Activated polymers and conjugates thereof

Macromolecular species such as enzymes, proteins, drugs, and solid supports which contain reactive carbonyl groups or groups which are readily converted to reactive carbonyl groups are modified by reaction with a compound of the formula ##STR1## in which R.sup.1 is H, C.sub.1 -C.sub.20 alkyl, phenyl, (C.sub.1 -C.sub.10 alkyl)-substituted phenyl, or phenyl-substituted C.sub.1 -C.sub.10 alkyl; R.sup.2 is C.sub.2 -C.sub.5 alkylene or (C.sub.1 -C.sub.4 alkoxy)-substituted C.sub.2 -C.sub.5 alkylene; R.sup.3 is ##STR2## R.sup.4 is H or C.sub.1 -C.sub.4 alkyl; n is at least 5; and m is zero or 1.

BACKGROUND OF THE INVENTION 
This invention relates to the modification of macromolecular species such 
as proteins, injectable compounds in general and solid supports by 
conjugation with polymers. This invention further relates to activated 
polymers for use in such conjugation. 
Polymers, such as polyethylene glycol, polypropylene glycol and analogs 
have been coupled to enzymes, drugs, polypeptides and poly(amino acids) of 
various kinds to modify their immunological and biochemical properties. 
Among the desirable changes achieved in this manner are the reduction or 
elimination of immunogenicity, induction of tolerance, an increased 
resistance to proteolytic degradation in the serum, a controlled or 
gradual release (in the case of a drug) arising from a slow rate of 
enzyme-induced dissociation from the polymer, an alteration in the tissue 
distribution (in the case of a drug, for instance) by varying its water 
solubility or its molecular size, a change in surface characteristics (in 
the case of a solid support), and, in the case of an enzyme, for instance, 
a change in kinetics, a shift in the pH and temperature optima, and 
altered reactivity to substrate. 
The goal of conjugation in most cases is to modify certain properties while 
retaining others (such as drug or enzyme activity for instance) 
substantially unchanged. In many cases, this selectivity varies with the 
locus of the conjugation site on the macromolecule, and the number of 
sites on a single macromolecule which take part in the conjugation. Most 
conjugations to date have been made at amino moieties on drugs or enzymes. 
In many cases, this presents a problem due to the wide distribution of 
reactive amino groups on the molecule. The result is often a lack of 
control over the degree or rate of conjugation, and a substantial 
interference with the desired activity of the macromolecule. In an 
immunoglobulin for instance, where the antigen-binding portions bear a 
large number of amino groups, conjugation at these groups can cause 
interference with the immunoglobulin s ability to bind antigen. 
SUMMARY OF THE INVENTION 
Novel compositions and methods are provided herein for the modification of 
macromolecular species by conjugation with polymers at reactive carbonyl 
groups on the species. Derivatized polymers for use in accordance with the 
invention are those of the formula 
##STR3## 
in which R.sup.1 is H or a protecting group which will prevent a coupling 
reaction from occurring at the end of the polymer to which it is attached; 
examples of such protecting groups are C.sub.1 -C.sub.20 alkyl, phenyl, 
(C.sub.1 -C.sub.10 alkyl)-substituted phenyl, or phenyl-substituted 
C.sub.1 -C.sub.10 alkyl; 
R.sup.2 is C.sub.2 -C.sub.5 alkylene or (C.sub.1 -C.sub.4 
alkoxy)-substituted C.sub.2 -C.sub.5 alkylene; 
R.sup.3 is 
##STR4## 
R.sup.4 is H or C.sub.1 -C.sub.4 alkyl; n is at least 5: and 
m is zero or 1. 
Further included within the scope of this disclosure are analogs and 
homologs of the above, varying for example in the identity and position of 
the substituents and in other ways readily occurring to those skilled in 
the art, which may be considered as equivalents of the above. 
Throughout this specification, the terms "alkyl, " "alkoxy," "alkylene" and 
the like denote both straight-chain and branched-chain structures. 
Conjugates formed as the reaction product of the macromolecular species 
with these derivatized polymers are those having the formula 
##STR5## 
in which R.sup.1, R.sup.2, R.sup.3, R.sup.4, n and m are as defined above. 
R.sup.5 is either --N.dbd. or --NH--, and A is the residue of the 
macromolecular species. The term "residue" is used herein in its 
conventional sense, meaning that portion of the molecule remaining after 
loss of the atoms removed from it in the process of conjugation. The 
residue may also vary with the type of linkage as represented by the 
R.sup.5 group. Likewise, the choice between the two alternatives for 
R.sup.5 will depend in part upon the reactive group on the macromolecular 
species and its neighboring atoms, and in part upon the conjugation 
reaction. The various possibilities and the appropriate selection among 
them in any given case will be readily apparent to those skilled in the 
art. 
