AGENTS DE RETICULATION A BASE DE POLYAMIDES NON IMMUNOGENE HYDROSOLUBLE

WATER SOLUBLE NON-IMMUNOGENIC POLYAMIDE CROSS-LINKING AGENTS

Abrégé français

La présente invention concerne des agents de réticulation à base de polyamides non immunogène hydrosoluble et leur emploi pour réticuler, polymériser, décorer et conjuguer des protéines, des polynucléotides et autres substrats biologiques afin de constituer des produits hydrosolubles non immunogènes. La présente invention concerne des protéines, des polynucléotides et autres substrats biologiques qu'il est possible de réticuler, conjuguer, polymériser ou décorer au moyen de polyamides hydrosolubles, pour constituer des produits essentiellement non immunogènes.

Abrégé anglais

The present invention relates to water-soluble nonimmunogenic polyamide cross-
linking agents and their use to cross-link, polymerize, decorate, and
conjugate proteins, polynucleotides and other biological substrates to form
substantially nonimmunogenic water-soluble products. The present invention
also relates to proteins, polynucleotides and other biological substrates
which are cross-linked, conjugated, polymerized or decorated with water-
soluble polyamides to form substantially nonimmunogenic products.

Revendications

- 43 -
CLAIMS:
1. A water-soluble, substantially
nonimmunogenic polyamide selected from the group
consisting of:
I Y-A-X-Y
II Z-B-X-Z
III Y-X-Z
(a) where terminus Y is OH or a carboxyl
coupling group;
(b) where terminus Z is H or a coupling
group attached to an amine group; and
(c) where X is a polyamide selected from:
(B-A)n, (A-B)n, and branched polyamides formed by
linking (B-A)n or (A-B)n to a central polyacid,
polyamine or polyamino acid; and
(d) where A is a .alpha.,.omega.-di-acid; B is a .alpha.,.omega.-
diamine; n is the number of amide repeat units in the
polyamide; and
(e) where the acid subunits of the amide
repeat units are two or more organic acids each of
said organic acids having fifteen or fewer atoms in
the chain and having one or more heteroatoms selected
from the group consisting of O other than carboxyl or
carbonyl O, S, P or tertiary N present as substituents
on or atoms in the chain bridged by a water-soluble
diamine having the formula -NH-R1-NH- where R1 is a
substituted or unsubstituted aliphatic chain having
from about 4 to about 5 carbon atoms; and
(f) where the amine subunits of the amide
repeat units are organic, water-soluble amines having
at least one primary amine group and having fifteen or
fewer atoms in the chain and having one or more
heteroatoms selected from the group consisting of O
other than carboxyl or carbonyl O, S, P or N present
as substituents on or atoms in the chain; and
(g) where n is from 2 to about 100.

- 44 -
2. A polyamide of Claim 1 wherein the water-
soluble diamine having the formula -NH-R1-NH- is 1,4
diaminobutane.
3. Two or more polyamides of Claims 1 or 2
linked by a central polyacid, polyamine or polyamino
acid to form branched, water-soluble polyamides.
4. A polyamide of Claims 1 or 2 reacted with a
substrate having a diagnostic or therapeutic
biological activity selected from the group consisting
of proteins including enzymes, haptens, and
antibodies; polypeptides; polynucleotides; steroids;
and carbohydrates; wherein the product of said
reaction is water-soluble, substantially
nonimmunogenic and retains a diagnostically or
therapeutically useful amount of the substrate's
biological activity.
5. A polyamide of Claims 1 or 2 decorating a
substrate having a diagnostic or therapeutic
biological activity selected from the group consisting
of proteins including enzymes, haptens, and
antibodies; polypeptides; polynucleotides including
probes; and carbohydrates; wherein the product of said
decorating is water-soluble, substantially
nonimmunogenic, and retains a diagnostically or
therapeutically useful amount of the substrate's
biological activity.
6. A polyamide of Claims 1 or 2 cross-linking
two or more substrates having a diagnostic or
therapeutic biological activity selected from the
group consisting of proteins including enzymes,
haptens, and antibodies; polypeptides; polynucleotides
including probes; and carbohydrates; wherein the
product of said cross-linking is water-soluble,
substantially nonimmunogenic and retains a
diagnostically or therapeutically useful amount of the
substrate's biological activity.

- 45 -
7. A polyamide of Claims 1 or 2 polymerizing
three or more substrates having a diagnostic or
therapeutic biological activity selected from the
group consisting of proteins including enzymes,
haptens, and antibodies; polypeptides; polynucleotides
including probes; and carbohydrates; wherein the
product of said polymerizing is water-soluble,
substantially nonimmunogenic and retains a
diagnostically or therapeutically useful amount of the
substrate's biological activity.
8. A polyamide of Claims 1 or 2 decorating a
product of Claim 7.
9. A polyamide of Claims 1 or 2 decorating a
product of Claim 6.
10. A product of Claim 7 wherein said
substrates are hemoglobin molecules and wherein said
polyamide is bis(maleimidoacyl) polyamide.
11. A product of Claim 7 wherein said
substrates are hemoglobin molecules and wherein said
polyamide is bis(N-oxa-succinimidyl) polyamide.
12. Water-soluble, substantially nonimmunogenic
branched or straight chain polyamides having number
average molecular weights of about 300 to about 20,000
grams per mole; comprising from 1 to about 100 amide
repeat units where each repeat unit comprises:
(a) a water-soluble organic acid having
fifteen or fewer atoms in the chain and having one or
more heteroatoms selected from the group consisting of
O other than carboxyl or carbonyl O, S, P or tertiary
N present as substituents on or atoms in the chain
bridged by a water-soluble diamine having the formula
-NH-R1-NH- where R1 is a substituted or unsubstituted
aliphatic chain having from about 4 to about 5 carbon
atoms; and
(b) covalently linked as an amide to;

- 46 -
(c) a water-soluble organic amine subunit
having at least one primary amino group and fifteen or
fewer atoms separating the amide functionalities in
the polyamide.
13. A polyamide of Claim 1 where terminus Y and
terminus Z are independently activated by reacting
said polyamide with bi- or polyfunctional protein
reagents selected from the group consisting of
dialdehydes, N-hydroxysuccinimide esters, activated
esters, functionalized acetals, bis-maleimides,
bifunctional imino esters, diepoxides, and
dicarboxylic acid chlorides.
14. A water-soluble, substantially
nonimmunogenic polyamide selected from the group
consisting of:
I Y-A-X-Y
II Z-B-X-Z
III Y-X-Z
(a) where terminus Y is OH or a carboxyl
coupling group;
(b) where terminus Z is H or a coupling
group attached to an amine group; and
(c) where X is a polyamide selected from:
(B-A)n, (A-B)n, and branched polyamides formed by
linking (B-A)n or (A-B)n to a central polyacid,
polyamine or polyamino acid; and
(d) where A is a .alpha.,.omega.-di-acid; B is a .alpha.,.omega.-
diamine; n is the number of amide repeat units in the
polyamide; and
(e) where the acid subunits of the amide
repeat units are organic acids having the formula
<IMG>
where m is from about 2 to about 4, X1 and X2 are
independently a heteroatom selected from the group

- 47 -
consisting of O other than carboxyl or carbonyl O, S,
P or N, and R2 is H or acetamide;
(f) where the amine subunits of the amide
repeat units are organic, water-soluble amines having
at least one primary amine group and having fifteen or
fewer atoms in the chain and having one or more
heteroatoms selected from the group consisting of
carboxyl or carbonyl O, S, P or N present as
substituents on or atoms in the chain; and
(g) where n is from 2 to about 100.
15. A polyamide of Claim 14 wherein X1 and X2
are both S.
16. A polyamide of Claim 14 wherein X1 and X2
are both O.
17. A polyamide of Claim 14 wherein m is 2, and
X1 and X2 are both O.
18. Two or more polyamides of Claim 14 or 17
linked by a central polyacid, polyamine or polyamino
acid to form branched, water-soluble polyamides.
19. A polyamide of Claims 14 or 17 reacted with
a substrate having a diagnostic or therapeutic
biological activity selected from the group consisting
of proteins including enzymes, haptens, and
antibodies; polypeptides; polynucleotides; steroids;
and carbohydrates; wherein the product of said
reaction is water-soluble, substantially
nonimmunogenic and retains a diagnostically or
therapeutically useful amount of the substrate's
biological activity.
20. A polyamide of Claims 14 or 17 decorating a
substrate having a diagnostic or therapeutic
biological activity selected from the group consisting
of proteins including enzymes, haptens, and
antibodies; polypeptides; polynucleotides including
probes; and carbohydrates; wherein the product of said
decorating is water-soluble, substantially

- 48 -
nonimmunogenic, and retains a diagnostically or
therapeutically useful amount of the substrate's
biological activity.
21. A polyamide of Claims 14 or 17 cross-
linking two or more substrates having a diagnostic or
therapeutic biological activity selected from the
group consisting of proteins including enzymes,
haptens, and antibodies; polypeptides; polynucleotides
including probes; and carbohydrates; wherein the
product of said cross-linking is water-soluble,
substantially nonimmunogenic and retains a
diagnostically or therapeutically useful amount of the
substrate's biological activity.
22. A polyamide of Claims 14 or 17 polymerizing
three or more substrates having a diagnostic or
therapeutic biological activity selected from the
group consisting of proteins including enzymes,
haptens, and antibodies; polypeptides; polynucleotides
including probes; and carbohydrates; wherein the
product of said polymerizing is water-soluble,
substantially nonimmunogenic and retains a
diagnostically or therapeutically useful amount of the
substrate's biological activity.
23. A polyamide of Claims 14 or 17 decorating a
product of Claim 22.
24. A polyamide of Claims 14 or 17 decorating a
product of Claim 21.
25. A product of Claim 23 wherein said
substrates are hemoglobin molecules and wherein said
polyamide is bis(maleimidoacyl) polyamide.
26. A product of Claim 23 wherein said
substrates are hemoglobin molecules and wherein said
polyamide is bis(N-oxa-succinimidyl) polyamide.
27. Water-soluble, substantially nonimmunogenic
branched or straight chain polyamides having number
average molecular weights of about 300 to about 20,000

