generate radicals in a bimolecular reaction,
the initiating system is termed a Type II photo initiator. Examples
of suitable alpha-cleavage homolytic free-radical photo initiators
(Type I) are benzoin derivatives, methylolbenzoin and
4-benzoyl-1,3-dioxolane derivatives, benzilketals,
.alpha.,.alpha.-dialkoxyacetophenones, .alpha.-hydroxy
alkylphenones, .alpha.-aminoalkylphenones, acylphosphine oxides
(under which also bisacylphosphine oxides), acylphosphine
sulphides, halogenated acetophenone derivatives, and the like.
[0096] Further examples of the photo-polymerization initiator are
1-hydroxycyclohexylphenyl ketone,
2,2-dimethoxy-2-phenylacetophenone, xanthone, fluorenone,
benzaldehyde, fluorene, anthraquinone, triphenylamine, carbazole,
3-methylacetophenone, 4-chlorobenzophenone,
4,4'-dimethoxybenzophenone, 4,4'-diaminobenzophenone, Michler's
ketone, benzoin propyl ether, benzoin ethyl ether, benzyl methyl
ketal, 1-(4-isopropylphenyl)-2-hydroxy-2-methylpropan-1-one,
2-hydroxy-2-methyl-1-phenylpropan-1-one, thioxanethone,
diethylthioxanthone, 2-isopropylthioxanthone, 2-chlorothioxanthone,
2-methyl-1-[4-(methylthio)phenyl]-2-morpholino-propan-1-one,
2,4,6-trimethylbenzoyldiphenylphosphine oxide,
bis-(2,6-dimethoxybenzoyl)-2,4,4-trimethylpentylphosphine oxide,
bis-(2,4,6-trimethylbenzoyl)-phenylphosphine oxide, camphorquinon,
eosine and the like. Mixtures of these photo-polymerization
initiators may also be used.
[0097] Examples of commercially available products of the
photo-polymerization initiator include IRGACURE 184, 369, 651, 500,
907, CGI1700, 1750, 1850, 819, 2959, CG24-61, Darocur I116, 1173
(manufactured by Ciba Specialty Chemicals Co., Ltd.), Lucirin
LR8728 (manufactured by BASF), Ubecryl P36 (manufactured by UCB),
and the like.
[0098] Further examples of Type II photo initiator are
triethylamine, diethylamine, N-methyldiethanoleamine, ethanolamine,
4-dimethylaminobenzoic acid, methyl 4-dimethylaminobenzoate, ethyl
4-dimethylaminobenzoate, isoamyl 4-dimethylaminobenzoate, and the
like. As commercially available products of the photosensitizer,
for example, Ebecryl P102, 103, 104, and 105 (manufactured by UCB)
are given. Use of mixtures is also possible.
[0099] In case the polymer is to be provided with a biomolecular
moiety, such moiety may be covalently attached to the polymer after
the polymerisation.
[0100] As indicated above, the invention also relates to an article
comprising a polymer according to the invention. The article or a
considerable part thereof may be made of a polymer of the invention
or a composition comprising such polymer, for instance in addition
to a bioactive agent, in particular a pharmaceutical that may be
released from the article.
[0101] Preferably at least part of a surface of the article
comprises the polymer.
[0102] If desired, the article can be provided with different
moieties Z at different parts of the article, for instance at
different parts of the surface. Thus different desired effects may
occur at different parts of the article (e.g. in vivo). For
instance, this may allow to control a growth direction for specific
cells, such as nerve cells in case the article is a nerve guide. If
desired, a part of the article may be provided with a biomolecular
moiety and another part with a protective group, thereby
stimulating a specific effect only at a targeted part of the
article.
[0103] In particular this may be desired at the surface of the
article. Accordingly, in a preferred embodiment at least a first
selected area of the surface of the article comprises a first
polymer or a first part of a polymer containing a biomolecular
moiety as moiety Z or as a part of moiety Z and wherein at least a
second area comprises a second polymer of the invention or a second
part of the same polymer containing a moiety different from said
biomolecular moiety, e.g. a hydrogen, a protecting group or a
different biomolecular moiety.
[0104] Such an article may in particular be prepared by [0105]
shaping the article using a compound or polymer according to the
invention, wherein R is a protecting group (preferably a
photocleavable group) or wherein R is hydrogen, [0106] selectively
removing the protecting (photocleavable) group at the area at which
the biomolecule is to be bound and attachment of the biomolecular
moiety directly or via a spacer to the carboxylic acid moiety.
[0107] R may in particular be hydrogen or a protective group in
case a biomolecular moiety is not desired for the intended purpose
or in case the compound/polymer/article is still to be subjected to
a treatment, such as a treatment which may be detrimental to the
biomolecular moiety. In the latter case the biomolecular moiety may
be bound to the compound/polymer/article after such treatment, if
desired. In particular, a protective group may be used to protect
the carboxylic acid from reacting with other reactive moieties in
the compound/polymer itself or with another molecule. A protective
group may also be used to allow or facilitate binding a
biomolecular moiety in a specific pattern. Suitable protective
groups include alkyls, in particular unsubstituted alkyls such as
methyl, ethyl and C3-C8 unsubstituted alkyls. Methyl and C3-C8
alkyls, in particular t-butyl, are preferred alkyls.
[0108] In case R is a protective group is may advantageously be
selected from photocleavable groups, as these as easily removable,
by using electromagnetic radiation, It is also possible to remove
such groups easily in a specific pattern, e.g. on a surface of an
article of the invention, by selective irradiation of specific
parts of the surface. Preferred examples of photocleavable groups
include those cited in Protective groups in Organic synthesis,
Theodora Greene, 3.sup.rd Edn Wiley ISBN 0 471-16019 (1999).
[0109] It is also possible to select a protective group that is
removable by acid treatment, e.g. by contacting with a
trifluoroacetic acid solution. An example of a protective group
removable by acid is t-butyl.
[0110] Preferably, the protecting group is a photocleavable
group.
[0111] Preferably the selective removing is accomplished by
selectively irradiating the surface of the polymer with
electromagnetic radiation.
[0112] The invention will now be illustrated by the following
examples without being limited thereto.
EXAMPLES
Materials
[0113] dl-Lactide and glycolide were purchased from PURAC.
L-lysine-diisocyanate tert-butylester was purchased from Symochem
(Eindhoven, The Netherlands). L-lysine-diisocyanate methyl ester
was provided by Kyowa Hakko Europe GmbH. Caprolactone was provided
by Solvaycaprolactone. Arg(Pmc)-Gly-Asp(O.sup.tBu)-O.sup.tBu and
Gly-Arg(Pmc)-Gly-Asp(O.sup.tBu)-Ser-(O.sup.tBu).sub.2 were
purchased from Chiralix (Nijmegen, The Netherlands). Pmc and
.sup.tBu stand for the protective groups
2,2,5,7,8-pentamethylchroman-6-sulfonyl and tertiary butyl,
respectively. All other chemicals were purchased from Aldrich. The
chemicals were used as such unless otherwise stated.
Instrumentation
[0114] NMR Advance 300 MHz spectrometer (Bruker), Agilent 1100 MSD
single quat LCMS, Perkin Elmer Spectrum and FTIR spectrometer were
used to characterize the chemical structure and purity. Silica gel
column chromatography (SGCC) was performed using Acros silica gel
(0.035-0.070 mm, pore diameter ca. 6 nm).
[0115] TLC was carried out on Merck precoated silica gel 60 F-254
plates. Compounds were visualized by UV or ninhydrin.
[0116] A Laminar flow cabinet (Clean Air DLF/R56), a incubator
(NAPCO model 6300), a Olympus CK2 microscope, equipped with a
monochrome CCD camera (ADIMEX Image Systems, MX5) connected to a
computer with Optimas image analysis software (BioScan Optimas)
were used.
Example 1
Preparation of Materials
Synthesis of p-(lactide-co-glycolide)1000 diol (1)
[0117] dl-Lactide (24.76 g, 17.2 mmol), glycolide (19.94 g, 17.2
mmol) and diethyleneglycol (5.306 g, 50 mmol) were melted at
150.degree. C. tin(II)-ethylhexanoate (13.9 mg) was added as a
catalyst. The reaction was allowed to proceed for 18 h upon which
the reaction mixture was cooled to room temperature to obtain
1.
[0118] .sup.1H-NMR (300 MHz, CDCl.sub.3, 22.degree. C., TMS):
.delta. (ppm)=5.25-5.18 (m, 5.3H, CH(lac)); 4.83-4.74 (m, 10.6H,
CH.sub.2(gly)); 4.30 (m, 6.7H, --(C.dbd.O)OCH.sub.2CH.sub.2O--,
--O(C.dbd.O)CH.sub.2OH, --O(C.dbd.O)CH(CH.sub.3)OH); 3.70 (m, 4H,
--(C.dbd.O)OCH.sub.2CH.sub.2O--); 2.79 (broad, 2H, --OH); 1.58 (m,
19.1H, CH.sub.3(lac))
Synthesis of p-(lactide-co-Glycolide)1000-(t-Bu-LDI-HEA).sub.2
(2)
[0119] Hydroxyethylacrylate (HEA, 1.16 g, 10 mmol) was added
dropwise to a solution of L-lysine-diisocyanate tert-butylester
(2.54 g, 10 mmol), Tin-(II)-ethylhexanoate (0.012 g, 0.028 mmol),
Irganox 1035 (0.012 g) in THF (17.4 gram) and dry air at controlled
temperature (<20.degree. C.). The reaction was monitored with
GPC w.r.t to the presence of HEA. After 18 hours 1(5 gram, 5 mmol)
was added at room temperature. The temperature was gradually
increased till 60.degree. C. until the IR vibrational stretch of
NCO group at v=2260 cm.sup.-1 disappeared. When the reaction was
complete, based on IR spectroscopy the solvent was evaporated. 2
was obtained without further purification as a slightly coloured
yellow oil.