The macromolecular species will be one bearing a carbohydrate or carboxyl 
group available for conjugation. Carbohydrate groups are first converted 
to aldehydes to prepare them for the reaction. This is done by 
conventional means well known to those skilled in the art. Examples 
include the use of galactose oxidase or periodate under mild conditions. 
Within the scope of these formulas, certain embodiments are preferred. For 
example R.sup.1 is preferably H or C.sub.1 -C.sub.10 alkyl, with C.sub.1 
-C.sub.5 alkyl more preferred and methyl particularly preferred. Likewise, 
R.sup.2 is preferably C.sub.2 -C.sub.5 alkylene, with --CH.sub.2 -CH.sub.2 
-- (ethylene) and --CH(CH.sub.3)--CH.sub.2 -- (isopropylene) more 
preferred and ethylene particularly preferred, Similarly, R.sup.4 is 
preferably H or methyl, with H particularly preferred. In further 
preferred embodiments, the group 
##STR6## 
occupies either a meta- or para- position with respect to R.sup.3 on the 
phenyl ring. The integer n is preferably 10 to 1000, and most preferably 
50 to 200. 
The following are examples of activated polymers within the scope of 
Formula I: 
.omega.-Methoxy-.alpha.-(p-aminobenzoyl)polyethylene glycol 
.omega.-Methoxy-.alpha.-(m-aminobenzoyl)polyethylene glycol alternative 
nomenclature: poly(oxy-1,2-ethanediyl), 
.alpha.-(4-aminobenzoyl)-.omega.-methoxy] 
.omega.-Methoxy-.alpha.-(p-aminobenzamido)polyethylene glycol 
.omega.-Methoxy-.alpha.-(o-aminobenzamido)polyethylene glycol 
.omega.-Methoxy-.alpha.-(p-aminophenylaminocarbonyl)polyethylene glycol 
.omega.-Methoxy-.alpha.-(m-aminophenylaminocarbonyl)polyethylene glycol 
.omega.-Methoxy-.alpha.-(p-aminophenylaminocarbonylamino)polyethylene 
glycol 
.omega.-Methoxy-.alpha.-(o-aminophenylaminocarbonylamino)polyethylene 
glycol 
.omega.-Methoxy-.alpha.-(p-hydrazinocarbonyl)benzamidopolyethylene glycol 
.omega.-Methoxy-.alpha.-(p-hydrazinocarbonyl)benzoyl polyethylene glycol 
.omega.-Methoxy-.alpha.-(m-hydrazinocarbonyl)benzoyl polyethylene glycol 
.omega.-Ethoxy-.alpha.-(p-aminobenzoyl)polyethylene glycol 
.omega.-Propoxy-.alpha.-(m-aminobenzoyl)polyethylene glycol 
.omega.-Phenoxy-.alpha.-(p-aminobenzamido)polyethylene glycol 
.omega.-n-Dodecyloxy-.alpha.-(m-aminophenylaminocarbonyl)polyethylene 
glycol 
.omega.-n-Octadecyloxy-.alpha.-(p-aminophenylaminocarbonylamino)polyethylen 
e glycol 
.omega.-p-(n-Octyl)phenoxy-.alpha.-(p-aminobenzoyl)polyethylene glycol 
.omega.-p-(n-Nonyl)phenoxy-.alpha.-(p-aminobenzoyl)polyethylene glycol

DETAILED DESCRIPTION OF THE INVENTION 
The compounds of the present invention are prepared by conventional 
techniques well known among those skilled in the art, selected in 
accordance with the desired substituent groups as listed above. 
Compounds where R.sup.3 terminates in a carbonyl group at the end adjacent 
to the phenyl group may be prepared by reacting a protected polyalkylene 
glycol with an appropriately substituted benzoyl halide. To achieve the 
--NH--C(O)-- linkage the protected polyalkylene glycol will be substituted 
with an amino group at the .alpha.-position. To achieve the --O--C(O)-- 
linkage, the protected polyalkylene glycol can be used directly. 
Compounds where R.sup.3 terminates in an amino group at the end adjacent to 
the phenyl group may be prepared by reacting a protected polyalkylene 
glycol with an appropriately substituted phenylisocyanate. To achieve the 
--NH--C(O)--NH-- linkage, an .alpha.-amino-substituted protected 
polyalkylene glycol is reacted with the phenyl isocyanate. To achieve the 
--O--CH.sub.2 -- linkage, the protected polyalkylene glycol is reacted 
with the appropriately substituted benzyl halide. 
Attachment of the protecting group may be achieved in any conventional 
manner as well. A number of suitable mono-protected polyalkylene glycols 
are commercially available and others are readily prepared by those 
skilled in the art. 
The macromolecular species to which the activated polymers attach may be 
any of a wide variety of species containing a carbonyl group available for 
reaction with an amine or species containing groups which are readily 
convertible to reactive carbonyl groups, such as carbohydrate moieties. 