- 49 -
grams per mole; comprising from 1 to about 100 amide
repeat units where each repeat unit comprises:
(a) organic acids having the formula
<IMG>
where m is from about 2 to about 4, X1 and X2 are
independently a heteroatom selected from the group
consisting of O other than carboxyl or carbonyl O, S,
P or tertiary N, and R2 is H or acetamide;
(b) covalently linked as an amide to;
(c) a water-soluble organic amine subunit
having at least one primary amino group and fifteen or
fewer atoms separating the amide functionalities in
the polyamide.
28. A polyamide of Claim 14 or 17 where
terminus Y and terminus Z are independently activated
by reacting said polyamide with bi- or polyfunctional
protein reagents selected from the group consisting of
dialdehydes, N-hydroxysuccinimide esters, activated
esters, functionalized acetals, bis-maleimides,
bifunctional imino esters, diepoxides, and
dicarboxylic acid chlorides.
29. A water-soluble, substantially
nonimmunogenic polyamide selected from the group
consisting of:
I Y-A-X-Y
II Z-B-X-Z
III Y-X-Z
(a) where terminus Y is OH or a carboxyl
coupling group;
(b) where terminus Z is H or a coupling
group attached to an amine group; and
(c) where X is a polyamide is selected
from the group consisting of: (B-A')n or (A'-B)n and
branched polyamides formed by linking (B-A')n or

- 50 -
(A'-B)n to a central polyacid, polyamine or polyamino
acid; and
(d) where B is a .alpha.,.omega.-diamine; n is the
number of amide repeat units in the polyamide; and A'
is a .alpha.,.omega.-di-acid having the formula
<IMG>
where Y1 has the formula
-OC-(CH2)p-NH-
where p is from 1 to about 4, m is from about 2 to
about 4, X1 and X2 are independently a heteroatom
selected from the group consisting of O other than
carboxyl or carbonyl O, S, P or tertiary N, and R3 is
a lower alkyl having from about 1 to about 2 carbon
atoms;
(e) where the amine subunits of the amide
repeat units are organic, water-soluble amines having
at least one primary amine group and having fifteen or
fewer atoms in the chain and having one or more
heteroatoms selected from the group consisting of O
other than carboxyl or carbonyl O, S, P or N present
as substituents on or atoms in the chain; and
(f) where n is from 2 to about 100.
30. A polyamide of claim 29 wherein X1 and X2
are both O, m is 2, and R3 is methyl.
31. Two or more polyamides of Claims 29 or 30
linked by a central polyacid, polyamine or polyamino
acid to form branched, water-soluble polyamides.
32. A polyamide of Claims 29 or 30 reacted with
a substrate having a diagnostic or therapeutic
biological activity selected from the group consisting
of proteins including enzymes, haptens, and
antibodies; polypeptides; polynucleotides; steroids;
and carbohydrates; wherein the product of said
reaction is water-soluble, substantially

- 51 -
nonimmunogenic and retains a diagnostically or
therapeutically useful amount of the substrate's
biological activity.
33. A polyamide of Claims 29 or 30 decorating a
substrate having a diagnostic or therapeutic
biological activity selected from the group consisting
of proteins including enzymes, haptens, and
antibodies; polypeptides; polynucleotides including
probes; and carbohydrates; wherein the product of said
decorating is water-soluble, substantially
nonimmunogenic, and retains a diagnostically or
therapeutically useful amount of the substrate's
biological activity.
34. A polyamide of Claims 29 or 30 cross-
linking two or more substrates having a diagnostic or
therapeutic biological activity selected from the
group consisting of proteins including enzymes,
haptens, and antibodies; polypeptides; polynucleotides
including probes; and carbohydrates; wherein the
product of said cross-linking is water-soluble,
substantially nonimmunogenic and retains a
diagnostically or therapeutically useful amount of the
substrate's biological activity.
35. A polyamide of Claims 29 or 30 polymerizing
three or more substrates having a diagnostic or
therapeutic biological activity selected from the
group consisting of proteins including enzymes,
haptens, and antibodies; polypeptides; polynucleotides
including probes; and carbohydrates; wherein the
product of said polymerizing is water-soluble,
substantially nonimmunogenic and retains a
diagnostically or therapeutically useful amount of the
substrate's biological activity.
36. A polyamide of Claims 29 or 30 decorating a
product of Claim 35.

- 52 -
37. A polyamide of Claims 29 or 30 decorating a
product of Claim 34.
38. A product of Claim 35 wherein said
substrates are hemoglobin molecules and wherein said
polyamide is bis(maleimidoacyl) polyamide.
39. A product of Claim 35 wherein said
substrates are hemoglobin molecules and wherein said
polyamide is bis(N-oxa-succinimidyl) polyamide.
40. Water-soluble, substantially nonimmunogenic
branched or straight chain polyamides having number
average molecular weights of about 300 to about 20,000
grams per mole; comprising from 1 to about 100 amide
repeat units where each repeat unit comprises:
(a) a water-soluble organic acid having
the formula
<IMG>
where Y1 has the formula
-OC-(CH2)p-NH-
where p is from 1 to about 4, m is from about 2 to
about 4, X1 and X2 are independently a heteroatom
selected from the group consisting of O other than
carboxyl or carbonyl O, S, P or tertiary N, and R3 is
a lower alkyl having from about 1 to about 2 carbon
atoms;
(b) covalently linked as an amide to;
(c) a water-soluble organic amine subunit
having at least one primary amino group and fifteen or
fewer atoms separating the amide functionalities in
the polyamide.
41. A polyamide of Claim 29 or 30 where
terminus Y and terminus Z are independently activated
by reacting said polyamide with bi- or polyfunctional
protein reagents selected from the group consisting of
dialdehydes, N-hydroxysuccinimide esters, activated

- 53 -
esters, functionalized acetals, bis-maleimides,
bifunctional imino esters, diepoxides, and
dicarboxylic acid chlorides.
42. A water-soluble, substantially
nonimmunogenic polyamide selected from the group
consisting of:
I Y-A-X-Y
II Z-B-X-Z
III Y-X-Z
(a) where terminus Y is OH or a carboxyl
coupling group;
(b) where terminus Z is H or a coupling
group attached to an amine group; and
(c) where X is a polyamide is selected
from the group consisting of: (B-A')n or (A'-B)n and
branched polyamides formed by linking (B-A')n or
(A'-B)n to a central polyacid, polyamine or polyamino
acid; and
(d) where B is a .alpha.,.omega.-diamine; n is the
number of amide repeat units in the polyamide; and A'
is a .alpha.,.omega.-di-acid having the formula
Y1-A-Y1
where Y1 has the formula - OC-(CH2)p-NH- ,where p is
from 1 to about 4; and where A is an acid subunit of
the amide repeat units having two or more organic
acids each of said organic acids having fifteen or
fewer atoms in the chain and having one or more
heteroatoms selected from the group consisting of O
other than carboxyl or carbonyl O, S, P or tertiary N
present as substituents on or atoms in the chain
bridged by a water-soluble diamine having the formula
-NH-R1-NH- where R1 is a substituted or unsubstituted
aliphatic chain having from about 4 to about 5 carbon
atoms;
(e) where the amine subunits of the amide
repeat units are organic, water-soluble amines having

- 54 -
at least one primary amine group and having fifteen or
fewer atoms in the chain and having one or more
heteroatoms selected from the group consisting of O
other than carboxyl or carbonyl O, S, P or N present
as substituents on or atoms in the chain; and
(f) where n is from 2 to about 100.

Description

WO95/17886~ S 6 ~ ~ PCT~S94/14821
WATER SOLUBLE NON-IMMUNOGENIC POLYAMIDE
5CROSS-LINKING AGENTS
CROSS REFERENCE TO RELATED APPLICATION
This is a continuation-in-part of copending U.S.
patent application number 7/981,447, filed ll-25-92.
Field of the Invention
The present invention relates to water-soluble,
substantially non;mml7~ogenic polyamide cross-linking
agents. The present invention also relates to
covalent binding of water-soluble polyamides to
proteins, polynucleotides and other biological
substrates to form substantially nonimmunogenic water-
soluble products. The present invention also relates
to proteins, polynucleotides and other biological
substrates which are cross-linked, conjugated,
polymerized or decorated with water-soluble polyamides
to form substantially non;mmllnogenic products.
Backaround of the Invention
Cross-linking reagents are used for a variety of
purposes, including the investigation of the spatial
arrangement and functions of various macromolecular
entities, the identification of binding sites
(receptors) for ligands, the preparation of affinity
matrices, and the modification and stabilization of
diverse macromolecular structures (Methods in
Enzymology, Volume 9l, pages 580 to 609 (1983)).
Cross-linkers have been designed to preserve
electrostatic charge; to alter electrostatic charge;
to decrease immunogenicity; to increase and decrease
susceptibility to proteolysis; to introduce
fluorescent labels, spin labels, radiolabels, and
electron-dense substituents; to attach several

WO9S/17886 2 ~ Z ~ - 2 - PCT~S94/14821 -
different types of carbohydrate moieties; to modify
enzyme specificity; and to introduce intramolecular
and/or intermolecular cross-links, both to couple
already associated species and to join various
proteins in order to combine the properties of both
into a single molecule (G. E. Means and R. E. Feeney,
Bioconjugate Chemistry, Volume l, page 2 to 12
(l990)). A large number of cross-linking reagents
have been developed to serve these and a variety of
other purposes. Many of these reagents are
commercially available.
Cross-linking of proteins and their
immobilization, either by attachment to an insoluble
support or by various other means, has been employed
to increase the stability of proteins or of certain
conformational relationships in proteins; to couple
two or more different proteins; to identify or
characterize the nature and extent of certain protein-
protein interactions or to determine distances between
reactive groups in or between protein subunits.
Proteins may be immobilized to facilitate their use
and their separation from other products. Cross-
linking therapeutic proteins or polypeptides has been
shown to decrease immunogenicity and to increase the
lifetime of the cross-linked product in the blood
stream.
In general, cross-linking agents consist of an
organic bridge between activated termini. The termini
bind to biological macromolecules to form a link.
Various organic bridges are recognized in the art,
including peptides, carbohydrates (e.g., dextran,
starch, and hydroxyethylstarch), fatty acids,
polyglycolides, polypeptides (e.g., gelatin or
collagen), polyalkylene units, and polymers such as
poly(vinylalcohol), polyvinylpyrrolidone, and
polyethylene glycol (also known as polyoxyethylene).