[0120] .sup.1H-NMR (300 MHz, CDCl.sub.3, 22.degree. C., TMS):
.delta. (ppm)=6.5-6.0 (6H, CH, acrylate), 5.5 (2H, NHCO), 5.3 (2H,
NHCO); 5.25-5.18 (m, H, CH(lac)); 4.83-4.74 (m, 2H, CH.sub.2(gly));
4.30 (m, 6.7H, --(C.dbd.O)OCH.sub.2CH.sub.2O--,
--O(C.dbd.O)CH.sub.2OH, --O(C.dbd.O)CH(CH.sub.3)OH); 4.3-4.1 (m,
CH.sub.2, CH (Lys), and CH.sub.2, HEA); 3.70 (m, 4H,
--(C.dbd.O)OCH.sub.2CH.sub.2O--); 3.1 (m, 4H, Lys); 1.8-1.3 (8H,
Lys, 12H, t-Butylester, m, 4H, CH.sub.2 (Lys)) 1.58 (m, 19.1H,
CH.sub.3(lac))
Synthesis of p-(Lactide-co-Glycolide)1000 diacrylate (3)
[0121] 1(50 gram, 50 mmol) and triethyleneamine (10.63 g, 0.105
mol) was dissolved in 100 mL tetrahydrofuran (THF).
Acryloylchloride (9.5 g, 0.105 mol) dissolved in THF (15 mL) was
added dropwise to the solution at controlled temperature
(<5.degree. C.). The reaction mixture was stirred at room
temperature for 18 hours. The THF was evaporated. Everything was
dissolved in 250 mL chloroform and washed successively with
H.sub.2O, 1N NaHCO.sub.3, brine. The resulting solution was dried
with NaSO.sub.4 and evaporated to dryness. 3 was obtained as a
slightly coloured yellow oil.
[0122] .sup.1H-NMR (300 MHz, CDCl.sub.3, 22.degree. C., TMS):
.delta. (ppm)=6.5-6.0 (6H, CH, acrylate), 5.25-5.18 (m, 9.1H,
CH(lac)); 4.83-4.74 (m, 15.9H, CH.sub.2(gly)); 4.30 (m, 4H,
--(C.dbd.O)OCH.sub.2CH.sub.2O--, --O(C.dbd.O)CH.sub.2OH,
--O(C.dbd.O)CH(CH.sub.3)OH); 3.70 (m, 4H,
--(C.dbd.O)OCH.sub.2CH.sub.2O--); 1.58 (m, 30H, CH.sub.3(lac))
Synthesis of p-(Lactide-co-Caprolactone)1000 diol (4)
[0123] dl-Lactide (37.41 g, 25.95 mmol), .epsilon.-caprolactone
(29.63 g, 25.9 mmol) and diethyleneglycol (7.959 g, 75 mmol) were
melted at 150.degree. C. Tin(II)-ethylhexanoate (21 mg) was added
as a catalyst. The reaction was allowed to proceed for 18 h upon
which the reaction mixture was cooled to room temperature to obtain
4.
[0124] .sup.1H-NMR (300 MHz, CDCl.sub.3, 22.degree. O, TMS):
.delta. (ppm)=5.25-5.18 (m, 5H, CH(lac)); 4.40-4.4 (m, 10H,
CH.sub.2(cap)); 4.30 (m, 4H, --(C.dbd.O)OCH.sub.2CH.sub.2O--,
--O(C.dbd.O)CH.sub.2OH, --O(C.dbd.O)CH(CH.sub.3)OH); 3.70 (m, 4H,
--(C.dbd.O)OCH.sub.2CH.sub.2O--); 3.4 (broad, 2H, --OH); 2.4 (m,
CH.sub.2 (cap) 1.58 (m, CH.sub.3(lac) and CH.sub.2(cap))
Synthesis of p-(Lactide-co-Caprolactone)1000-(t-Bu-LDI-HEA).sub.2
(5)
[0125] Hydroxyethylacrylate (2.23 g, 20 mmol) was added dropwise to
a solution of L-lysine-diisocyanate tert-butylester
(tert.-butyl-LDI) (5.08 g, 20 mol), tin-(II)-ethylhexanoate (0.023
g, 0.056 mmol), Irganox 1035 (0.023 g) in toluene (17.4 gram) and
dry air at controlled temperature (<20.degree. C.). The reaction
was monitored with GPC w.r.t to the presence of HEA. After 18 hours
4 (10 gram, 5 mmol) was added at room temperature. The temperature
was gradually increased till 60.degree. C. until the IR vibrational
stretch of NCO group at v=2260 cm.sup.-1 disappeared. When the
reaction was complete, based on IR spectroscopy the solvent was
evaporated. 5 was obtained without further purification as a
slightly colored yellow oil.
[0126] .sup.1H-NMR (300 MHz, CDCl.sub.3, 22.degree. C., TMS):
.delta. (ppm)=6.5-6.0 (6H, CH, acrylate), 5.5 (2H, NHCO), 5.3 (2H,
NHCO); 5.25-5.18 (m, H, CH(lac)); 4.40 (m, 10H, CH.sub.2(cap));
4.30 (m, 6.7H, --(C.dbd.O)OCH.sub.2CH.sub.2O--,
--O(C.dbd.O)CH.sub.2OH, --O(C.dbd.O)CH(CH.sub.3)OH); 4.3-4.1 (m,
CH.sub.2, CH (Lys), and CH.sub.2, HEA); 3.70 (m, 4H,
--(C.dbd.O)OCH.sub.2CH.sub.2O--); 3.1 (m, 4H, Lys); 2.4 (m,
CH.sub.2 (cap) 1.58 (m, CH.sub.3(lac) and CH.sub.2(cap)); 1.8-1.3
(8H, Lys, 12H, t-Butylester, m, 4H, CH.sub.2 (Lys)) 1.58 (m, 19.1H,
CH.sub.3(lac))
Synthesis of p-(lactide-co-glycolide)1500 triol (6)
[0127] Trimethylolpropane (10 gram) was recrystalised in
ethylacetate (25 mL) to dry the sample. dl-Lactide (25.22 g, 17.5
mmol), glycolide (20.31 g, 17.5 mmol) and trimethylolpropane (4.47
g, 33.3 mmol) were melted at 150.degree. C. Tin(II)-ethylhexanoate
(14 mg) was added as a catalyst. The reaction was allowed to
proceed for 18 h upon which the reaction mixture was cooled to room
temperature to obtain 6.
[0128] .sup.1H-NMR (300 MHz, CDCl.sub.3, 22.degree. C., TMS):
.delta. (ppm)=5.4-5.0 (m, 8.2, CH(lac)); 4.82-4.70 (m, 16.9H,
CH.sub.2(gly)); 4.5-4.0 (m, 9.0H, --O(C.dbd.O)CH.sub.2OH,
--O(C.dbd.O)CH(CH.sub.3)OH) and CH.sub.3CH.sub.2C(CH.sub.2O
--).sub.3); 3.0 (broad, 3H, --OH); 1.57 (m, 31.6H, CH.sub.3(lac)
and CH.sub.3CH.sub.2C(CH.sub.2O --).sub.3); 0.90 (t, 3H,
CH.sub.3CH.sub.2C(CH.sub.2O --).sub.3)
Synthesis of p-(Lactide-co-Glycolide)1500 triacrylate (7)
[0129] 6 (10 gram, 6.6 mmol) and triethylamine (0.71 gram, 7 mmol)
were dissolved THF (100 mL). Acryloylchloride (0.67 g, 7 mmol)
dissolved in THF (25 mL) was added dropwise at controlled
temperature (<5.degree. C.). The reaction mixture was stirred at
room temperature for 18 hours. The THF solvent was evaporated. The
residue was dissolved in 250 mL chloroform and washed successively
with H.sub.2O, 0.1 N NaHCO.sub.3, brine. The resulting solution was
dried with NaSO.sub.4 and evaporated to dryness. 7 was obtained as
a slightly colored yellow oil.
[0130] .sup.1H-NMR (300 MHz, CDCl.sub.3, 22.degree. C., TMS):
.delta. (ppm)=6.5-6.0 (6H, CH, acrylate), 5.4-5.0 (m, 8.2,
CH(lac)); 4.82-4.70 (m, 16.9H, CH.sub.2(gly)); 4.5-4.0 (m, 9.0H,
--O(C.dbd.O)CH.sub.2OH, --O(C.dbd.O)CH(CH.sub.3)OH) and
CH.sub.3CH.sub.2C(CH.sub.2O --).sub.3); 3.0 (broad, 3H, --OH); 1.57
(m, 31.6H, CH.sub.3(lac) and CH.sub.3CH.sub.2C(CH.sub.2O
--).sub.3); 0.90 (t, 3H, CH.sub.3CH.sub.2C(CH.sub.2O --).sub.3)
Synthesis of p-(Lactide-co-Glycolide)1500-(t-Bu-LDI-HEA) 3 (8)
[0131] Hydroxyethylacrylate (1.16 g, 10 mmol) was added dropwise to
a solution of L-lysine-diisocyanate tert-butylester (2.54 g, 10
mol), Tin-(II)-ethylhexanoate (0.037 g, 0.084 mmol), Irganox 1035
(0.012 g) in tetrahydrofuran (17.4 gram) and dry air at controlled
temperature (<20.degree. C.). The reaction was monitored with
GPC w.r.t to the presence of HEA. After 72 hours 6(10 gram, 5 mmol)
was added at room temperature. The temperature was gradually
increased till 60.degree. C. until the IR vibrational stretch of
NCO group at v=2260 cm.sup.-1 disappeared. When the reaction was
complete, based on IR spectroscopy the solvent was evaporated. 8
was obtained without further purification as a slightly colloured
yellow oil.
[0132] .sup.1H-NMR (300 MHz, CDCl.sub.3, 22.degree. C., TMS):
.delta. (ppm)=6.5-6.0 (6H, CH, acrylate), 5.5 (2H, NHCO), 5.3 (2H,
NHCO); 5.4-5.0 (m, 8.2, CH(lac)); 4.82-4.70 (m, 16.9H,
CH.sub.2(gly)); 4.5-4.0 (m, 9.0H, --O(C.dbd.O)CH.sub.2OH,
--O(C.dbd.O)CH(CH.sub.3)OH) and CH.sub.3CH.sub.2C(CH.sub.2O
--).sub.3); 4.3-4.1 (m, CH.sub.2, CH (Lys), and CH.sub.2, HEA);
3.70 (m, 4H, --(C.dbd.O)OCH.sub.2CH.sub.2O--); 3.1 (m, 6H, Lys);
1.8-1.3 (12H, Lys, 18H, t-Butylester, m, 6H, CH.sub.2 (Lys)) 1.58
(m, 19.1H, CH.sub.3(lac))
Synthesis of PEG600-(t-Bu-LDI-HEA) 2 (9)
[0133] Hydroxyethylacrylate (4.8 g, 40 mmol) was added dropwise to
a solution of the L-lysine-diisocyanate tert-butylester (10.2 g, 40
mmol), tin-(II)-ehtylhexanote (50 mg) and Irganox 1035 (50 mg) at
controlled temperature (<20.degree. C.). The reaction was
monitored with GPC w.r.t to the presence of HEA. After 18 hours
[0134] Polyethyleneglycol Mn=600 (12 gram, 20 mmol) was added at
room temperature. The temperature was gradually increased till
60.degree. C. until the IR vibrational stretch of NCO group at
v=2260 cm.sup.-1 disappeared. When the reaction was complete, based
on IR spectroscopy the solvent was evaporated. 9 was obtained
without further purification as a slightly coloured yellow oil.