WO9Y17886 2 1 5 6 9 2 4 PCT~S94/14821
Commercially available homobifunctional and
heterbifunctional cross-linking agents range in size
from about 6 to 16 A. Their solubility in water
decreases with chain length. Yet the efficiency of
5 cross-linking is increased with chain length as steric
hindrance is reduced.
Peptides composed of three to nine amino acid
residues are commonly used as cross-linking agents.
However, these suffer from the following
disadvantages: the chemistries used in peptide
synthesis are complex, involving selective blocking
and deblocking of functional groups and specific
coupling conditions. Care must be taken not to
racemize the amino acid components. Peptides must be
chosen carefully so that they have no biological
activity. Finally, they are subject to enzymatic
hydrolysis, which limits their period of utility,
particularly during circulation in vivo.
Synthetic polymers are being developed for use
as cross-linking agents. A synthetic polymer cross-
linker desirably has the following characteristics:
(l) The polymer must be water-soluble and exhibit a
narrow, definite molecular weight distribution. (2)
It should provide attachment/release sites or the
possibility of the incorporation of such sites. (3)
The polymer should be compatible with the biological
environmental, i.e., non-toxic, non-antigenic, and not
provocative in any other respect. (4) It should be
biodegradable or eliminated from the organism after
having fulfilled its function (Duncan and Kopecek,
Advances in Polymer Science, Volume 97, pages 53 to
l~l (19~34)~.
The conjugation of biologically active
polypeptides with water-soluble polymers such as PEG
is well-known. The coupling of biologically active
and pharmaceutically active peptides and polypeptides

WO95117886 2~ ~9~ ~ PCT~S94114821 -
to PEG and similar water-soluble polvmers is disclosed
by U.S. Patent No. 4,179,377 to Davis et al.
Polypeptides modified with PEG are disclosed as
exhibiting dramatically reduced immunogenicity and
antigenicity. The PEG conjugates also exhibit a wide
range of solubilities and low toxicity, and have been
shown to remain in the bloodstream considerably longer
than the corresponding native compounds yet are
readily excreted. The PEG conjugates have also been
shown not to interfere with enzymatic activity in the
bloodstream or the conformation of the polypeptides
conjugated thereto. Accordingly, a number of PEG-
conjugates of therapeutic proteins have been developed
exhibiting reduced immunogenicity and antigenicity and
longer clearance times, while retaining a substantial
portion of the protein's physiological activity.
Attention has also focused upon the conjugation
of PEG with therapeutic drugs. Gnanov et al.,
"Macromolecules," 17, pages 945 to 952 (1984) observed
that the attachment of PEG to various drugs led to
prolonged pharmacological activity.
U.S. Patent No. 5,122,614 to Zalipsky describes
the use of polyethylene gycol as a cross-linking
agent. U.S. Patent No. 5,053,520 to Bieniarz
describes polyamino acid based coupling agents which
are not water-soluble. U.S. Patent No. 4,182,695 to
Horn describes protein bound to polyamides. Russian
Patent Application No. SU 1659433 discloses water-
soluble polyamides with luminescent groups in the
30 chain. U.S. Patent No. 5,110,909 to Dellacherie
discloses water-soluble macromolecular conjugates of
hemoglobin. PCT Application WO 92/08790 to Cargill
discloses the use of polyamide polymers bonded to a
linker group which is bonded to a protein.
Many potentially therapeutic proteins have
undesirable characteristics such as short half life in

2~92~
WO95117886 PCT~S94/14821
-- 5
vivo, poor solubility, vulnerability to enzymatic
degradation in vivo, or immunogenicity. The
polyamides of the present invention when coupled to
such proteins overcome these disadvantages.
Summarv of the Invention
The present invention is water-soluble,
substantially non;mml1nogenic polyamides having number
average molecular weights of about 300 to about 20,000
grams per mole; where the amide repeat units are
comprised of: (i) a water-soluble organic acid subunit
having at least one carboxylate group and fifteen or
fewer atoms separating the amide functionalities in
the polyamide; covalently linked as an amide to (ii) a
water-soluble organic amine subunit having at least
one primary amino group and fifteen or fewer atoms
separating the amide functionalities in the polyamide.
In other words, the polyamide of the present
invention is a water-soluble, substantially
non;mml~nogenic polyamide selected rom the formulas I,
II, and III:
I Y-A-X-Y
II Z-B-X-Z
III Y-X-Z
(i) where terminus Y is OH or a carboxyl coupling
group; (ii) where terminus Z is H or a coupling group
attached to an amine group; and (iii) where X is a
polyamide selected from: (B-A)n, (A-B)n, (B-A')n or
(A'-B)n and branched polyamides formed by linking
(B-A)n, (A-B)n (B-A')n or (A'-B)n to a central
polyacid, polyamine or polyamino acidi and (iv) where
A is a ~,~-di-acid; B is a ~,~-diamine; A' is a ~,~-
amino acid having the formula
YlOC-CH2-CH-S-(CH2)m-Xl-CH2-CH2-X2-(CH2)m-S-CHCH2-COY
R3 R3

WO9S/17886 2 ~ 6 - PCT~S94/14821 -
where Y1 has the formula
- OC-(CH2)p-NH-
where p is from 1 to about 4, m is from about 2 to
about 4, X1 and X2 are independently a heteroatom
selected from the group consisting of O other than
carboxyl or carbonyl O, S, P or tertiary N, and R3 is
a lower alkyl having from about 1 to about 2 carbon
atoms; n is the number of amide repeat units in the
polyamide; and (v) where the acid subunits of the
amide repeat units are (a) organic acids having
fifteen or fewer atoms in the chain and having one or
more heteroatoms selected from the group consisting of
O other than carboxyl or carbonyl O, S, P or tertiary
N present as substituents on or atoms in the chain, or
(b) two or more of such organic acids bridged by
water-soluble organic diamines; and (vi) where the
amine subunits of the amide repeat units are organic,
water-soluble amines having at least one primary amine
group and having fifteen or fewer atoms in the chain
and having one or more heteroatoms selected from the
group consisting of O other than carboxyl or carbonyl
O, S, P or tertiary N present as substituents on or
atoms in the chain; and (vii) where n is from 2 to
about 100.
The present invention includes one or more such
polyamides used to cross-link, conjugate, decorate or
polymerize proteins, antibodies, haptens,
polypeptides, polynucleotides or other biological
substrates. The cross-linked, conjugated, polymerized
or decorated product is water-soluble, substantially
non;mmnnogenic and retains all or a useful portion of
the physiological activity of the substrate.

WO95/17886 21 5 6 ~ 2~ PCT~S94114821
Brief 3escri~tisn of the Drawinas
Figure 1 shows the polycondensation of ethylene
glycol bis(methoxycarbonylmethyl ether) and 1,4-
diaminobutane.
Figure 2 shows the reaction conditions, product
characteristics, and yield of the reactions shown in
Figure 1.
Figure 3 shows the experimental data, including
oxygen binding function of diaspirin cross-linked
hemoglobin polymerized and decorated with PAS-2400.
Figure 4 shows the size exclusion
chromatographic profiles of diaspirin cross-linked
hemoglobin polymerized and decorated with PAS-2400.
Figure 5 shows the reverse phase HPLC profiles
of diaspirin cross-linked hemoglobin polymerized and
decorated with PAS-2400.
Figure 6 depicts the components of polyamide
synthesis.
Figure 7 depicts the synthesis of BMDAB (a
polyamide component).
Figure 8 depicts the polycondensation of BMDAB
with diamine, to form a polyamide.
Figure 9 depicts the synthesis of polyamide
activated esters PAS-3037 and PAS-4200.
Figure 10 depicts the synthesis of maleimide-
capped polyamide, designated PAM-4080.
Figure 11 depicts the size exclusion profiles
following polymerization of diaspirin cross-linked
hemoglobin with PAS-3037.
Figure 12 depicts size exclusion chromatography
- following polymerization of diaspirin cross-linked
hemoglobin with PAS-4200.
- Figure 13 depicts the reverse phase HPLC
profiles following polymerization of diaspirin cross-
linked hemoglobin with PAS-4200.

WO95/17886 1 ~ ~9 ~1 PCT~S94/14821 -
Figure 14 depicts the size exclusion
chromatography profiles following polymerization of
diaspirin cross-linked hemoglobin with PAM-4080.
Figure 15 depicts reverse phase HPLC following
polymerization of diaspirin cross-linked hemoglobin
with PAM-4080.
Figure 16 shows the experimental data foIlowing
polymerization of diaspirin cross-linked hemoglobin
with PAS-3070.
Figure 17 shows the experimental data following
polymerization of diaspirin cross-linked hemoglobin
with PAS-4080.
Figure 18 shows the experimental data following
polymerization of diaspirin cross-linked hemoglobin
with PAM-4080.
Detailed Descri~tion of the Invention
The polyamides of the present invention are
substantially non-immunogenic, water-soluble
polyamides having number average molecular weights of
about 300 to about 20,000 grams per mole. The amide
repeat units of these polyamides are composed of a
water-soluble organic acid subunit having at least one
carboxylate group and fifteen or fewer atoms
separating the amide functionalities in the polymer,
covalently linked as an amide to a water-soluble
organic amine subunit having at least one primary
amino group and fifteen or fewer atoms separating the
amide functionalities in the polymer. These
polyamides may be employed directly or after
activation, for the purposes of cross-linking,
conjugating, polymerizing and/or decorating biological
substrates such as proteins, polypeptides, antibodies,
haptens, carbohydrates or polynucleotides to give
products which are water-soluble, substantially
non1mm~]nogenic, and which retain all or a useful

~ WO95/17886 2 ~ ~ ~ 9 2 ~1 PCT~S94/14821
_ g _
portion of the substrate's physiological activity.
They may also be used to attach substrates to
detection agents or solid matrices.
The term "substantially non-immunogenic"
S indicates that the polyamide does not elicit a humoral
or cell-mediated immune response, either in vivo or in
vitro.
The term "water-soluble" indicates that the
polyamide has a solubility in water that exceeds 500
mg per 100 mL. The term also indicates that the
polyamide does not act as a detergent and does not
form aggregates such as micelles in water.
The term "activation" means converting a group
located on a polyamide terminus to a more reactive
coupling group.
The polyamide may be linear or branched.
The term "substrate" means the molecule to which
the polyamide of the present invention is bound.
Substrates include but are not limited to proteins
such as enzymes, growth factors, antibodies or blood
proteins; polynucleotides such as complementary DNA
fragments; steroids and hormones; immunoconjugates;
carbohydrates; and conjugates of any of these
substrates. The substrate may also be a solid support
or bead. Substrates include molecules having
therapeutically useful biological activity.
As used herein, a substrate is said to be
"decorated" when multiple polyamides are bound to the
substrate by one terminus of each polyamide and all
other termini of the polyamide are not bound to a
different substrate molecule.
The water-soluble polyamides of this invention
may be prepared by methods known in the art. Known
methods for the preparation of polyamides are
incorporated here by reference as useful methods for
the preparation of the polyamides of the present

WO95/17886 PCT~S94/14821 -
2~ 24 - 10 -
invention. N. Ogata et al., Polymer Journal, Volume
5, pages 186ff (1973) and N. Ogata and Y. Hosoda,
Journal Polymer Science, Polymer Lett. Ed., Volume 12,
pages 355ff (1974) describe the polycondensation of
diesters activated by ether or hydroxyl groups with
diamines. N. Ogata et al., Journal Polymer Science,
Polymer Chemistry Ed., Volume 14, pages 783ff (1976),
N. Ogata et al., Polymer Journal, Volume 11, pages 827
to 833 (1979), and H. Sato, et al., Makromol
Chemistry, Volume 182, page 755 to 762 (1981) describe
the polycondensation of activated diesters containing
ether, thioether or hydroxyl groups with diamines. D.
Kieley and T-H. Lin have also described
polyhydroxypolyamides and a process for making same,
U.S. Patent No. 4,833,230. N. Ogata and Y. Hosoda,
Journal Polymer Science, Polymer Chemistry Ed., Volume
18, pages 1159 to 1162 (1978) describe the synthesis
of water-soluble polyamides by polycondensation in
solutions of ethylene glycol dimethoxycarbonylmethyl
ether and hexamethylene diamine.
The acid subunits of the amide repeat units are
selected from the group of organic acids having
fifteen or fewer atoms in the chain and having
heteroatoms (O, S, P, N) present either as
substituents on or atoms in the chain. Alternatively,
the acid subunits of the amide repeat units may
consist of two or more such organic acids joined to
bridging water-soluble, organic diamines. The amine
subunits of the amide repeat units are selected from
among the group of organic amines having fifteen or
fewer atoms in the chain and having heteroatoms (O, S,
P, N) present as substituents on or atoms in the
chain. Polyamides of similar and/or dissimilar
structure may be linked by a central polyacid,
polyamine or polyamino acid to form branched, water-
soluble polyamides.

21~692~
WO95/17886 PCT~S94/14821
In one embodiment of the present invention, X is
a polyamide selected from (B-A) n~ (A-B) n~ and branched
~ polyamides formed by linking (B-A) n or (A-B) n to a
central polyacid, polyamine or poly(amino acid). In
this embodiment, the acid subunits of the polyamide
repeat units are two or more organic acids each of
said organic acids having fifteen or fewer atoms in
the chain and having one or more heteroatoms selected
from the group consisting of O other than carboxyl or
carbonyl 0, S, P or tertiary N present as substituents
on or atoms in the chain bridged by a water-soluble
diamine having the formula -NH-Rl-NH- where Rl is a
substituted or unsubstituted aliphatic chain having
from about 4 to about 5 carbon atoms. In a preferred
embodiment of the present invention, the water-soluble
diamine having the formula -NH-Rl-NH- is l,4-
diaminobutane.
In another embodiment of the present invention
where X is a branched or unbranched polyamide selected
from (B-A) n or (A-B~ n~ the acid subunits o~ the amide
repeat units are organic acids having the formula
-OC-CH-CH2-S-(CH2)m-Xl-CH2-CH2-X2-(CH2)m-S-CH2CH-CO-
R2 R2
where m is from about 2 to about 4, Xl and X2 are
independently a heteroatom selected from the group
consisting of O other than carboxyl or carbonyl O, S,
P or tertiary N, and R2 is H or acetamide. In these
polyamides, the sulfur group is located in a position
~ to each of the terminal carboxyl groups. In a
preferred embodiment of the present invention, Xl and
X2 are both 0. In a more preferred embodiment of the
present invention, Xl and X2 are both O and m is 2.
Compounds in accordance with this aspect of the
present invention have several advantageous features
when used with substrates having therapeutic

WO95/17886 ~ 2 ~ PCT~S94/14821 -
- 12 -
biological activity. These compounds are less
reactive which permits greater control following
activation to m; n i mi ze hydrolysis. This, in turn,
optimizes coupling with protein substrates. These
polyamides are also effective oxygen radical
quenchers.
In yet another embodiment of the present
invention when X is a branched or unbranched polyamide
having the formula (B-A')n or (A'-B)n, the acid
subunits of the amide repeat units are organic acids
having the formula
Yloc-cH2-cH-s-(cH2)m-xl-cH2-cH2-x2-(cH2)m-s-cHcH
R3 R3
where Yl has the formula
- OC-(CH2)p-NH-
where p is from l to about 4, m is from about 2 to
about 4, Xl and X2 are independently a heteroatom
selected from the group consisting of O other than
carboxyl or carbonyl O, S, P or tertiary N, and R3 is
a lower alkyl having from about l to about 2 carbon
atoms. In a preferred embodiment of the present
invention, Xl and X2 are both O. In a more preferred
embodiment of the present invention, Xl and X2 are
both O, m is 2, and R3 is methyl.
In accordance with the present invention, A' is
an a, ~-di-acid having the formula Yl-A-Yl, where Y
has the formula -OC(CH2)p-NH- where A is an a, ~-di-
acid as described herein above.
Any of the known coupling chemistries may be
used to activate polyamides of this invention to
decorate, link, polymerize and/or conjugate
substrates. Many examples of such coupling
chemistries are given in "Chemistry of Protein
Conjugation and Cross-linking," ~. Wong, CRC Press,
Inc. (l99l) which is incorporated by reference herein.

21~6924
WO95117886 ' PCT~S94114821
- 13 -
Such chemistries include reacting the polyamides
with bi- or poly- functional protein reagents such as
~ dialdehydes, N-hydroxysuccinimide esters,
functionalized acetals, bis-maleimides, bifunctional
imino esters, diepoxides, and dicarboxylic acid
chlorides. The choice of coupling chemistries will
depend upon the substrate molecule being cross-linked,
conjugated, polymerized and/or decorated. The
coupling chemistry should be selected so that it does
not alter the biological or chemical activity of the
substrate molecule.
Generally, to decorate a substrate molecule,
between about 4 and 50 moles of polyamide should be
used per mole of substrate. Larger substrate
molecules will require a greater proportion of
polyamide. To primarily conjugate, cross-link or
polymerize a substrate without decorating requires the
knowledge of a chemist skilled in the art as to the
chemistries of the coupling agents, the reactive
groups on the particular substrate, the size of the
substrate, the size of the polyamide, concentration of
the substrate, and general reaction parameters.
As will be appreciated readily by those of skill
in the art, substrates such as amino acids, peptides,
proteins, nucleotides, polynucleotides, pharmaceutic
agents, and diagnostic agents have functional groups
which may be covalently bound to the pendant
functional groups of the polyamide backbone and
functionalized derivatives thereof. Those of ordinary
skill in the art having the benefit of this disclosure
will comprehend the synthetic approaches that may be
employed to covalently join the polyamide and the
substrate. The order of reaction is not important.
The pendant functional group(s~ of the polyamide may
be activated appropriately, if so required, and then
attached to the substrate. Likewise, the substrate

WO95/17886 2 ~ ~ G g ~ 4 PCT~S94/14821 -
- 14 -
may be activated appropriately, if necessary, and then
attached to the polyamide.
For example, amino, hydroxy, carbonyl, carboxyl,
or thiol substituents are commonly found as part of
the structure of amino acid, peptide, protein,
nucleotide, polynucleotide, and diagnostic agent
compounds. Moreover, the polyamide may be synthesized
to incorporate reactive termini such as these
substituents. The substrate may be joined to the
polyamide by chemistries such as those cited below, or
by other chemistries such as those disclosed in
Bodanszky and Bodanszky, "The Practice of Peptide
Synthesis," Springer-Verlag, New York, (1984);
Lundblad, "Chemical Reagents for Protein
Modification," CRC Press, Boca Raton, Florida, (l99l);
Mosbach "Methods in Enzymology, Volume XLIV,
Immobilized Enzymes," Academic Press, New York,
(1976); or U~lm~nn and Peyman, "Antisense
Oligonucleotides: A New Therapeutic Principle,"
Chemical Reviews, Volume 90, No. 4, pages 543 to 585
(June l990).
For example, biotin is recognized as a
diagnostic probe that is selectively retained by
complexation with avidin. Biotin contains a carboxyl
group that may be activated as a succinimidyl ester
and attached to a polyamide having a amino terminus.
Either prior to or following covalent bonding to
biotin, the other terminus of the polyamide may be
covalently bonded to a peptide, protein or other
biochemical agent. Under these conditions, the
polyamide serves as a spacer group that concurrently
maintains or increases the aqueous solubility of the
product. The biochemical agent is thereby labeled
with a diagnostic probe that is positioned at the end
of the polyamide spacer to facilitate interaction with
avidin.

21~6~24
WO95/17886 PCT~S94/14821
- 15 -
Similarly, deferoxamine is a pharmaceutic agent
that is used therapeutically as an antidote to iron
~ poisoning. The duration of therapeutic action of
deferoxamine is short, because it is rapidly excreted
via the kidney. It has been recognized that if
deferoxamine is conjugated to a larger molecular
weight entity such as a dextran or albumin, it will be
retained in the vascular circulation for longer
periods of time. In accordance with the present
invention, a polyamide may be used as a spacer group
that concurrently maintains or increases the aqueous
solubility of the product. One terminus of the
polyamide may be converted to a carbonyl functional
group and attached to the amino substituent of
deferoxamine by reductive amination, and the other
terminus of the polyamide may be converted to an
activated ester (e.g. a succinimidyl ester) and
attached to albumin. Through this conjugation, the
duration of vascular circulation of conjugated
de~eroxamine is lengthened and the agent retains its
chelating abilities.
All the components of the polyamides of the
present invention are selected so as to preserve
water-solubility. They are water-soluble, hydrophilic
over the entire chain length. The length o~ the
polyamide is chosen to facilitate interaction between
the substrate and the polyamide. The cross-linking of
a large substrate will require a longer polyamide
since it will minimi ze steric interactions between two
large substrate molecules.
An immunogenic substrate should generally be
highly decorated and should have relatively long chain
polyamides.
In reacting the polyamides of the present
invention to biologically active substrates, such as
enzymes, care is taken to avoid destroying the

WOgS/17886 2 ~ ~ ~ 92~:; PCT~S94/14821 ~
- 16 -
activity of the substrate. One skilled in the art
will understand that varying the degree of decoration
and/or polymerization will allow one to prepare a
product having a useful biological activity.
The polyamides of the present invention are not
polymers of a-amino acids, so they are not subject to
enzymatic hydrolysis.
In addition, the polyamides of the present
invention may be used to render substrates soluble in
organic solvents such as methanol, ethanol or
acetonitryl.
The polyamides of the present invention may be
used as polymerization agents. In one example
described fully below, the t~rm;n; of a polyamide have
been modified as maleimide groups, suitable for
reaction with thiol substituents of proteins.
Bis(maleimide) polyamide was employed to polymerize
human hemoglobin via the cysteine-~93 thiol residues
of that protein. Similarly, in another example below,
the t~rm;ni of a polyamide were converted to
bis(succinimidyl) esters or bisaldehydes, suitable for
reaction with amino substituents of proteins. Both
the bis(succinimidyl) polyamide and the bisaldehyde
polyamide have been employed to polymerize human
hemoglobin via the a-aminO groups of lysine residues
of the protein.
In another embodiment the polyamides of the
present invention may be used to link probes (e.g.,
fluorescent, radioactive, etc.) to a substrate to be
detected.
In the examples that follow, we use the
following nomenclature for our polyamides: since the
backbone is a polyamide, the letters PA will apply;
the letter designating the coupling group will follow,
M for maleimide and S for N-hydroxysuccinimide; a
hyphen will separate the alphabetic code from the