[0135] .sup.1H-NMR (300 MHz, CDCl.sub.3, 22.degree. C., TMS):
.delta. (ppm)=6.5-6.0 (6H, CH, acrylate), 5.5 (2H, NHCO), 5.3 (2H,
NHCO), 4.3-4.1 (m, CH.sub.2, CH (Lys), and CH.sub.2, HEA) and (m,
4H, --(C.dbd.O)OCH.sub.2CH.sub.2O--, 3.6 (s, CH.sub.2, PEG600 and
(m, 4H, --(C.dbd.O)OCH.sub.2CH.sub.2O--); 3.1 (m, 4H, Lys), 1.8-1.3
(8H, Lys, 12H, t-Butylester, m, 4H, CH.sub.2 (Lys))
Synthesis of p-(Lactide-co-Glycolide)1000-(m-LDI-HEA).sub.2
(10)
[0136] Hydroxyethylacrylate (HEA, 6.0 g, 50 mmol) was added
dropwise to a solution of L-lysine-diisocyanate methylester
(Me-LDI) (10.6 g, 50 mmol), Tin-(II)-ethylhexanoate (0.020 g, 0.050
mmol), Irganox 1035 (0.060 g) in Tetrahydrofuran (100 mL) and dry
air at controlled temperature (<20.degree. C.). The reaction was
monitored with GPC w.r.t to the presence of HEA. After 18 hours 1
(25 gram, 25 mmol) was added at room temperature. The temperature
was gradually increased until the IR vibrational stretch of NCO
group at v=2260 cm.sup.-1 disappeared. When the reaction was
complete, based on IR spectroscopy the solvent was evaporated. 10
was obtained without further purification as a slightly colloured
yellow oil.
[0137] .sup.1H-NMR (300 MHz, CDCl.sub.3, 22.degree. C., TMS):
.delta. (ppm)=6.5-6.0 (6H, CH, acrylate), 5.5 (2H, NHCO), 5.3 (2H,
NHCO); 5.25-5.18 (m, H, CH(lac)); 4.83-4.74 (m, 2H, CH.sub.2(gly));
4.30 (m, 6.7H, --(C.dbd.O)OCH.sub.2CH.sub.2O--,
--O(C.dbd.O)CH.sub.2OH, --O(C.dbd.O)CH(CH.sub.3)OH); 4.3-4.1 (m,
CH.sub.2, CH (Lys), and CH.sub.2, HEA); 3.70 (m, 4H,
--(C.dbd.O)OCH.sub.2CH.sub.2O-- and 3H, methylester); 3.2 (m, 4H,
Lys); 1.8-1.3 (8H, Lys, m, 4H, CH.sub.2 (Lys)) 1.58 (m, 19.1H,
CH.sub.3(lac))
Synthesis of p-(Lactide-co-Caprolactone)1545 diol (11)
[0138] dl-Lactide (51.9 g, 36.1 mmol), .epsilon.-caprolactone (41.2
g, 36.1 mmol) and diethyleneglycol (6.846 g, 64 mmol) were melted
at 150.degree. C. Tin(II)-ethylhexanoate (29 mg) was added as a
catalyst. The reaction was allowed to proceed for 18 h upon which
the reaction mixture was cooled to room temperature to obtain
11.
[0139] .sup.1H-NMR (300 MHz, CDCl.sub.3, 22.degree. O, TMS):
.delta. (ppm)=5.25-5.18 (m, 5H, CH(lac)); 4.40-4.4 (m, 10H,
CH.sub.2(cap)); 4.30 (m, 4H, --(C.dbd.O)OCH.sub.2CH.sub.2O--,
--O(C.dbd.O)CH.sub.2OH, --O(C.dbd.O)CH(CH.sub.3)OH); 3.70 (m, 4H,
--(C.dbd.O)OCH.sub.2CH.sub.2O--); 3.4 (broad, 2H, --OH); 2.4 (m,
CH.sub.2 (cap) 1.58 (m, CH.sub.3(lac) and CH.sub.2(cap))
Synthesis of p-(Lactide-co-Caprolactone)1545 diacrylate (12)
[0140] 11 (100 gram, 64.7 mmol) and triethyleneamine (14.36 g,
0.142 mol) was dissolved in 100 mL tetrahydrofuran.
Acryloylchloride (12.8 g, 0.141 mol) dissolved in THF (50 mL) was
added dropwise to the solution at controlled temperature
(<5.degree. C.). The reaction mixture was stirred at room
temperature for 18 hours. The THF was evaporated. Everything was
dissolved in 250 mL chloroform and washed successively with
H.sub.2O, 1N NaHCO.sub.3, brine. The resulting solution was dried
with NaSO.sub.4 and evaporated to dryness. 12 was obtained as a
slightly coloured yellow oil.
[0141] .sup.1H-NMR (300 MHz, CDCl.sub.3, 22.degree. C., TMS):
.delta. (ppm)=6.5-6.0 (6H, CH, acrylate), 5.25-5.18 (m, 5H,
CH(lac)); 4.40-4.4 (m, 10H, CH.sub.2(cap)); 4.30 (m, 4H,
--(C.dbd.O)OCH.sub.2CH.sub.2O--, --O(C.dbd.O)CH.sub.2OH,
--O(C.dbd.O)CH(CH.sub.3)OH); 3.70 (m, 4H,
--(C.dbd.O)OCH.sub.2CH.sub.2O--); 3.4 (broad, 2H, --OH); 2.4 (m,
CH.sub.2 (cap) 1.58 (m, CH.sub.3(lac) and CH.sub.2(cap))
Synthesis of p-(Lactide-co-Caprolactone)1000-(m-LDI-HEA).sub.2
(13)
[0142] Hydroxyethylacrylate (6.0 g, 50 mmol) dissolved in 25 mL THF
was added dropwise to a solution of L-lysine-diisocyanate
methylester (10.6 g, 50 mol), Tin-(II)-ethylhexanoate (0.021 g,
0.050 mmol), Irganox 1035 (0.060 g) in tetrahydrofuran (50 mL) and
dry air at controlled temperature (<20.degree. C.). The reaction
was monitored with GPC w.r.t to the presence of HEA. After 18 hours
4 (25 gram, 25 mmol) dissolved in 50 mL Tetrahydrofuran was added
at room temperature. The temperature was gradually increased till
60.degree. C. until the IR vibrational stretch of NCO group at
v=2260 cm.sup.-1 disappeared. When the reaction was complete, based
on IR spectroscopy the solvent was evaporated. 13 was obtained
without further purification as a slightly colloured yellow
oil.
[0143] .sup.1H-NMR (300 MHz, CDCl.sub.3, 22.degree. C., TMS):
.delta. (ppm)=6.5-6.0 (6H, CH, acrylate), 5.5 (2H, NHCO), 5.3 (2H,
NHCO); 5.25-5.18 (m, H, CH(lac)); 4.40 (m, 10H, CH.sub.2(cap));
4.30 (m, 6.7H, --(C.dbd.O)OCH.sub.2CH.sub.2O--,
--O(C.dbd.O)CH.sub.2OH, --O(C.dbd.O)CH(CH.sub.3)OH); 4.3-4.1 (m, CH
(Lys), and CH.sub.2, HEA); 3.70 (m, 4H,
--(C.dbd.O)OCH.sub.2CH.sub.2O--); 3.1 (m, 4H, Lys and 3H,
methylester); 2.4 (m, CH.sub.2 (cap) 1.58 (m, CH.sub.3(lac) and
CH.sub.2(cap)); 1.8-1.3 (8H, Lys, m, 4H, CH.sub.2 (Lys)) 1.58 (m,
19.1H, CH.sub.3(lac))
Synthesis of t-Bu-LDI-(HEA).sub.2 (14)
##STR00005##
[0145] Hydroxyethylacrylate (HEA, 9.13 g, 78 mmol) dissolved in
toluene (15 mL) was added dropwise under a dry atmosphere to a
solution of L-lysine-diisocyanate tert-butylester (10 g, 39 mmol),
tin-(II)-ethylhexanoate (0.086 g), Irganox 1035 (89 mg) in toluene
(50 mL) at controlled temperature (<5.degree. C.). The
temperature was gradually increased till 60.degree. C. until the IR
vibrational stretch of NCO group at v=2260 cm.sup.-1 disappeared.
When the reaction was complete, based on IR spectroscopy the
solvent was evaporated. 14 was obtained without further
purification as a colourless oil.
[0146] .sup.1H-NMR (300 MHz, CDCl.sub.3, 22.degree. C., TMS):
.delta. 6.5-6.0 (6H, CH, acrylate), 5.4 (2H, NHCO), 4.9 (2H, NHCO);
4.4 (m, CH.sub.2, HEA); 4.3 (m, CH (Lys),); 3.1 (m, 4H, Lys);
1.8-1.3 (6H, CH.sub.2, Lys and 12H, t-Butylester).
Synthesis of LDI-(HEA).sub.2 (15)
##STR00006##
[0148] 14 (18.3 gram, 37.6 mmol), trifluoroacetic acid (TFA, 36
gram) and dichloromethane (10 g) were stirred at 35.degree. C. for
18 h. The deprotection reaction was complete based on .sup.1H NMR
(disappearance tert-butyl ester at 1.39 ppm). The reaction mixture
was dissolved in 250 mL dichloromethane and 200 mL of water. While
stirring, the mixture was brought to pH=2 with aq. 1N NaHCO.sub.3
solution. The CH.sub.2Cl.sub.2 layer was washed 6 times with 200 mL
water; during each extraction to pH was brought to 2 using aq. 1N
NaHCO.sub.3 solution. The organic phase was concentrated in vacuo
to give 15 as a colourless oil. The TFA had been completely removed
as confirmed by F-NMR (internal standard
4,4'-difluorobenzophenone).