21~92~
WO95/17886 PCT~S94/14821
- 17 -
approximate molecular weight. In a preferred
embodiment of the present invention the polyamide is
~ bis(maleimidoacyl) polyamide. In a more preferred
embodiment of the present invention the polyamide is
bis(maleimidoglycyl) polyamide. Thus, a polyamide
identified as PAM-3800 is a polyamide bis(maleimide)
having a molecular weight of about 3800 Daltons.
DESIGN AND SYNTHESIS OF POLYAMIDES
10In the following examples the polyamide
condensation products are characterized in three ways.
Size exclusion chromatographic (SEC) analysis is
completed using a SuperoseTM 12 column and 50 mM
phosphate, pH 6.5, mobile phase delivered at a flow
rate of 0.~ mL~min. with detection at 220 nm; this
analysis confirms that polymerized products were
formed and permits approximation of molecular weights
and the range of molecular weights of the components
in the product mixture. Thin-layer chromatographic
(TLC) analysis permits separation and characterization
of end-group functionality of the components in the
product mixture. The structure of each component is
assigned on the basis of relative migration (Rf) and
reactivity toward ninhydrin spray reagent. Under the
TLC conditions, polyamides with diester end-groups
have the largest Rf, followed by components with mono-
ester/mono-amine end-groups, and di-amine end-groups,
respectively. Only components having an amine end-
group are reactive toward ninhydrin. The structure of
the mono- and di-esters is confirmed by base-catalyzed
hydrolysis and TLC of the resulting products; under
these conditions esters are hydrolyzed to acids and
the Rf of the material decreases. Finally, the
molecular weight is estimated by amino end-group
analysis using fluorescamine. Precisely and
accurately weighed polyamide samples are dissolved in

2 ~
WO95/17886 ~ ' PCT~S94/14821 -
- 18 -
methanol/phosphate buffer, derivatized by adding
fluorescamine dissolved in acetone, and then analyzed
by flow injection with a HPLC system equipped with a
fluorescence detector. Equivalent weights are
determined by comparison of responses for standard
solutions of diaminohexane/PEG/ethyl acetate in
methanol/phosphate buffer. Equivalent weights are
converted to number average molecular weights based on
the average number of amines per molecule.
Alternatively, the NMR spectrum can be used to
estimate the number average molecular weights of the
polyamides, as follows: the first step is to divide
the structure of the polyamide into end groups and
repeating units. Then the molecular weight of each
part is calculated. Next one identifies unique
components in each part and correlates the
corresponding NMR resonance with that component.
Polyamides have a number of well-resolved resonances
that can be correlated with specific functional
groups. For example, the two pairs of two hydrogens
on the succinate group in PAS-4200 give rise to
(triplet) resonances at about 2.53 and 2.92 ppm having
integrals of 2.197 and 2.605 units, respectively.
Similarly, the internal methylene groups of the
butanediamine residue give rise to a broad resonance
at 1.5 ppm having an integrated area of 16.034 units.
There are two succinate residues in the end
groups of the polyamide derivative: therefore, the
resonance at about 2.53 ppm and the one at 2.92 ppm
each results from four hydrogens. The average area
response is ~2.197 + 2.605)/2 or 2.401 units per four
hydrogens on each succinate. Similarly, the two
internal methylene groups of the butanediamine residue
in the repeating unit contain four hydrogens. The
observation that the integrated area of the latter
resonance (16.034 units) is larger than that of the

~ WO95/17886 2 ~ ~ 6 9 ~ 4 PCT~S94/14821
- 19 -
four-hydrogen response for either type of succinate
hydrogen indicates that there are multiple
~ butanediamine residues within the repeat units in the
polymer. We can estimate the value of the multiple by
~ 5 ratioing the integrated areas: 16.034/2.401 or
approximately 7. Thus, there are seven repeat groups
in the polyamide. The molecular weight of the
polyamide is the sum of the molecular weights of each
of the end groups (416.44 and 198.14, respectively)
and the multiple seven times the molecular weight of
the repeat unit (7 x 504.57 or 3532). The sum is
4146.57 or about 4200 Da. This value was also
obtained independently by end-group analysis of the
polyamide bisamine precursor of PAS-4200 using
fluorescamine.
SYnthesis of PAS-2400
~xam~le l(a)
PolYcondensation of Ethvlene ~1YCO1
bis(methoxycarbonvlmethvl) and 1,4-diaminobutane.
Ethylene glycol bis(methoxycarbonylmethyl) ether
(EDE), which has an ether group as a substituent ~ to
each ester group, was condensed with 1,4-diaminobutane
(DAB) to produce polyamides. See Figure 1. Two poly-
condensation methods were used: the solution methodand the melt method.
In general, the polycondensations were completed
as follows. For the solution method, EDE and DAB in
the desired molar ratio were dissolved in methanol,
and the solution was heated at 30C for seventy two
hours or at 65C for twenty four hours. The solvent
was evaporated and the residue was treated with
acetone and repeatedly evaporated to remove residual
methanol. Trituration of the residue with acetone
afforded a solid. In the melt method, a mixture of
EDE and DAB was heated at 120C under vacuum with

W O 95/17886 - PCTrUS94114821
- 2 0 -
magnetic stirring to remove methanol. After one to
two hours the mixture was dissolved in methanol. The
solution was evaporated to dryness and the residue was
triturated with acetone to give polyamide product.
Analysis of the reaction mixtures by SEC
confirmed that polymerized products were formed. TLC
analysis (stationary phase: silica gel; eluent: 2-
propanol / NH40H / H20, 7:1:2, by volume) of the
product showed three spots having Rf values of 0.1,
0.4, and 0.7, respectively. The structures of the
corresponding polyamides were assigned on the basis of
reactivity toward ninhydrin and base as a, ~-
diaminopolyamide (designated I in the figure), ~-
amino-~-esterpolyamide (designated II), and ~,~-
diesterpolyamide (designated III), respectively(Figure 1). In addition, product III is ninhydrin-
negative while products I and II are ninhydrin-
positive, indicating the products has at least one
primary amine group. Finally, products II and III can
be hydrolyzed with dilute aqueous NaOH, whereas I
cannot, indicating products II and III contain at
least one ester group.
The yield of these products depends on the molar
ratio of DAB to EDE. A molar ratio of one gives I as
the major product. A molar ratio of DAB/EDE greater
than one gives polyamide II as the major product. In
contrast, III became the major product with a molar
ratio of DAB/EDE of less than one. The results of
polycondensation of EDE and DAB are summarized in
Figure 2. The experimental data indicate that ~-
amino-~-ester polyamide II having a number average
molecular weight (MW) of about 2,400 Dalton is best
produced by the solution method at 30C. ~,~-
Diamino-polyamide I having a MW in the range of 1,300
to 1,500 Dalton could be prepared either by the
solution or the melt method employing a DAB/EDE molar

WO95/17886 2 15 6 9 2`4 . PCT~S94114821
- 21 -
ratio of l.3 to l.5. ~,~-Diester-polyamides III were
obtained in good yield by the melt method with
equimolar EDE and DAB. Because DAB is a volatile
compound, DAB is gradually removed from the reaction
mixture when the melt method is utilized, leaving EDE
in large excess. Conseauently III is obtained as the
major product.
Exam~le l(b)
Conversion of ~olyamide III to an activated
cross-linkina aaent.
Crude diester III (Figure l), obtained by
condensation of EDE and DAB, was hydrolyzed with
dilute sodium hydroxide to the corresponding di-acid.
After hydrolysis, the reaction mixture was treated
with AG50W-X8 resin (BioRad) to remove sodium ion and
by-products I and II. The di-acid was obtained in a
pure state as judged by TLC. The di-acid was treated
with dicyclohexylcarbodiimide (DCC) and N-
hydroxysuccinimide (NHS) in chloroform to convert it
to the corresponding polyamide bis(N-
hydroxysuccinimide ester) (designated PAS-2400).
Exam~le 2
PolYmerization of Hemoalobin with PAS-2400.
A typical polymerization of diaspirin cross-
linked hemoglobin (designated DCLHb) with PAS-2400 was
completed as follows. DCLHb was prepared according to
the method described in U.S. Patent No. 5,128,452. A
solution of DCLHb in O.l M HEPES of about pH 7 to 8
was deoxygenated by successive vacuum / nitrogen
cycles for one and a half hours at room temperature.
PAS-2400 was dissolved in deoxygenated water, and the
solution was added immediately to the DCLHb solution.
The reaction mixture was stirred at room temperature
under nitrogen, and the reaction was monitored by size

~ ~ } ~ ~ i
WO95/17886 2 ~ 2 4 PCT~S94/14821 -
- 22 -
exclusion chromatography using TSK-G4000SW brand and
TSK-G3000SW brand columns connected in series with
mobile phase consisting of 2-propranol/50mM phosphate
buffer, pH 6.5 (l:9, v/v), delivered at a flow rate of
l mL/minute detection at 280 nm. The latter method
demonstrated that the polymerization was completed in
less than thirty minutes and that polymerization was
accompanied by decoration. The solution was cooled to
5C and a solution of l M NAC (N-acetyl-L-cysteine)
(molar ratio of NAC/Hb about 5:l) was added. The
solution was stirred at 5C under nitrogen overnight
and then dialyzed against lactated Ringer's solution
to give the final product. Experimental data are
summarized in Figures 3, 4, and 5. Note: In the
figures NHS-PA 6 is an alternate designation for PAS-
2400.
The data indicate the following. The yield of
oligomer is increased with increasing ratios of PAS-
2400 to DCLHb. SEC elution times of DCLHb monomer
decrease with increasing molar ratios of PAS-2400,
indicating that PAS-2400 decorates DCLHb.
Polymerization is fast; it was complete in less than
thirty minutes. However, competitive hydrolysis of
the polymerization agent is also fast. As the
solution pH is increased, higher yields of high
molecular weight polymers are obtained. For example,
five eguivalents of PAS-2400 give 7%, 17%, and gel,
respectively, of high molecular weight polymers at
values of pH of 7.0, 7.5, and 8.0, respectively.
P50 values and n values of DCLHb polymerized
with PAS-2400 are in the range of 29 to 33 mm Hg and
l.8 to 2.l, respectively. P50 is the oxygen partial
pressure at which hemoglobin is half saturated while
the "n" value is a measure of the cooperativity of
oxygen binding. The Pso of human hemoglobin in red
blood cells is about 28. Thus, the excellent oxygen-

WO95/17886 ~ 1 5 6 9 2 4 PCT~S94/14821
binding function of DCLHb is maintained in thesepolymers. RP-HPLC analysis (Figure 5) indicates than
~ both a~ - and ~-chains are modified. However, ~-
chains apparently are more extensively modified than
are ~-ch~;n~.
Thus, PAS-2400 can be used to produce decorated,
polymerized DCLHb. The short reaction time (thirty
minutes) is favorable for large-scale synthesis. Two
to four equivalents of PAS-2400 at pH 7.0 are suitable
for polymerization. The hemoglobin maintains its
biological activity, i.e., oxygen binding and, as
described below is nonimml7nogenic.
Exam~le 3
Methods for the Synthesis of Lonaer PolYamides.
Longer polyamides are obtained if the lengths of
the component acid and amine are increased, i.e.,
polymerization with adipic acid (six carbons) or 1,6-
hexanediamine (six carbons) yields longer polymers
than does polymerization with succinic acid (four
carbons) or 1,4-butanediamine (four carbons). However,
increases in chain length using hydrocarbon components
would reduce the aqueous solubility of the protein.
With this in mind, we synthesized polyamides
from diester EDE and each of two longer diamines:
ethylene glycol bis(3-aminopropyl) ether (EGBE; MW
176) and diethylene glycol bis(3-aminopropyl) ether
(DGBE; MW 220). See Figure 6. The SEC retention
times of each of the polyamides suggested these
products had higher molecular weights, but the
products were waxy and had low melting points.
Purification of such products by crystallization is
extremely difficult.
To mi n i mi ze these shortcomings we combined three
concepts to select appropriate activated esters for
the synthesis of longer polyamides. First, we

WO95/17886 2 ~ 5 ~ 3 2 ~1 PCT~S94ti4821 ~
- 24 -
identified components that are di-acids having ~-ether
links; these di-acids are easily converted to
activated diesters. Our initial di-acid of choice was
diglycolic acid. Second, we converted one end of this
di-acid to an amide by reacting two equivalents of di-
acid with one equivalent of diamine; this generated a
new and longer di-acid that we can use as a component
for longer polyamides. Our first di-acid of choice
was 1,4-(carboxymethoxyacetamido) butane, which we
used as the activated methyl diester BMDAB (MW 348).
Insertion of the methylene (hydrocarbon) groups
reduced the flexibility of the molecule sufficiently
to render it a crystalline solid and retention of the
ether link preserved the solubility in water. Third,
we increased the length of the diamine component in a
way that will maintain water solubility; thus, we used
ethylene glycol bis(3-aminopropyl) ether (EGBE) and
diethylene glycol bis(3-aminopropyl) ether (DGBE) as
the diamine components in polyamide synthesis.
The activated diester building block, BMDAB, was
obtained in two steps (Figure 7). DAB (1,4-diaminobu-
tane) was allowed to react with two equivalents of
glycolic anhydride in N,N-dimethylformamide (DMF) to
give an almost quantitative yield of BCDAB [1,4-
bis(carboxymethoxyacetamido) butane]. The latter was
esterified in methanol in the presence of aqueous HCl
or HCl in dioxane solution. The advantage of using
HCl in solution is the ease of carrying out the
reaction, especially in a large scale synthesis, and
the observation that an exact amount of HCl can be
employed to avoid the formation of by-products.
Gaseous HCl was tried, but a by-product was detected
in the product mixture.
In contrast to the polycondensation of EDE and
DAB by the solution method, which gives the ~-
methylester-~-aminopolyamide as the major product

~ 56924
WO95/17886 PCT~S94/14821
- 25 -
when a molar ratio of EDE to DAB of 1 was used, the
polycondensation of equimolar ~uantities of BMDAB and
EGBE or DGBE or of molar ratios of BMDAB to DGBE of
greater than 1 (Figure 8) gave mixtures containing
substantial amounts of three products: an a-ester-~-
amine (reactive to ninhydrin; hydrolyzed by base); an
a,~-diamine (reactive to ninhydrin); and a a,~-
diester (unreactive to ninhydrin). Unfortunately, thepresence of large amounts of other products made the
purification of a desired product tedious. However,
we found that the use of an excess of diamine (e.g., a
molar ratio of diamine to BMDAB of 1.3) gave the a,~-
bisamine polyamide as the major product containingonly very small amounts of the monoamine by-product.
This latter procedure is therefore preferred to
produce the polyamide backbone of the polymerization
reagents.
Exam~le 4
Conversion to Activated Polvmerization A~ents:
Pol~amide bis(N-hvdroxvsuccinimide) ester.
The first attempt to synthesize polyamide bis(N-
hydroxysuccinimide) ester (designated PAS-3070) was a
three step synthesis from BMDAB and EGBE (Figure 9).
First, EGBE and BMDAB in a molar ratio of 1.3 to 1.0
were condensed by the solution method using methanol
as solvent at 65C for 24 hours to give a slightly
orange solution. The product could be decolorized by
adding decolorizing charcoal (NoritTM A) to the
solution, filtering, and evaporating to dryness. A
white product (Figure 9, 2a), having a MW of 2700, was
isolated by crystallization from methanol-acetone.
The product was not stable and turned yellow during
storage. Second, conversion of the white product to
the corresponding bis(2-carboxyethylcarbonyl)
polyamide (Figure 9, 3a) was completed by reaction of

WO95/17886 ~ S 69~4 PCT~S94114821
(2a) with succinic anhydride in DMF (a small amount of
methanol was added to enhance to the solubility of
2a). The reaction produced a yellow product mixture
containing bis(2-carboxyethylcarbonyl)polyamide (3a)
as the major product and two minor by-products: the
methyl ester of (2a) and an unknown by-product
containing a free amino group as indicated by TLC.
Therefore, the mixture was treated with sodium
hydroxide to convert the methyl ester to bis(2-
carboxyethylcarbonyl)polyamide, (3a) and then stirredwith cation exchange resin (AG50W-X8) to absorb
polyamide amine by-product. After removal of the
resin by filtration, the filtrate, which contained a
single product as indicated by TLC, was concentrated.
Pure product bis(2-carboxyethylcarbonyl)polyamide (3a)
was obtained by crystallization from methanol/acetone.
Third, conversion of the pure product to the activated
diester (4a) (designated PAS-3070) was accomplished by
treatment with N-hydroxysuccinimide in the presence of
dicyclohexylcarbodiimide (DCC) in DMF. The polyamide
bis(succinimide ester) was soluble in water but the
coupling groups were slowly hydrolyzed. Therefore
when dissolved in water the activated polymerization
agent was used without delay.
The synthesis described above has several
drawbacks. For example, the isolated white product,
(2a) is not stable; it is oxidized during storage to
an unknown yellow product which could not be removed
readily by crystallization. Furthermore,
recrystallization of bis(2-carboxyethylcarbonyl)
polyamide (3a) in methanolJacetone converts some of
the di-acid to the corresponding methyl ester(s). To
avoid these drawbacks, our preferred synthetic
strategy is as follows. First, steps 1 and 2 (Figure
9) were carried out as an integrated process in which
product 3 in Figure 9, obtained from Norit A

WO95/17886 21 5 ~ 9 2 4 PCT~S94/14821
- 27 -
treatment, is not isolated but is allowed to react
;mme~; ately with succinic anhydride to mask the amino
~ groups which tend to be oxidized to colored product.
Such a "one-pot" synthesis increased the yield of 3 in
Figure 9, because all crude 2 is used for the second
step instead of the 50-60~ of isolated product 2 that
was converted in the method described above. In
addition, the use of methanol as crystallization
solvent for 3 was excluded to avoid the formation of
methyl ester of 3.
Exam~le 5
Svnt~esis of PAS-4200.
PA5-4200 (Figure 9, 4b) was prepared using the
inteqrated approach above.
~xam~le 6
Conversion to Activated Polvmerization Aqents:
Polyamide Bis(maleimido~ro~ionate).
Synthesis o~ polyamide bis(maleimidopropionate)
(designated PAM-4080) was completed as a "one-pot"
two-step synthesis which is summarized in Figure lO.
Crude polyamide bisamine (Figure lO, 2B) was obtained
by heating BMDAB and DGBE in refluxing methanol for 24
hours followed by decolorizing with Norit A and was
immediately treated with N-hydroxysuccinimido-3-
maleimidopropionate (SMP, Figure lO, 5) to give 6b.
The first attempt to carry out the latter step by
mixing 2b and 5 in a molar ratio of l:2 in chloroform
in the presence of triethylamine gave a higher
molecular weight product. Since SMP is a bifunctional
cross-linking reagent, it could polymerize 2b under
these conditions. Increasing the SMP/2b molar ratio
to 4.5 and the slow addition of 2b in chloroform
containing triethylamine to a solution of SMP in
chloroform eliminated the polymerization of 2b by SMP.

WO95/17886 2 ~ ~ g ~ 2 il PCT~S94/14821 -
- 28 -
Thus, crude product 6b, obtained by this procedure,
was mixed with cation-exchange resin (AG50W-X8) to
remove unreacted polyamide amine and the purified
polyamide polymerization agent 6b (designated PAM-
4080) was obtained by crystallization of crude productfrom methanol/acetone. In contract to PAS
derivatives, PAM derivatives are stable in water.
Examle 7
Polvmerization of DCLHb with Polvamide
Pol~merization Reaqents.
A typical polymerization of DCLHb with PAS
derivatives of the type described in Examples 4 and 5
above or PAM derivatives of the type described in
Example 6 above was completed as follows. A solution
of DCLHb (lO g/dL for PAS and 20 g/dL for PAM) was
deoxygenated by successive vacuum/nitrogen cycles for
l.5 hours at room temperature. Polyamide reagent in
deoxygenated water was added immediately to the DCLHb
solution. The reaction mixture was stirred at room
temperature under nitrogen and the course of the
reaction was followed by SEC. Polymerization was
completed within 2 to 3 hours for PAS derivatives and
overnight for PAM derivatives. The reaction mixture
was cooled to 5C; the solution pH was adjusted to
8.0 with l molar HEPES pH 9.0 and a solution of l M N-
acetyl-L-cysteine, pH 9.0 (molar ratio NAC/DCLHb of 5)
was added. The solution was stirred at 5C under
nitrogen overnight and then dialyzed against lactated
Ringer's solution to give the ~inal product.
Experimental results are summarized in Figures ll to
18.
Polymerization of DCLHb with the activated ester
PAS derivatives may be summarized as follows. (a) The
degree of polymerization and the yield of oligomers
increased with the molar ratio of PAS used. (b)