[0149] .sup.1H-NMR (300 MHz, CDCl.sub.3, 22.degree. C., TMS):
.delta. 6.5-6.0 (6H, CH, acrylate), 5.4 (2H, NHCO), 4.9 (2H, NHCO);
4.4 (m, CH.sub.2, HEA, and m, CH (Lys),); 3.1 (m, 4H, Lys); 1.8-1.3
(6H, CH.sub.2, Lys).
Synthesis of LDI-(HEA).sub.2-Arg(Pmc)-Gly-Asp(O.sup.tBu)-O.sup.tBu
(16)
##STR00007##
[0151] Diisopropylethylamine (0.125 g, 0.97 mmol) was added to a
solution of 15 (0.379 g, 0.88 mmol) in dichloromethane (22 mL) at
0.degree. C. Successively, 1-hydroxy-7-azabenzotriazole, (0.131 g,
0.97 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide
hydrochloride (0.186 g, 0.97 mmol) and
Arg(Pmc)-Gly-Asp(O.sup.tBu)-O.sup.tBu (0.702 g, 0.97 mmol) were
added and the reaction mixture was stirred for 1 h at 0.degree. C.
and 17 h at ambient temperature. The mixture was concentrated under
reduced pressure and the resulting residue taken up in 105 mL
EtOAc, washed with aq. HCl (pH=2.5, 3.times.100 mL), saturated aq.
NaHCO.sub.3(2.times.100 mL) and brine (100 mL). The organic phase
was dried (Na.sub.2SO.sub.4) and evaporated to dryness. 16 was
obtained in impure form as a white solid in a yield of 85% based on
15. The solid was purified by column chromatography on silica using
EtOAc/MeOH (95/5, v/v) as the eluent giving pure 16 as a white
powder in 53% yield based on 15.
[0152] .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta. (ppm) 7.8-7.25
(3H, m, arom. Pmc), 6.32 (3H, m, acryloyl+NH), 6.08 (3H, m,
acryloyl+NH), 5.74 (2H, d, acryloyl), 4.61 (1H, m,
C.sup..alpha.-Arg or C.sup..alpha.-Asp or C.sup..alpha.-Lys), 4.49
(1H, m, C.sup..alpha.-Arg or C.sup..alpha.-Asp or
C.sup..alpha.-Lys), 4.23 (9H, 2.times.CH.sub.2CH.sub.2 HEA,
C.sup..alpha.-Arg or C.sup..alpha.-Asp or C.sup..alpha.-Lys), 3.92
(2H, s, CH.sub.2-Gly), 3.33 (2H, m, CH.sub.2-N.sup..epsilon.-Lys or
CH.sub.2--C(NH.sub.2).dbd.NH), 3.07 (2H, m,
CH.sub.2--N.sup..epsilon.-Lys or CH.sub.2--C(NH.sub.2).dbd.NH),
2.90-1.50 (25H, m, CH.sub.2-Asp, CH.sub.2--CH.sub.2-Arg,
CH.sub.2--CH.sub.2--CH.sub.2-Lys, 3.times.CH.sub.3 Pmc,
CH.sub.2CH.sub.2 Pmc), 1.35 (18H, s, 6.times.CH3 tBu), 1.20 (6H, s,
C(CH.sub.3).sub.2 Pmc). HPLC-MS: [M+H].sup.+=1138 (as
calculated).
Synthesis of LDI-(HEA).sub.2-Arg-Gly-Asp (17)
##STR00008##
[0154] 16 (3.45 g, 3.03 mmol) was charged in a Schlenck reactor
under a nitrogen atmosphere and the reactor was brought under
reduced pressure and flushed five times with nitrogen.
Subsequently, trifluoroacetic acid (95 mL) was dosed under a
nitrogen atmosphere. After 30 min an aliquot was withdrawn from the
reaction mixture and analyzed with HPLC indicating complete
deprotection. The TFA was removed under reduced pressure and the
product was precipitated and thoroughly washed with anhydrous
diethyl ether. The product was dried on the air giving 2.25 g of
pure 17 as a white solid (98% yield based on 16).
[0155] .sup.1H-NMR (300 MHz, MeOD): .delta. (ppm) 6.43 (2H, d,
acryloyl), 6.23 (1H, d, acryloyl), 6.17 (1H, d, acryloyl), 5.91
(2H, d, acryloyl), 4.8 (2H, m,
C.sup..alpha.-Arg/C.sup..alpha.-Asp), 4.5-4.2 (9H,
2.times.CH.sub.2CH.sub.2 HEA, C.sup..alpha.-Lys), 3.93 (2H, s,
CH.sub.2-Gly), 3.23 (2H, m, CH.sub.2--N.sup..epsilon.-Lys), 3.11
(2H, m, CH.sub.2--C(NH.sub.2).dbd.NH), 2.90 (2H, d, CH.sub.2-Asp),
2.05-1.15 (10H, CH.sub.2--CH.sub.2-Arg,
CH.sub.2--CH.sub.2--CH.sub.2-Lys). HPLC-MS: [M+H].sup.+=759 (as
calculated).
Synthesis of H-Arg(Pmc)-OtBu-HEA-6-amino-hexanoate (18)
##STR00009##
[0157] To a solution of 571 mg (2.09 mmol) of HEA-6-amino-hexanoate
in 22 mL dichloromethane at 0.degree. C. were added
N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide.HCl (441 mg, 2.30
mmol), 1-hydroxy-7-azabenzotriazole (313 mg, 2.30 mmol),
N,N-diisopropylethylamine (384 .mu.I, 2.30 mmol), Arg-(Pmc)-OtBu
(1.093 g, 2.20 mmol) in this order. The reaction mixture was
stirred overnight at ambient temperature and subsequently diluted
with 100 mL of EtOAc and washed with aq. HCl (0.5 M, 3.times.25 mL)
and brine (2.times.25 mL). The organic layer was dried with
Na.sub.2SO.sub.4 and concentrated in vacuo. The product was
purified by column chromatography on silica gel using
EtOAc/n-heptane (33%.fwdarw.0% n-heptane) as the eluent. This
yielded (18) as a white solid (0.437 g, 0.58 mmol).
[0158] .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. 6.48 (dd, J=17.2
and 1.4 Hz, 1H), 6.27 (d, J=8.1 Hz, 1H), 6.12 (dd, J=17.1 and 10.6
Hz, 1H), 6.13-6.09 (m, 1H), 5.85 (dd, J=10.4 and 1.4 Hz, 1H), 4.95
(t, J=5.2 Hz, 1H), 4.48-4.37 (m, 1H), 4.35-4.25 (m, 4H), 3.32-3.11
(bs, 2H), 3.14 (q, J=6.62 Hz, 2H), 2.62 (t, J=6.9 Hz, 2H), 2.58 (s,
3H), 2.57 (s, 3H), 2.28-2.15 (m, 2H), 2.10 (s, 3H), 1.78 (t, J=6.9
Hz, 2H), 1.68-1.48 (m, 12H), 1.46 (s, 9H), 1.29 (s, 6H)
Synthesis of H-Arg(Pmc)-OtBu-LDI-(HEA).sub.2 (19)
##STR00010##
[0160] To a solution of 1.24 g (2.88 mmol) of LDI-(HEA).sub.2 in 30
mL dichloromethane at 0.degree. C. were added
N-(3-dimethylaminopropyl)-W-ethylcarbodiimide.HCl (608 mg, 3.17
mmol), 1-hydroxy-7-azabenzotriazole (439 mg, 3.17 mmol),
N,N-Diisopropylethylamine (532 .mu.I, 3.17 mmol), Arg-(Pmc)-OtBu
(1.53 g, 3.02 mmol) in this order. The reaction mixture was stirred
overnight at ambient temperature, diluted with 100 mL of EtOAc and
washed with aq. HCl (0.5 M, 3.times.30 mL) and brine (2.times.30
mL). The organic layer was dried (Na.sub.2SO.sub.4) and
concentrated in vacuo. The product was purified by column
chromatography on silica gel using EtOAc/n-heptane (33%.fwdarw.0%
n-heptane) as the eluent. This yielded (19) as a white solid (2.0
g, 2.21 mmol).
[0161] .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. 7.08 (d, J=7.3 Hz,
1H), 6.42 (d, J=17.2 Hz, 2H), 6.13 (dd, J=10.3 and 17.2 Hz, 2H),
6.05-5.96 (m, 1H), 5.85 (d, J=10.6 Hz, 2H), 5.81-5.76 (m, 1H), 5.13
(t, J=5.3 Hz, 1H), 4.47-4.44 (m, 1H), 4.37-4.24 (m, 8H), 4.23-4.13
(m, 1H), 3.26-3.11 (m, 4H), 2.62 (t, J=6.9 Hz, 2H), 2.57 (s, 3H),
2.55 (s, 3H), 2.10 (2, 3H), 1.79 (t, J=6.8 Hz, 2H), 1.87-1.35 (m,
12H), 1.44 (s, 9H), 1.30 (s, 6H).
Synthesis of p-(lactide-co-glycolide)1550 diol (20)
[0162] dl-Lactide (51.6 g, 0.358 mol), glycolide (41.5 g, 0.358
mmol) and diethyleneglycol (6.85 g, 6.45 mmol) were melted at
150.degree. C. Tin(II)-ethylhexanoate (29 mg) was added as a
catalyst. The reaction was allowed to proceed for 18 h upon which
the reaction mixture was cooled to room temperature to obtain
20.