~¦ WO 95/17886 2 15 6 9 2 4 PCT/US94/14821
-- 29 --
Concurrent with increases in the molar ratio of PAS
used, the elution time of DCLHb monomer decreased,
suggesting that decoration of DCLHb by PAS is
occurring. (c) Polymerization was fast; it was
5 complete within 2 to 3 hours. (d) The SEC profiles of
polymeric product obtained by employing five
equivalents of PAS-3070 and three equivalents of PAS-
4200 are very similar. This also demonstrates that
longer reagents facilitate polymerization of DCLHb.
(e) Four equivalents of PAS-3070 and 2.5 equivalents
of PAS-4200 gave the best product mixtures under these
experimental conditions. (f) PAS derivatives do not
affect the P50 values of DCLHb: the P50 values of
polymerized product are in the range of 29 to 36 mm
15 Hg. (g) RP-HPLC analyses (Figure 13) indicate that
both $- and o~a-chains are modified by PAS, but oca--
ch~; n.s to a lesser extent than $-chains.
DCLHb polymerization by PAM derivatives may also
be summarized. (a) As was true of the PAS
20 derivatives, the yield o~ oligomer increased with the
number of molar equivalents of PAM used. (b) Elution
times of the monomer decreased with the number of
molar equivalents of PAM used; thus, decoration of
DCLHb by PAM is likely. (c) Two equivalents of PAM
25 give the best product mixtures. (d) RP-HPLC profiles
(Figure 15) suggest that reagent reacted specifically.
A specific $'-peak, which could be a modified $-peak,
was detected at all ratios of PAM tested. Specific
reaction with the subunits was also supported by the
30 decrease in titrable thiol residues. Reagent PAM is
expected to bind specifically to cysteine-$93
residues, and about 65% and 90% of thiol groups are
modified when 1 and 2 ec[uivalents of PAM are used,
respectively. (e) The binding of PAM to the cysteine
35 residue results in a decrease in Pso values of the
polymerized products to 18 to 20 mm Hg. (f) ococ-

WO 95/17886 2 ~ 5 ~ ~ 2 ~ PCT/US94/14821 1~
- 30 -
~h~;ns are also modified, but much less extensively
than the ~-~h~, n.~ .
BIQLOGICAL TESTING
In examples 8 through 12 we quenched the
polyamide PAM-4200 by reaction with N-acetyl-L-
cysteine and tested a sterile, non-pyrogenic solution
of the polyamide (PAM-4080) in Ringer's lactate
solution. The polyamide concentration was 5 g/dL of
solution. The pH of the polyamide solution was
adjusted to physiologic values. The osmolality of the
solution was within the physiologic range. The
concentration of the polyamide was selected to exceed
projected use levels by at least an order of
magnitude.
Exam~le 8
In vitro exposure of isolated mammalian cells.
CCL 1 NCTC 929 (clone of strain L cells, mouse
connective tissue) were cultured aseptically in
sterile media until confluency. The L-929 cell
concentration was adjusted to about 1. 3 x 105
cells/mL, and aliquots were transferred to wells of a
tissue culture plate. The plates were covered and
incubated for approximately twenty four hours. Then
the culture medium was aspirated from each well and
aliquots of the test article solution and dilutions
having PAM-4080 concentrations of 2.5 and 1 g/dL,
respectively, were added to duplicate wells of the
prepared plates. After incubation of the plates for
approximately forty eight hours, the wells were
stained with 2% crystal violet stain. The toxicity
was rated on a scale from 0 to 4+, where a rating of 0
corresponded to the presence of discrete
intracytoplasmic granules and the absence of cell
lysis and a rating of 4+ corresponded to nearly

2 ~ 2 4
WO9S/17886 - 31 - PCT~S94/14821
complete destruction of the cell layers. At the
highest concentration, a moderate toxicity rating of
2+ applied. At the two lower concentrations, a
toxicity rating of 0 applied, i.e., the polyamide
caused no adverse biological response.
No toxicity was observed at the lower doses and
moderate toxicity was observed at the highest dose.
Accordingly, the polyamides of the present invention
are expected to be nontoxic when ~mini stered as
conjugates of therapeutically useful substrates.
.~xam~le 9
Acute toxicitv testin~ in rodents. Doses of 500
or 1500 mg of quenched PAM-4080/kg body weight were
infused at a rate of 1 mL/kg/min. into the tail vein
of male, Sprague-Dawley rats. Each test group
consisted of six ~nim~l s; six undosed animals served
as controls. All ~nim~ls were monitored for seventy
two hours for signs of overt toxicity; none were
observed. The ~nim~l s were sacrificed. No evidence
of toxicity was seen at the time of necropsy. Tissues
from the liver, kidney, lung were subjected to
histopathological analysis. No adverse histopathology
findings were noted.
F.xample 10
Com~atibilitY with human ervthrocytes. To
determine the biocompatibility of PAM-4080 with human
erythrocytes, the stock polyamide solution was diluted
five-fold with lactated Ringer's solution. A volume
of this preparation was mixed with an equal volume of
heparinized human blood, vortexed gently, and placed
in an incubator (37C) overnight. After an incubation
period of 16 hours, a 100-~L ali~uot of the
supernatant was removed from the top of the test
sample; care was taken not to disturb the sedimented

WO95/17886 ~b~2~:-` PCT~S94/14821 -
- 32 -
red cells below. The aliquot was mixed with 5000 ~L
of SEC mobile phase, filtered through a 0.2 ~m pore-
size filter and injected on a SuperoseTM 12 column for
SEC analysis for native hemoglobin. The experimental
data indicated that less than 0.1% hemolysis had
occurred. This amount of hemolysis was considered
negligible.
Ex~mnle ll
Com~atibilitv with human ~eri~heral blood
mononuclear cells (monocvtes). The potential of PAM-
4080 for causing white blood cell activation was
evaluated. The stock polyamide solution was diluted
five-fold with lactated Ringer's solution. A volume
of this preparation was mixed with an equal volume of
peripheral blood mononuclear cell preparation and
vortexed gently. An aliquot of this test preparation
was removed and diluted with trypan blue. Toxicity was
determined by microscopic detection of cells that
could no longer exclude the dye. Percent viability
was measured by a ratio of live/dead cells. PAM-4080
caused no decrease in cell viability. The remaining
test preparation was placed in an incubator (37C)
overnight. After an incubation period of 16 hours,
cytokines were analyzed by pipetting an aliquot of the
sample into microtiter wells and quantitation by
ELISA. The concentrations of Tumor Necrosis Factor
(TNF), Interleukin-l~ and Interleukin-6 determined
were no different from those found by exposure of
human monocytes to lactated Ringer's solution. Thus,
PAM-4080 is compatible with human monocytes.

WO95/17886 21~ 6 9~24 PCT~S94114821
Exam~le 12
Com~atibilitv of PA-DCLHb with human ~eri~heral
blood mononuclear cells (monocYtes). The potential of
polyamide decorated and polymerized DCLHb (PA-DCLHb)
to induce cytokine production by human monocytes was
evaluated. Lactated Ringer's solution was used as the
control article. The test articles were seven
different preparations of PA-DCLHb in lactated
Ringer's solution. Test and control solutions were
made by mixing a volume of each test and control
article with an equal volume of peripheral blood
mononuclear cell preparation. After incubation of
each resulting test and control solution at 37C for
about 16 hours, an aliquot of each sample was
transferred into separate wells of microtiter plates
and the concentrations of Tumor Necrosis Factor (TNF),
Interleukin-l~ and Interleukin-6 were quantitated by
ELISA. The concentrations of each cytokine determined
are shown in the Table below. The experimental data
indicate that induction of TNF, IL-l, and IL-6 are low
and in some cases comparable to Ringers. In summary,
PA-DCLHb appears to be very compatible with human
monocytes.

WO95/17886 2 ~ ~ ~ 9 ~ i PCT~S94/14821 -
- 34 -
CYTOKINE CONCENTRATION, ng~mL
TEST ARTICLE
TNF IL-1~ IL-6
Lactated Ringer's 0.044 i 0.006 i c
Solution (Control) 0.023 0.005 Detection
Limit (DL)
PA-DCLHb 0.154 i 0.080 i < DL
(Preparation 1) 0.027 0.040
PA-DCLHb 0.249 i 0.073 i < DL
(Preparation 2) 0.081 0.028
PA-DCLHb 0.161 i 0.054 i < DL
(Preparation 3) 0.055 0.011
PA-DCLHb 0.173 i 0.058 i < DL
(Preparation 4) 0.043 0.012
PA-DCLHb 0.139 i 0.049 + < DL
(Preparation 5) 0.027 0.009
PA-DCLHb 0.159 + 0.042 + < DL
(Preparation 6) 0.012 0.026
PA-DCLHb 0.144 i 0.048 i < DL
(Preparation 7) 0.050 0.028
Fxample 13
Cvtokine Induction bY PAS-DCLHb.
Samples of six PAS-DCLHb product mixtures were
submitted for cytokine testing. The products selected
were prepared by polymerization of 3 g/dL DCLHb in
0.lM HEPES buffer at pH 7Ø Each sample was diluted
to a DCLHb concentration of about 1 g/dL and passed
through an END-XTM endotoxin-removing filter. The
filtrate was tested for cytokine induction using the
method described in Example 12.
TEST SAMPLE TNF- , IL-1~, IL-6,
ng/mL nq/mL ng/mL
Lactated Ringer's 0.044 0.006 0.044
Solution
PAS-DCLHb (:::) 0.1,4 0.080 0.008
PAS-DCLH~ (~ ) o.^~c 0.07 0
PAS-DCLH~ ( ::) 0.:~: 0.0 L_ O . 001
PAS-DCLH~ (~::) 0. 'l- 0.0 0.0:7
PAS-DCLH~ (7::) O. 3r~ O.OL r` O.O 6
PAS-DCLH~ (~::) 0. 5~ 0.0~2 0.0_~
PAS-DCLH~ ( 0:1) 0. 4~ 0.0~8 0.O

WO95/17886 2 1 5 6 9 2 4 PCT~S94/14821
PAS-DCLHb (3:l) is the least decorated and polymerized
product mixture, whereas PAS-DCLHb (lO:l) is the most
~ extensively decorated and polymerized product mixture.
The extent of decoration and polymerization increases
with the molar ratio of PAS employed. However, the
PAS-Hb products, irrespective of the extent of
decoration or polymerization, all yield low TNF- and
IL-l responses. None of the samples show an IL-6
response.
Exam~le 14
Svnthesis of Thio-containinq ~olvamide
As a part of this work, five experimental
parameters were studied: (l) the molar ratio of
monomers (diester and diamine); the solvent; the
reaction temperature; the order of mixing; and the
method of product isolation. The objective of these
studies was the identification of reaction conditions
that would reproducibly maximize the yield of polymers
having specific molecular weights. The molar ratios
of diester to diamine were varied from l:l to l:l.4.
The solvents studied included chloroform and DMF. The
basic catalyst was varied from 2,6-lutidine (a mild
base) to triethylamine (a strong base) to no added
catalyst. The temperature ranged from -50 C to room
temperature (approximately 23 C). The diester was
added to the diamine or vice versa. The rate of
mixing was controlled by varying the rate of stirring
of the solution, by varying the rate of addition, or
by controlling the solubility of one of the components
(the diester). It was found that product isolation
was facilitated and polymer yield was improved by
conversion of a polyamide bis(diamine) to a polyamide
bis(succinate).
As a result of this study, it was found that
water-soluble polyamides having molecular weights of