[0163] .sup.1H-NMR (300 MHz, CDCl.sub.3, 22.degree. C., TMS):
.delta. (ppm)=5.25-5.18 (m, 5.3H, CH(lac)); 4.83-4.74 (m, 10.6H,
CH.sub.2(gly)); 4.30 (m, 6.7H, --(C.dbd.O)OCH.sub.2CH.sub.2O--,
--O(C.dbd.O)CH.sub.2OH, --O(C.dbd.O)CH(CH.sub.3)OH); 3.70 (m, 4H,
--(C.dbd.O)OCH.sub.2CH.sub.2O--); 2.79 (broad, 2H, --OH); 1.58 (m,
19.1H, CH.sub.3(lac))
Synthesis of p-(Lactide-co-Glycolide)1550 diacrylate (21)
[0164] 20 (100 gram, 65 mmol) and triethyleneamine (14.36 g, 0.141
mol) was dissolved in 100 mL tetrahydrofuran. Acryloylchloride
(12.8 g, 0.141 mol) dissolved in THF (50 mL) was added dropwise to
the solution at controlled temperature (<5.degree. C.). The
reaction mixture was stirred at room temperature for 18 hours. The
THF was evaporated. Everything was squenched in 2500 mL
ethylacetate. The triethylamine.HCl salt precipitated well. This
was isolated via filtration. The ethylacetate layer was washed
successively with 2 times 150 mL brine, 150 mL NaHCO.sub.3, and 2
times 150 mL water. The resulting solution was dried with
NaSO.sub.4 and evaporated to dryness. 21 was obtained as a slightly
coloured yellow oil.
[0165] .sup.1H-NMR (300 MHz, CDCl.sub.3, 22.degree. C., TMS):
.delta. (ppm)=6.5-6.0 (6H, CH, acrylate), 5.25-5.18 (m, 9.1H,
CH(lac)); 4.83-4.74 (m, 15.9H, CH.sub.2(gly)); 4.30 (m, 4H,
--(C.dbd.O)OCH.sub.2CH.sub.2O--, --O(C.dbd.O)CH.sub.2OH,
--O(C.dbd.O)CH(CH.sub.3)OH); 3.70 (m, 4H,
--(C.dbd.O)OCH.sub.2CH.sub.2O--); 1.58 (m, 30H, CH.sub.3(lac))
Synthesis of p-(Glycolide-co-Caprolacton)1000-(m-LDI-HEA).sub.2
(22)
[0166] Hydroxyethylacrylate (HEA, 6 g, 50 mmol) was added dropwise
to a solution of L-lysine-diisocyanate methylester (10.6 g, 50
mmol), Tin-(II)-ethylhexanoate (0.020 g, 0.049 mmol), Irganox 1035
(0.060 g) in THF (50 mL) and dry air at controlled temperature
(<20.degree. C.). The reaction was monitored with GPC w.r.t to
the presence of HEA. After 18 hours p-(glycolide-co-Caprolacton)
1000-diol (25 gram, 25 mmol) dissolved THF (50 mL) was added at
room temperature. The temperature was gradually increased till
60.degree. C. until the IR vibrational stretch of NCO group at
v=2260 cm.sup.-1 disappeared. When the reaction was complete, based
on IR spectroscopy the solvent was evaporated. 22 was obtained
without further purification as a slightly coloured yellow oil.
[0167] .sup.1H-NMR (300 MHz, CDCl.sub.3, 22.degree. C., TMS):
.delta. (ppm)=6.5-6.0 (6H, CH, acrylate), 5.6 (2H, NHCO), 5.4 (2H,
NHCO); 4.7 (m, 2H, CH.sub.2(gly)); 4.6 (m, 10H, CH2(cap)); 4.30 (m,
H, --(C.dbd.O)OCH.sub.2CH.sub.2O--, --O(C.dbd.O)CH.sub.2OH,
--O(C.dbd.O)CH(CH.sub.3)OH); 4.1 (m, CH.sub.2, CH (Lys), and
CH.sub.2, HEA); 3.70 (m, 4H, --(C.dbd.O)OCH.sub.2CH.sub.2O-- and
3H, methylester)); 3.1 (m, 4H, Lys); 2.4 (m, CH.sub.2 (cap));
1.8-1.3 ((CH.sub.2(cap)); m, 8H CH2 (Lys))
Synthesis of p-(Glycolide-co-caprolactone)1550 diol (23)
[0168] Caprolactone (46.2 g, 0.41 mol), glycolide (46.9 g, 0.41
mol) and diethyleneglycol (6.846 g, 64.5 mmol) were melted at
150.degree. C. Tin(II)-ethylhexanoate (32.8 mg) was added as a
catalyst. The reaction was allowed to proceed for 18 h upon which
the reaction mixture was cooled to room temperature to obtain
23.
[0169] .sup.1H-NMR (300 MHz, CDCl.sub.3, 22.degree. C., TMS):
.delta. (ppm)=4.7 (m, 2H, CH.sub.2(gly)); 4.6 (m, 10H, CH2(cap));
4.30 (m, H, --(C.dbd.O)OCH.sub.2CH.sub.2O--,
--O(C.dbd.O)CH.sub.2OH, --O(C.dbd.O)CH(CH.sub.3)OH); 4.1 CH.sub.2,
HEA); 3.70 (m, 4H, --(C.dbd.O)OCH.sub.2CH.sub.2O--); 2.4 (m,
CH.sub.2 (cap)); 1.8-1.3 ((CH.sub.2(cap))
Synthesis of p-(Glycolide-co-caprolactone)1550-diacrylate (24)
[0170] 24 (100 gram, 65 mmol) and triethyleneamine (14.36 g, 0.141
mol) was dissolved in 100 mL tetrahydrofuran. Acryloylchloride
(12.8 g, 0.141 mol) dissolved in THF (50 mL) was added dropwise to
the solution at controlled temperature (<5.degree. C.). The
reaction mixture was stirred at room temperature for 18 hours. The
THF was evaporated. Everything was squenched in 2500 mL
ethylacetate. The triethylamine.HCl salt was removed via
decantation. The ethylacetate layer was washed successively with
250 mL brine, 250 mL NaHCO.sub.3, and 250 mL water. The resulting
solution was dried with NaSO.sub.4 and evaporated to dryness. 24
was obtained as a slightly colored yellow oil.
[0171] .sup.1H-NMR (300 MHz, CDCl.sub.3, 22.degree. C., TMS):
.delta. (ppm)=6.5-5.8 (6H, CH, acrylate), 4.7 (m, 2H,
CH.sub.2(gly)); 4.6 (m, 10H, CH.sub.2(cap)); 4.30 (m, H,
--(C.dbd.O)OCH.sub.2CH.sub.2O--, --O(C.dbd.O)CH.sub.2OH,
--O(C.dbd.O)CH(CH.sub.3)OH); 4.1 (CH.sub.2, HEA); 3.70 (m, 4H,
--(C.dbd.O)OCH.sub.2CH.sub.2O--); 2.4 (m, CH.sub.2 (cap)); 1.8-1.3
((CH.sub.2(cap))
Synthesis of (MeO-PEG750).sub.2-m-Lys (25)
[0172] L-lysine-diisocyanate methylester (1.5 g), Irganox1035 (2
mg) and Tin(II) ethylhexanoate were dissolved in dry toluene (5
mL). To this mixture MeO-PEG750-0H (10.9 g) in 10 mL toluene was
added drop wise until on IR .lamda.=2243 cm.sup.-1 disappeared.
When the reaction was complete, based on IR spectroscopy the
solvent was evaporated. 25 was obtained without further
purification (10.81 g).
[0173] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. ; 1.0-1.8 (m, 6H,
CH.sub.2 (Lys)), 3.1-3.2 (m, 2H, CH.sub.2 (Lys)), 3.4 (s, 6H, OMe
(Peg)), 3.5-3.8 (m, 126H, CH.sub.2 (Peg), OMe (Lys), 4.2 (m, 4H,
(2.times.) CH.sub.2--OMe (Peg)), 4.3 (m, 1H, .alpha.H) 4.9 (m, 1H,
NH), 5.4 (d, 1H NH).
[0174] .sup.13CNMR (75.5 MHz, CDCl.sub.3)6; 22.0, 28.9, 31.3, 39.9,
51.5, 51.7, 53.3, 58.4, 60.9, 63.1, 63.6, 68.8, 69.0, 69.6, 69.8,
71.3, 72.1, 155.2, 155.8, 171.9
Synthesis of (MeO-PEG750).sub.2-Lys (26)
[0175] 25 (9.81 g) was dissolved in 10 mL dioxane. To this solution
7.1 mL 1M NaOH was added. The reaction was complete after stirring
at 40.degree. C. for 30 min. according to TLC (5% MeOH/DCM). After
stirring the solvent was evaporated in vacuo and the residue was
dissolved in water, acidified with 1N HCl and extracted with DOM.
The resulting solution was dried (MgSO.sub.4) and evaporated to
dryness and after column chromatography (5% MeOH/DCM) 26 was
obtained as white gel in 90% yield (8.5 g).
[0176] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.; 1.0-1.5 (m, 6H,
CH.sub.2 (Lys)), 2.8 (m, 2H, CH.sub.2 (Lys)), 3.0 (s, 6H, OMe
(Peg)), 3.3-3.6 (m, 118H, CH.sub.2 (Peg), OMe (Lys), 4.0 (m, 5H,
(2.times.) CH2-OMe (Peg) and .alpha.H (Lys)), 5.6 (bs, 1H, NH), 5.8
(d, 1H, NH).
[0177] .sup.13C NMR (75.5 MHz, CDCl.sub.3) .delta. ; 22.0, 28.9,
31.2, 39.9, 53.0 53.6, 58.4, 60.7, 63.0, 63.4, 68.9, 71.9, 155.2,
155.7, 172.6
Synthesis of Boc-Glycine-o-nitrobenzyl (27)
[0178] Boc-Glycine (2 g, 11.4 mmol) was dissolved in 30 mL DOM.
DMAP (1.39 g), 0-nitrobenzyl alcohol (1.74 g, 11.4 mol and lastly
DCC (2.35 g, 11.4 mmol) were added. The reaction was stirred
overnight at room temperature. After the precipitate was filtered
off the solvent was evaporated in vacuo and re-dissolved in EtOAc.
The organic layer was washed successively with 1N KHSO.sub.4,
H.sub.2O, 1N NaHCO.sub.3 and brine. The resulting solution was
dried (MgSO.sub.4) and evaporated to dryness and after column
chromatography (1:1 EtOAc/Hexane) 27 was obtained as yellow solid
in 98% yield (3.55 g).
[0179] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. ; 1.2 (s, 9H,
Boc), 1.3 (d, 3H, CH.sub.3), 4.0 (d, 2H, CH2 (Gly)), 5.0 (bs, 1H,
NH), 5.5 (q, 1H, CH (benzyl)), 7.2-7.3 (m, 1H, arom-H), 7.3-7.4 (m,
2H, arom-H), 8.1 (m, 1H, arom-H)
Synthesis of HCl.NH.sub.2-Glycine-o-nitrobenzyl (28)
[0180] 27 was dissolved in EtOAc and an excess of HCl/EtOAc was
added. After 2 h of stirring at room temperature the reaction was
complete according to TLC. The precipitate 28 was filtrated and
washed with ether. Then the filtrate was coevaporated with
tert-butanol to eliminate the residual HCl salts.
[0181] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.; 1.2 (s, 9H, Boc),
1.3 (d, 3H, CH.sub.3), 3.9 (d, 2H, CH2 (Gly)), 5.1 (bs, 1H, NH),
6.4 (q, 1H, CH (benzyl), 7.2-7.3 (m, 1H, arom-H), 7.3-7.4 (m, 2H,
arom-H), 8.1 (m, 1H, arom-H)
Synthesis of (MeO-PEG750).sub.2-Lys-Gly-o-nitrobenzyl (29)
[0182] 26 (400 mg.apprxeq.0.24 mmol) was dissolved in DMF (2 mL).
To this mixture 28 (230 mg) with DIPEA (160 .mu.L) in 3 mL DMF was
added followed by the addition of DCC. The reaction was stirred
over night at room temperature. Then, 10 mL of DOM was added and
the organic layer was successively washed with 1N KHSO.sub.4,
H.sub.2O, 1N NaHCO.sub.3 and brine. The resulting solution was
dried (MgSO.sub.4) and evaporated to dryness and after column
chromatography to obtain 29 (5% MeOH/DCM)
[0183] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.; 1.0-2.0 (m, 9H,
CH.sub.3 and CH.sub.2 (Lys)), 3.1 (m, 2H, CH.sub.2 (Lys)), 3.4 (s,
6H, OMe (PEG)), 3.3-3.9 (m, 120H, CH.sub.2 (Peg), OMe (Lys),
4.0-4.4 (m, 7H, (2.times.) CH.sub.2-OMe (Peg), .alpha.H (Lys) and
CH.sub.2 (Gly)), 5.1 (bs, 1H, NH), 5.7 (bs, 1H, NH), 6.2 (m, 1H,
CH), 7.0 (bs, 1H, NH), 7.4-7.5 (m, 1H, arom-H), 7.5-7.7 (m, 2H,
arom-H), 8.0-8.1 (m, 1H, arom-H)
Synthesis of (MeO-PEG750).sub.2-Lys-Gly (30)
[0184] 29 (20 mg, 0.01 mmol) was dissolved in MeOH (2 mL) in a
reaction tube. While stirring the mixture was exposed to a beam of
UV light (254 nm). The reaction was followed with TLC (2:1
EtOAc/Hexane) and after 20 min the reaction was completed. The
solvent was evaporated in vacuo and the residue was dissolved in
water and washed with EtOAc. Then the water-layer was acidified
with 1N HCl and extracted with DOM. The resulting solution was
dried (MgSO.sub.4), filtered and evaporated to dryness. Compound 30
was obtained as a white gel in quantitative yield (18 mg, 0.01
mmol).
[0185] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. ; 1.0-2.0 (m, 6H,
CH.sub.2 (Lys)), 3.1 (m, 2H, CH.sub.2 (Lys)), 3.4 (s, 6H, OMe
(Peg)), 3.3-3.9 (m, 120H, CH.sub.2 (Peg), 4.0-4.4 (m, 7H,
(2.times.) CH.sub.2-OMe (Peg), .alpha.H (Lys) and CH.sub.2 (Gly)),
5.5 (bs, 1H, NH), 5.7 (bs, 1H, NH), 7.1 (bs, 1H, NH).
[0186] .sup.13C NMR (75.5 MHz, CDCl.sub.3) .delta. ; 21.8, 28.7,
31.5, 39.8, 40.4, 53.2, 54.0, 58.2, 60.7, 62.9, 63.4, 68.6, 69.5,
71.1, 155.2, 155.7, 170.1, 171.6
Synthesis of (MeO-PEG750).sub.2-Lys-Gly-Fmoc-Lys(NH.sub.3Cl)-OMe
(31)
[0187] Compound 30 (50 mg, 0.028) and Fmoc-Lys(NH.sub.3Cl)-OMe (43
mg, 0.11) were dissolved in H.sub.2O (3 mL). To this mixture DIPEA
(4.8 .mu.l) and EDC (21 mg, 0.11) were added. After 2 h 10 mL
H.sub.2O was added to the mixture and was extracted with DCM. The
resulting solution was dried (MgSO.sub.4), filtered and evaporated
to dryness. Compound 31 was obtained as a white gel in 90% yield
(54 mg, 0.025 mmol).
[0188] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. ; 1.0-2.0 (m, 12H
CH.sub.2 (Lys)), 3.1 (m, 2H, CH.sub.2 (Lys)), 3.4 (s, 6H, OMe
(Peg)), 3.3-3.9 (m, 126H, CH.sub.2 (Peg), OMe (Lys)), 4.0-4.4 (m,
11H, (2.times.) CH.sub.2--OMe (Peg), (2.times.) .alpha.H (Lys) and
CH2 (Gly), CH (Fmoc) and CH.sub.2 (Fmoc)), bs (1H, NH), 6.0 (bs,
2H, (2.times.) NH), 7.1-8.4 (m, 11H, (3.times.) NH, arom-H
(Fmoc))
Synthesis of PEG.sub.600(LDI-HEA).sub.2) with UV masking group
(32)
[0189] The gel (cured PEG.sub.600(LDI-HEA).sub.2) was dried and
weighed (84.1 mg, 0.079 mmol) and put in a syringe with 2 mL of
H.sub.2O. The syringe was wrapped in aluminium foil to keep the
reaction mixture in the dark. 28 (66 mg, 4 eq), DIPEA (54 .mu.l, 4
eq) and EDC (59 mg, 4 eq) were added. After 1 night shaking at room
temperature the excess of reagents was washed away with water.
After the gel was dried 92.3 mg of gel was obtained in small
fragments (92%).
Synthesis of
LDI-(HEA).sub.2-Gly-Arg(Pmc)-Gly-Asp(O.sup.tBu)-Ser-(O.sup.tBu).sub.2
(33)
##STR00011##
[0191] To a cooled solution (0.degree. C.) of 2.40 g (5.5 mmol)
LDI-(HEA).sub.2 in 100 mL CH.sub.2Cl.sub.2 was added 0.96 g (5.0
mmol, 0.9 equiv.) N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide
hydrochloride, 0.68 g (5.0 mmol; 0.9 equiv.)
1-hydroxy-7-azabenzotriazole and 0.87 mL (0.64 g, 5.0 mmol, 0.9
equiv.) N,N-diisopropylethylamine (DiPEA). Subsequently, 4.62 g
(5.0 mmol, 0.9 equiv.)
Gly-Arg(Pmc)-Gly-Asp(O.sup.tBu)-Ser-(O.sup.tBu).sub.2 was added and
the reaction mixture was stirred at ambient temperature. After 18 h
the reaction mixture was concentrated under reduced pressure and
the residue purified by column chromatography on silica gel using
EtOAc/MeOH 95/5 (v/v) as the eluent furnishing pure 33 as a white
solid (2.8 g, 42% yield). The product was analyzed by HPLC and
.sup.1H-NMR.
[0192] .sup.1H-NMR (300 MHz, DMSO-d6): .delta. (ppm) 8.26 (1H, t,
J=5.1 Hz, NH), 8.21-8.12 (3H, m, 3.times.NH), 7.97 (1H, d, J=7.8
Hz, NH), 7.92 (1H, d, J=8.0 Hz, NH), 7.49 (1H, d, J=7.8 Hz, NH),
7.24 (1H, t, J=5.5 Hz, NH), 6.92 (1H, bs, NH), 6.52 (1H, bs, NH),
6.37 (2H, m, acryloyl), 6.20 (2H, m, acryloyl), 5.98 (2H, m,
acryloyl), 4.73 (1H, q, C.sup..alpha.-Asp), 4.33-4.24 (6H, m,
2.times.O--CH.sub.2--CH.sub.2--O+C.sup..alpha.-Ser+C.sup..alpha.-Arg),
4.23-4.16 (4H, m, 2.times.O--CH.sub.2--CH.sub.2--O), 3.94 (1H, q,
C.sup..alpha.-Lys), 3.80-3.71 (6H, m,
2.times.C.sup..alpha.-Gly+C.sup..beta.-Ser), 3.05 (2H, q,
C.sup..epsilon.-Lys), 2.96 (2H, q, C.sup..delta.-Arg), 2.70-2.56
(4H, m, CH.sub.2CH.sub.2 Pmc), 2.49 (s, 6H, 2.times.CH.sub.3 Pmc),
2.06 (3H, s, CH.sub.3 Pmc), 1.80 (2H, t, C.sup..beta.-Asp),
1.73-1.43 (10H, m, CH.sub.2--CH.sub.2-Arg,
CH.sub.2-CH.sub.2--CH.sub.2-Lys), 1.42 (6H, s, C(CH.sub.3).sub.2
Pmc), 1.39 (9H, s, tBu), 1.28 (9H, s, tBu), 1.13 (9H, s, tBu).
Synthesis of LDI-(HEA).sub.2-Gly-Arg-Gly-Asp-Ser (34)
##STR00012##
[0194]
LDI-(HEA).sub.2-Gly-Arg(Pmc)-Gly-Asp(O.sup.tBu)-Ser-(O.sup.tBu).sub-
.2 (33) (0.76 g, 0.57 mmol) was charged in a sealed Schlenck
reactor under a nitrogen atmosphere and at ambient temperature. The
reactor was brought under a reduced pressure of 100 mbar.
Trifluoroacetic acid (TFA, 5.55 mL) was dosed via a syringe
followed by 0.45 mL of 1,3-dimethoxybenzene (to act as scavenger)
and the reaction mixture was stirred at ambient temperature. The
solution turned from colorless to pink. After 2 h an aliquot was
withdrawn from the reaction mixture and analyzed by HPLC showing
that the deprotection reaction was not complete. Subsequently, an
additional 8.8 mL of TFA was added, the reaction mixture stirred
for another 2 h under a reduced pressure of 100 mbar and the TFA
removed under reduced pressure. To a solution of the resulting
residue in 2.2 mL MeOH was added 200 mL of n-heptane and the
resulting white precipitate was isolated by filtration giving pure
34 as a white solid (0.50 g, 0.56 mmol, 99% yield based on 33). The
identity of the product was confirmed with .sup.1H-NMR and HPLC-MS
([M+H].sup.+=902, as calculated).