~ ` ~
WO95/17886 " ~ " PCT~S94/14821 -
approximately 2800 and 5700 could be prepared
selectively by polycondensation of TBB (3,3_-
thiodipropionate active ester) with DGBE [diethylene
glycol bis(3-aminopropyl ether); the diamine] under
the conditions described in the Table. In both cases,
the polycondensation reaction re~uired 4-24 hours,
depending on scale, and the yield of polyamide
approximated or exceeded 50%.
Table. Optimum conditions for the
polycondensation of TBB and DGBE to yield water-
soluble polyamides having specific molecular weights

2~ 692~
WO95/17886 ; PCT~S94/14821
- 37 -
Svnthesis of DiPhenvl (2,3-dihvdro-2-thioxo-3-
benzoxazoYl)~hos~honate (DDTBP). To a solution of 2-
mercaptobenzoxazole (251.6 g, 1.66 mole) and
triethylamine (191.4 g, 1.89 mol) in 1 liter of
toluene was added dropwise at room temperature with
stirring a solution of diphenyl chlorophosphate (505.1
g, 1.88 mole) in 400 mL toluene. After the addition
was completed, the solution was stirred for an
additional 1.5 hours, during which time a precipitate
formed. Thin-layer chromatographic analysis (TLC;
silica gel; eluent: ethyl ether/hexane, 1:1, v/v) of
the reaction mixture indicated complete reaction. The
solid was removed by filtration and washed with
toluene (3 x 100 mL). The combined filtrates were
evaporated to a residual oil. The oil was taken up in
500 mL of chloroform and the resulting solution was
heated at reflux for about an hour. The solution was
passed through a pad of silica gel. The pad was
washed with chloroform until TLC analysis of the
~iltrate indicated that the product was completely
eluted from the silica gel. The chloroform solution
was evaporated, leaving as a solid the desired
product. Hexane (1.5 L) was added, and the solid was
collected by filtration and dried under vacuum. The
product, 582.0 g of a white solid identified by the
acronym DDTBP, was characterized by a melting point of
80-83 C (uncorrected) and the following NMR
resonances.
1H-NMR (CDCl3): 7.21 (m, 4), 7.35 (m, 9) and
7.91 (m, 1) ppm.
13C-NMR (CDC13): 109.9 (s, C9), 114.8 (s, C8),
120.1-120.3 (overlapping singlets, C2), 125.5 (s, C7),
126.3 (s, C10), 129.9-130.0, overlapping singlets,
C3), 147.5 (s, C5), 149.5 (s, C1), and 180.2 (s, C11)
ppm.

~ .
WO95/17886 6 9 2 1 PCT~S94/14821
31P-NMR (CDCl3): -16.17 ppm (with a minor
impurity at -8.9 ppm)
SYnthesis of N,N~-(3 3~-
thiodi~ro~ionvl)bis(benzoxazoline-2-thione) (TBB). To
a stirred solution of 3,3'-thiodipropionic acid (891
- mg, 5 mmol) and triethylamine (1.5 mL) in chloroform
(20 mL) was added DDTBP (4.22 g, 11 mmol). The
solution was stirred at room temperature and monitored
by TLC (silica gel; eluent: ethyl ether/hexane, 1:1,
v/v). With time, the product began to precipitate from
solution. Methanol (100 mL) was added to facilitate
complete precipitation of the product, which was
isolated by filtration and air-dried. About 1.7 g (76%
of theoretical) of the product, N,N'-(3,3'-
thiodipropionyl)bis(benzoxazoline-2-thione) or TBB,
was obtained as a white solid having a melting point
(uncorrected) of 147-149-C.
1H-NMR (CDCl3): 3.07 (t, H1), 3.89 (t, H2),
7.27, 7.32, 7.37 (H4, Hs, H6) and 8.08 (t, H3) ppm.
13C-NMR (CDCl3): 22.65 (C2), 39.91 (C1), 109.58,
116.4 (C4, Cs), 125.5, 126.17 (C3, C6), 129.6 (Cg),
146.41 (C7), 172.22 (C1o), and 178.46 (Cg) ppm.
Svnthesis of PATS-2800, a water-soluble ~olYamide
havina a number averaae molecular weiaht of about 2800
Daltons. A slurry of TBB (5.34 g, 12 mmol) in 60 mL
chloroform was stirred at a moderate rate while being
cooled to 0 in an external ice-bath as a solution of
DGBE (3.17 g, 1.4 mmol, a 1.2:1 molar ratio relative
to TBB) in 12 mL chloroform was added. The resulting
mixture was stirred for 15 minutes at 0-5 C and then
the ice-bath was removed. Stirring continued at
ambient temperatures overnight. Thin-layer
chromatographic analysis indicated that a polyamide
bis(amine) had formed.

215~924
WO95/17886 PCT~S94114821
- 39 -
A solution of succinic anhydride (850 mg) in 3 mL
DMF was added gradually to the polymer solution until
thin-layer chromatographic analysis indicated all the
polyamide bis(amine) had been converted to the
corresponding polyamide bis(carboxylic acid).
Volatile materials were removed from the reaction
mixture by evaporation to dryness under vacuum, and
the residue was taken up in 20 mL DMF. Then volatile
materials were removed by evaporation under vacuum.
The residue was dissolved in a second, 20-mL portion
of DMF. Ethyl acetate was added slowly to the
resulting solution; precipitation of the product
appeared to be complete after the addition of about
300 mL of ethyl acetate. The product was collected by
filtration, washed with ethyl acetate and dried under
high vacuum. About 2.27 g (47% of theoretical) of
water-soluble polyamide (identified by the acronym
PATS) having a number average molecular weight (based
on NMR analysis) of 2800 Daltons and a size exclusion
chromatographic (SEC) retention time of 38.8 minutes
was obtained.
SYnthesis of PATSS-2800, an activated, water-
soluble ~olvamide havina a number avera~e molecular
wei~ht of about 2800 Daltons. N,N'-Disuccinimidyl
carbonate (1.18 g, 6.42 mmol~ was added to a stirred
solution of PATS-2800 (1.96 g, 0.7 mmol) in a mixture
of 10 mL DMF and 30 mL chloroform. The mixture was
stirred at room temperature and the reaction was
followed by TLC (silica gel; eluent:
CHCl3/MeOH/NH4OH/H2O, 10:3:0.2:0.2, by volume). After
3,5 hours, the reaction mixture was filtered to remove
insoluble materials. The filtrate was concentrated
under vacuum to dryness. The residue was dissolved in
15 mL DMF and the solution was diluted slowly with
ethyl acetate (300 mL) to precipitate the product, a
bis(N-oxysuccinimidyl) polyamidedicarboxylate which is

WO95/17886 ~ l 56~ PCT~S94/14821
- 40 -
identified by the acronym PATSS-2800. The product
(1.52 g, 77% yield) was isolated by filtration and
dried under high vacuum.
1H-NMR (CDCl3): 1.71 (m, Hb); 2.39 (m, Hg); 2.52
(t, Hs or Ht); 2.76 (m); 2.78 (Hf, Hh); 2.90 (t, Hs or
Ht); 3.27 (m, Ha); 3.47-3.57 (m, Hc, Hd); 6.78 (CONH)
ppm.
13C-NMR (CDCl3): 25.5 (Cr); 26.7; 30.3 (Cs, Ct);
27.7 (Cg); 28.9 (Cb); 36.4 (Cf); 37.5 (Ca); 69.5;
69.9; 70.3 (Cc, Cd); 168.1, 169.0; 169.8; and 171.2
(C=O) ppm. (NOTE: Not all resonances have been
correlated with specific structural sites.)
Absorbance ratioing indicated the number average
molecular weight of the polymer was about 2800
Daltons.
Svnthesis of PATS-5700, a watex-soluble olYamide
havina a number averaae molecular weiaht of about S700
Daltons. To a slurry of TBB (5.34 g, 12 mmol) in 60
mL DMF that was being stirred at a moderately slow
rate was added a solution of DGBE (3.44 g, 1.68 mmol,
a 1.3:1 molar ratio relative to TBB) in 6 mL DMF. The
solid gradually dissolved and the mixture became warm.
The resulting solution was stirred at room temperature
overnight. A precipitate gradually separated from the
solution. The precipitate (2.44 g; about 47% of
theoretical) was isolated by filtration and was found
to have a SEC retention time of about 35.8 minutes and
was characterized by TLC as a polyamide monoamine
monocarboxylate having an average molecular weight of
about 5700 Daltons. The filtrate was found to contain
a polymer (1.14 g; 22% of theoretical) having a SEC
retention time of about 38.6 minutes (i.e., the
polyamide bis(amine) had a number average molecular
weight of about 2800 Daltons).

WO95/17886 ~ ~ ~ 6 ~ ~ 4 PCT~S94/14821
- 41 -
SYnthesis of PATSS-5700, an activated water-
soluble ~olvamide havina a number averaae molecular
weiaht of about 2800 Daltons. The above precipitate
(2.44 g) was dissolved in 60 mL chloroform and about 3
mL methanol was added to ensure complete dissolution.
Then volatile materials were removed under vacuum to
ensure complete removal of residual DMF. The solid
was dissolved in a second, 60-mL portion of
chloroform. To the resulting solution was added
succinic anhydride (108 mg) in 1 mL DMF. Reaction was
monitored by TLC. Stirring was continued until all
polyamide amine had been converted to polyamide
bis(carboxylic acid). To this solution was added
successively triethylamine (about 400 ~L), 10 mL DMF
and 730 mg N,N'-disuccinimidyl carbonate. The
mixture was stirred at room temperature and the
reaction was followed by TLC. After 2.5 hours,
volatile materials were removed under vacuum and the
residual solid was dissolved in 10 mL DMF. The
solution was slowly diluted with ethyl acetate (100
mL) to precipitate the product, bis(N-oxysuccinimidyl)
polyamidedicarboxylate, identified by the acronym
PATSS-5700. About 2.36 g of product was isolated by
filtration and dried under vacuum.
13C-NMR (CDC13): 25.5 (Cr); 26.8; 30.4 (Cs, Ct);
27.7 (Cg); 29.0 (Cb); 36.5 (Cf); 37.6 (Ca); 69.6;
70.0; 70.4 (Cc, Cd); 168.2, 169.1; 169.9; and 171.3
(C=O) ppm. (NOTE: Not all resonances have been
correlated with specific structural sites.)
Absorbance ratioing indicated the number average
molecular weight of the polymer was about 5700
Daltons.
As will be readily appreciated, numerous
variations and combinations of the features set forth
above can be utilized without departing from the
present invention as set forth in the claims. All

~ 4' PCT~S94/14821 -
Wo95/17886
- 42 -
such variations are intended to be included in the
scope of the following claims.