[0195] HPLC method for monitoring the deprotection reaction:
analytical HPLC was performed on an HP1090 Liquid Chromatograph
using an Inertsil ODS-3 (150 mm length, 4.6 mm ID) column at
40.degree. C. UV detection was performed at 220 nm using a UVVIS
204 Linear spectrometer. The gradient program was: 0-20 min linear
gradient from 5% to 98% buffer B; 20.1-25.0 min 98% buffer B;
25.1-30 min 5% buffer B. Buffer A: 0.5 mL/L methane sulfonic acid
(MSA) in H.sub.2O; buffer B: 0.5 mL/L MSA in acetonitrile. The flow
was 1 mL/min from 0-25.1 min, 2 mL/min from 25.2-29.8 min and 1
mL/min from 29.8-30 min. Injection volumes were 20 .mu.L. HPLC-MS
was performed on an Agilent 1100 series system using the same
column and identical flow conditions as for analytical HPLC.
Retention times:
LDI-(HEA).sub.2-Gly-Arg(Pmc)-Gly-Asp(O.sup.tBu)-Ser-(O.sup.tBu).sub.2:
23.98 min; LDI-(HEA).sub.2-Gly-Arg-Gly-Asp-Ser: 9.11 min.
[0196] .sup.1H-NMR (300 MHz, DMSO-d6): .delta. (ppm) 12.5 (2H, bs,
2.times.COOH), 8.30-8.17 (2H, m, 2.times.NH), 8.13 (1H, t, NH),
8.00-7.91 (2H, m, 2.times.NH), 7.49-7.38 (2H, m, 2.times.NH), 7.23
(1H, t, NH), 6.95 (3H, bs, 3.times.NH), 6.38 (2H, d, acryloyl),
6.19 (2H, m, acryloyl), 5.98 (2H, d, acryloyl), 5.01-4.95 (1H, m,
NH), 4.67 (1H, q, C.sup..alpha.-Asp), 4.36-4.23 (6H, m,
2.times.O--CH.sub.2--CH.sub.2--O+C.sup..alpha.-Ser+C.sup..alpha.-Arg),
4.23-4.14 (4H, m, 2.times.O--CH.sub.2--CH.sub.2--O), 3.92 (1H, q,
C.sup..alpha.-Lys), 3.82-3.57 (6H, m,
2.times.C.sup..alpha.-Gly+C.sub..beta.-Ser), 3.10 (2H, q,
C.sup..epsilon.-Lys), 2.94 (2H, q, C.sup..delta.-Arg), 1.80-1.69
(2H, m, C.sub..beta.-Asp), 1.69-1.44 (10H, m,
CH.sub.2--CH.sub.2-Arg, CH.sub.2--CH.sub.2--CH.sub.2-Lys). HPLC-MS:
[M+H].sup.+=902. (as calculated).
Example 2
Photolitographic Patterning of PEG.sub.600(LDI-HEA).sub.2) with UV
Masking Group (32)
[0197] Gel 32 was put between two glass cover slips and was covered
with water. With a 405 nm laser of a confocal microscope 50 .mu.m
squares were irradiated for 20 times with 100% laser intensity.
Afterwards the gels were shaken in a flask for 24 h either with
MeOH or EtOH to remove nitrosobenzeacetone.
[0198] FIG. 1 shows:
[0199] Top row: A) Confocal microscopy picture of the gel with
cleavable groups B) Irradiation of a 50 .mu.m square in the gel,
the cleaved group is fluorescent.
[0200] Bottom row A) Blanco gel viewed trough a confocal microscope
B) After irradiating a 50 .mu.m square in the gel no fluorescence
is observed.
Example 3
Degradation Experiments (Series I)
[0201] A clear 75 w % formulation in THF of the oligomers presented
in the table below
TABLE-US-00002 Irgacure 2959 Sample THF (mg) (gram) (Gram) 1 w %
wrt. Nr. Sample 75 w % 25 w % Sample 1 PEG600- 3.233 1.08 35
(t-Bu-LDI-HEA).sub.2 2 p-(Lac-Cap)1000- 2.713 0.90 38
(t-Bu-LDI-HEA).sub.2 3 p-(Lac-Gly)1000- 3.771 1.26 40 diacrylate 4
p-(Lac-Gly)1500- 2.155 0.72 24 triacrylate 5 p-(Lac-Gly)1000- 2.622
0.87 29 (t-Bu-LDI-HEA).sub.2 6 p-(Lac-Gly)1500- 2.099 0.70 24
(t-Bu-LDI-HEA).sub.3 7 PEG600-diacrylate 4.021 1.34 51
Coating Preparation
[0202] The formulation was applied onto tin float glass plate with
the coating doctor blade designed to give 100 .mu.m thick wet
coating. This wet film was cured with UV (1 J/cm.sup.2) from D-bulb
and speed 20 m/s at 22.degree. C. The coatings were dried for 4
hours at 60.degree. C. in the vacuum oven (200 mbar). The resulting
cured and dried coating has a film thickness of 50-60 .mu.m. The
coatings were used as such.
Sample Preparation for Weight Loss Experiment in RVS Steel
Sieves
[0203] Cured film (.about.200 mg) was placed in sieves with a mesh
size of 350-370 .mu.m. The gel fraction of these coatings was
determined via washing with chloroform. Subsequently the coatings
were degraded at 37.degree. C. in an aqueous phosphate saline
buffer solution (PBS: pH 7.4 via dissolving 0.2 g KCl, 0.2 g
KH.sub.2PO.sub.4, 8 g NaCl and 1.15 g NaHPO.sub.4 in 1 litre of
water). Every 2-3 days the buffer was changed with fresh buffer.
Before adding the fresh buffer the sieves were washed 3 times with
15 mL water, dried overnight at 60.degree. C. and weighed. The
degradation was followed by monitoring weight loss, as shown in
FIGS. 2-4.
Example 4
Degradation Experiments (Series II)
[0204] A clear 90 w % formulation in THF of the oligomers presented
in the table below
TABLE-US-00003 Irgacure 2959 THF (mg) Sample (Gram) 1 w % wrt. Nr.
Sample (gram) 10 w %) Acrylates 1 p-(Lac-gly)1545- 8.89 0.99 88
diacrylate 2 p-(Lac-gly)1000- 2.46 0.27 25 (m-LDI-HEA).sub.2 3
p-(Gly-cap)1545- 7.98 0.89 78 diarcylate 4 p-(Gly-cap)1000- 5.53
0.61 55 (m-LDI-HEA).sub.2 5 p-(Lac-Cap)-1545- 8.08 0.90 78
diacrylate 6 p-(Lac-Cap)-1000- 5.34 0.59 53 (m-LDI-HEA).sub.2
Coating Preparation
[0205] The formulation was applied onto tin float glass plate with
the coating doctor blade designed to give 200 .mu.m thick wet
coating. This wet film was cured with UV (2 J/cm.sup.2) from D-bulb
and speed 20 m/s at 22.degree. C. The coatings were dried for 4
hours at 60.degree. C. in the vacuum oven (200 mbar). The resulting
cured and dried coating has a film thickness of 150 .mu.m. The
coatings were used as such.
Sample Preparation for Weight Loss Experiment in RVS Steel
Sieves
[0206] Cured film (.about.200 mg) was placed in sieves with a mesh
size of 350-370 .mu.m. The gel fraction of these coatings was
determined via washing with chloroform. Subsequently the coatings
were degraded at 37.degree. C. in an aqueous phosphate saline
buffer solution or enzym phosphate buffer solution (PBS: pH 7.4 via
dissolving 0.2 g KCl, 0.2 g KH.sub.2PO.sub.4, 8 g NaCl and 1.15 g
NaHPO.sub.4 in 1 litre of water, enzym PBS: 28.6 mg of cholesterol
esterase was dissolved in 1000 mL PBS buffer).
[0207] Every 2-3 days the buffer was changed with fresh buffer.
Before adding the fresh buffer the sieves were washed 3 times with
15 mL water, dried overnight at 60.degree. C. and weighed. The
degradation was followed by monitoring weight loss, as shown in
FIGS. 5-10.
Example 5
Dynamic Mechanical Measurements in Tensile of Coatings
[0208] The materials were delivered as films on a glass plate. The
samples for the measurements were punched out of the film. The
thickness was measured with the calibrated Heidenhain thickness
meter. The dynamic mechanical measurements were done in accordance
with ASTM D5026 on equipment of the firm Rheometrics called RSA-III
(Rheometrics Solids Analyser III) at a frequency of 1 Hz and over a
temperature area of -130.degree. C. tot 250.degree. C. with a
heating speed of 5.degree. C./min. During the measurements the
storage modulus (E'), the lost modulus (E'') and the tangent delta
(tan .delta.) as function of temperature were defined.
[0209] Deviation from the ASTM D5026 were: [0210] Allowed
temperature deviation .+-.2.degree. C. (in standard .+-.1.degree.
C.) [0211] Allowed force deviation .+-.2% (in norm standard .+-.1%)
[0212] Allowed frequency deviation .+-.2% (in standard .+-.1%)
[0213] Heating speed 5.degree. C./min. (in standard 1 to 2.degree.
C./min.)
Test Conditions Tensile Test:
Tensile Test:
Machine: Zwick 1484.
Tensile bar: Conform DIN 53504 S3a.
[0214] Force cell: 10N
Strain: Optical Extensometer (L0.+-.20 mm).
[0215] Length between clamps: 35 mm. Test speed: 50 mm/min Clamps:
20N clamp
Mechanical Properties (Series II)
[0216] A clear formulation of the oligomers presented in the table
below was prepared.
TABLE-US-00004 Caprolactone Irgacure acrylate 2959 Amount (SR495)
(mg) (gram) (gram) (1 w % wrt Sample 60 w % 40 w % arcylates) 1
p-(lactide-caprolactone 10.22 6.81 170 50/50)1545-diacrylate 2
p-(lactide-caprolactone 9.91 6.60 165 50/50)1000-(m-LDI-HEA)2
Coating Preparation
[0217] The formulation was applied onto tin float glass plate with
the coating doctor blade designed to give 200 .mu.m thick coating.
This film was cured with UV (1 J/cm.sup.2) from D-bulb and speed 20
m/s at 22.degree. C. The resulting cured coating has a film
thickness of 180-200 .mu.m. The coatings were used as such.
DMA Results
TABLE-US-00005 [0218] E-mod (37.degree. C.) .epsilon.-break Tg nr.
Material [MPa] [%] [.degree. C.] 1 p-(lactide-caprolactone
50/50)1545- 5.55 30 -30 diacrylate 2 p-(lactide-caprolactone
50/50)1000- 3.99 42 -24 (m-LDI-HEA).sub.2
FIG. 11 shows a graphical representation of the tensile test.
Example 6
[0219] H-Arg(PMC)-OtBu-hexanoate-HEA (18, Monoacrylate, MA) and
H-Arg(PMC)-OtBu-hexanoate-LDI-(HEA).sub.2 (19, Diacrylate, DA) were
formulated in PTGL1000-(TDI-HEA).sub.2 and PEG600-diacrylate as
presented below.
TABLE-US-00006 PTGL1000- Irgacure 2959 Monoacrylate Diacrylate
(TH)2 (mg) Nr. (mg, mmol) (mg, mmol) (mg) 1 w % wrt. Sample 1 135,
0.18 1720 19 2 68, 0.09 1723 18 3 82, 0.09 1714 17 4 82, 0.09 1725
17
TABLE-US-00007 PEG600- Irgacure 2959 Monoacrylate Diacrylate
diacrylate (mg) Nr. (mg, mmol) (mg, mmol) (mg) 1 w % wrt. Sample 5
113, 0.15 1527 17 6 56, 0.075 1500 16 7 68, 0.075 1534 17 8 68,
0.075 1818 16
Coating Preparation
[0220] The formulation was applied onto tin float glass plate with
the coating doctor blade designed to give 100 .mu.m thick wet
coating. This wet film was cured with UV D-bulb and speed 17.5 m/s
at 22.degree. C. Different intensities were applied to yield in
different acrylate conversion. Formulations 1-4 were cured with the
intensities of 0.04 J/cm.sup.2, 0.20 J/cm.sup.2 and 2.0 J/cm.sup.2.
Formulations 5-8 were cured with the intensities of 0.09
J/cm.sup.2, 0.44 J/cm.sup.2 and 2.0 J/cm.sup.2. The coatings are
used as such. Coatings 1-8 were placed in a vial together with
acetonitril. After 1 hour the extractables were measured with HPLC.
The results are shown in the FIGS. 12 and 13.
Example 7
[0221] A clear 50 w % formulation in MeOH of the oligomers
presented in the table below
TABLE-US-00008 RGD- Irgacure 2959 LDI(HEA)2 (mg) Amount (17) MeOH
(1 w % wrt Sample (gram) (gram) gram arcylates) 1 PTGL1000- 6.60 --
6.75 66 (TDI-HEA)2 2 PTGL1000- 6.70 0.332 7.14 67 (TDI-HEA)2
[0222] Coatings of PTGL1000-(TDI-HEA).sub.2 control and
PTGL1000-(TDI-HEA)2/RGD-LDI-(HEA).sub.2 on glass cover slips could
not be used for cell culture experiments, because the coating was
not resistant to sterilization conditions (with 70% EtOH). Coating
of the polymers on plastic coverslips was much better. The
formulation was applied onto Thermanox.RTM. PET cover slips
(diameter 13 mm) via spin coating (10 sec, 28 rpm). These cover
slips were cured with UV (2 J/cm.sup.2) from D-bulb and speed 20
m/s at 22.degree. C. The cover slips and coatings were used as
such.
[0223] All experiments were carried out using Fibroblasts from
human foreskin. Culture 24-well plates were purchased from
Corning/Costar. (cat#3524). The thermanox plastic coverslips were
purchased from NUNC (cat#174950). As control gelatin, 1% (w/v)
water, .+-.200 .mu.I/2 cm2 (Merck, cat#104070) incubated for 1 hour
at RT was used. Cyclic RGD: Cyclo(-Arg-Gly-Asp-D-Phe-Val) was
purchased from Bachem (cat# H-2574) and dissolved in sterile water
(10 mg/). The serum free culture medium contains M199
Cambrex/BioWhittaker, cat# BE12-117F, 100 IU/penicillin, 100
.mu.g/streptomycin (Invitrogen/Gibco, cat#15140-122).
[0224] The plastic cover slips have the tendency to float in the
medium in contrast to glass cover slips. Therefore the cover slips
had to be "glued" to the bottom of the wells, using paraffin: a
droplet (or two) of melted paraffin was applied half on the cover
slip and half on the bottom of the well (this was done using a
wooden stick). The paraffin was allowed to set for 30 minutes at
RT. Then 0.5 mL of 70% (v/v) ethanol was added to the wells and
incubated for 30 minutes at RT After this period, the now sterile
cover slips were washed 5 times with 1 mL of M199 medium
(+pen/strep), (one time they were left for 1 hour at RT). The cover
slips with the coatings were now ready to use.
[0225] The uncoated cover slips were now incubated with gelatin or
vitronectin (1 hour, RT), after which they were washed one more
time.
[0226] Cells were cultured at 37.degree. C., 5% CO.sub.2/95% air,
in a humid environment. The cells were seeded (0.5 ml/well) in
"high" density, approx. 10000 cells/well Fibroblasts were seeded in
complete M199 or M199 containing only pen/strep (serum free
medium). (in the latter case cells were washed once with the serum
free medium before seeding). To half the cells cyclic RGD was added
(50 .mu.g/ml final conc) so the cyclic RGD was present during
attachment of the cells. Photographs were taken after: approx. 16
hours (overnight)
[0227] To study the effect of c-RGD this peptide was added to the
cells (50 .mu.g/mL) before they were seeded (so the cyclic RGD was
present during attachment of the cells). Cells were cultured at
37.degree. C., 5% CO.sub.2/95% air, in a humid environment.
[0228] The experiments under serum-free conditions were only
carried out using fibroblasts. The
PTGL1000-(TDI-HEA).sub.2/RGD-LDI-(HEA).sub.2 coating showed a
significant better cell attachment as compared to control polymer,
suggesting that the RGD-moiety on the polymer is able to interact
with the cells and improve attachment. The morphology of cells
grown on PTGL1000-(TDI-HEA).sub.2/RGD-LDI-(HEA).sub.2 coating was
better than that of cells grown on PTGL1000-(TDI-HEA).sub.2
coating. FIG. 14 shows photographs, illustrating this.
Example 8
[0229] A clear 50 w % formulation of the oligomers in THF are
presented in the table below
TABLE-US-00009 GRGDS- Daracure Amount LDI(HEA)2 THF 1173 Sample
(gram) (gram) gram (mg) 1 PEG600-(m-LDI-HEA).sub.2 2.75 -- 6.75 40
2 PEG600-(m-LDI-HEA).sub.2 2.50 0.200 2.70 40
[0230] The formulations of PEG600-(m-LDI-HEA).sub.2 and
PEG600-(m-LDI-HEA).sub.2/GRGDS-LDI-(HEMA).sub.2 was applied onto
Thermanox.RTM. PET cover slips (diameter 13 mm, Thermanox Plastic
NUNC, cat#174950) via spin coating (5 sec, 3000 rpm). These cover
slips were cured with UV (5 J/cm.sup.2) from D-bulb under nitrogen
atmosphere and a speed of 18 m/s at 22.degree. C. The cover slips
and coatings were used as such.
[0231] All experiments were carried out using Fibroblasts from
human foreskin. The 24-well culture plates were purchased form
Corning/Costar (cat#3524). The Thermanox Plastic coverslips were
purchased from (NUNC, cat#174950). As a control Gelatin, 1% (w/v)
water, .+-.200 .mu.I/2 cm2 (Merck, cat#104070) incubated for 1 hour
at RT was used. The Cyclic RGD: Cyclo(-Arg-Gly-Asp-D-Phe-Val)
(Bachem, cat# H-2574) was dissolved in sterile water (10 mg/ml) and
used as such. The serum free culture medium contains: M199
Cambrex/BioWhittaker, cat# BE12-117F, 100 IU/ml penicillin, 100
.mu.g/ml streptomycin (Invitrogen/Gibco, cat#15140-122). The serum
containing medium contains 199 (Cambrex/BioWhittaker, cat#
BE12-117F), 10% human serum, 10% NewBornCalf Serum (NBCS), 150
.mu.g/ml ECGF (Endothelial Cell Growth Factor), 2 mM L-Glutamin, 5
U/ml heparin, 100 IU/ml penicillin and 100 .mu.g/ml streptomycin As
fixative 2% formaldehyde+0.2% glutaraldehyde in water was used.
[0232] The coverslips were "glued" to the bottom of the wells,
using paraffin. The paraffin was melted and 3-4 droplets were
applied half on the coverslip and half on the bottom of the well
(this was done using a wooden stick). The paraffin was allowed to
set for 30 minutes at RT. Then 0.5 ml of 70% (v/v) ethanol was
added to the wells and incubated for 30 minutes at RT. After this
period, the now sterile coverslips were washed 3 times with 1 ml of
M199 medium (+pen/strep), (one time they were left for 1 hour at
RT)
[0233] During this one hour incubation of the coated coverslips,
the uncoated coverslips were incubated with gelatin or vitronectin
(1 hour, RT) after which all coverslips were washed one more time.
The coverslips were now ready for use.
[0234] Cells were cultured at 37.degree. C., 5% CO.sub.2/95% air,
in a humid environment. The cells were seeded (0.5 ml/well) in
"high" density, approx. 30000 cells/well Fibroblasts were seeded in
complete M199 or M199 containing only pen/strep (serum free
medium). (in the latter case cells were washed once with the serum
free medium before seeding). To half the cells cyclic RGD was added
(50 .mu.g/ml final conc) so the cyclic RGD was present during
attachment of the cells. Photographs were taken after: approx. 16
hours (overnight)
[0235] The PEG600-(m-LDI-HEA).sub.2/GRGDS-(LDI-HEA).sub.2 coating
showed a significant better cell attachment under serum free and
serum containing conditions as compared to control polymer
PEG600-(m-LDI-HEA).sub.2, suggesting that the GRGDS-moiety on the
polymer is able to interact with the cells and improve attachment.
FIG. 15 shows photographs, illustrating this.
* * * * *