The macromolecular species may be enzymes, coenzymes, immunoglobulins, 
immunotoxins and other conjugates, proteins and drugs in general, and 
polymeric solid supports such as microbeads and chromatographic support 
media. Enzymes and coenzymes are of particular interest. Examples are 
L-asparaginase, uricase, and catalases. The coupling reaction itself may 
be effected in accordance with conventional procedures for Schiff base 
reactions or amide bond formation. Well known to those skilled in the art, 
The following examples are offered for purposes of illustration, and are 
intended neither to define nor limit the invention in any manner. These 
examples illustrate the preparation of compounds within the scope of the 
present invention. In these examples, the following abbreviations are 
used: 
IR: infrared spectra; values given in cm.sup.-1 ; "s"=strong, "vs"=very 
strong, "w"=weak "sh"=shoulder, 
TLC: thin-layer chromatography: values given in R.sub.f (ratio to the 
front), 
UV/VIS: ultraviolet/visible spectroscopy range; values given in relative 
absorption, 
EXAMPLE 1 
A. Preparation of .omega.-Methoxy-.alpha.-(p-nitrobenzoyloxy)-polyethylene 
Glycol 
This compound was prepared as a precursor to 
.omega.-methoxy-.alpha.-(p-aminobenzoyl)polyethylene glycol, whose 
preparation is described in part B which follows. 
.omega.-Methoxy-.alpha.-hydroxypolyethylene glycol with an average 
molecular weight of approximately 5000 (0.90 g, 0.18 mMol) and 
p-nitrobenzoyl chloride (34 mg, 0.18 mMol) were refluxed in 25 mL of dry 
methylene chloride for 5.5 hours, then stirred for two and one-half days 
at room temperature. Solvent was then removed in vacuo and the residue 
dried in vacuo to give a white solid weighing 0.85 g. IR analysis yielded 
the following: 
IR: 1722 (C.dbd.O), 1530 (NO.sub.2) 
B. Preparation of .omega.-Methoxy-.alpha.-(p-aminobenzoyl)polyethylene 
Glycol 
The product of part A of this Example (0.85 g) was dissolved in warm 
methanol (100 mL) and treated with 10% Pd/C (200 mg, caution: sparks) and 
hydrogenated at 1 atmosphere for three days. The catalyst was filtered off 
and the solvent removed in vacuo to give a white solid weighing 0.72 g. 
The structure was confirmed as that of 
.omega.-methoxy-.alpha.-(p-aminobenzoyloxy)polyethylene glycol by TLC and 
IR as follows: 
TLC (90/10 CH.sub.2 Cl.sub.2 /CH.sub.3 OH): one rather streaky spot at 
R.sub.f 0.22-0.37 (I.sub.2 visible, not strongly UV positive); ninhydrin 
positive (starting material is ninhydrin negative) 
IR: 1695 (C.dbd.O), 1600, no absorbance at 1530 cm.sup.-1 
This material (55 mg) was dialyzed against H.sub.2 O (3500 molecular weight 
cutoff) for 16 days at 4.degree. C. then lyophilized. The UV/VIS spectrum 
and IR were basically unchanged as a result. 
EXAMPLE 2 
A. Preparation of .omega.-Methoxy-.alpha.-(m-nitrobenzamido)-polyethylene 
Glycol 
This compound was prepared as a precursor to 
.omega.-methoxy-.alpha.-(m-aminobenzamido)polyethylene glycol, whose 
preparation is described in part B which follows. 
.omega.-Methoxy-.alpha.-aminopolyethylene glycol with an average molecular 
weight of approximately 5000 (5.0 g, 1 mMol) in 75 mL of dry toluene was 
treated with 2.0 g (10.7 mMol) of m-nitrobenzoyl chloride at 80.degree. C. 
in a constant temperature bath under a nitrogen atmosphere for 18 hours. 
The reaction mixture was concentrated to dryness in vacuo, then dissolved 
in H.sub.2 O and dialyzed against H.sub.2 O using 3500 molecular weight 
cutoff dialysis tubing for five days. A small amount of crude material 
which did not dissolve well in H.sub.2 O remained at all times. The 
aqueous solution was concentrated to dryness in vacuo at 50.degree. C. and 
dried in vacuo at room temperature to give a beige-colored solid weighing 
2.4 g. Its structure was verified as that of the title compound by IR, 
UV/VIS and TLC as follows: 
IR (KBr): 2890 (s), 1730 (C.dbd.O), 1540 (NO.sub.2), 1475, 1370, 1350, 
1285, 1250, 1120 (vs), 975, 850, 
UV/VIS: (CH.sub.3 OH) 254 (0.18), 218 (0.48); (CH.sub.2 Cl.sub.2) 254 
(0.96), 220 (0.12). 
B. Preparation of .omega.-Methoxy-.alpha.-(m-aminobenzamido)-polyethylene 
Glycol 
The product of part A of this Example (2.4 g) was dissolved in 100 mL of 
warm ethanol, cooled, treated with 500 mg of 10% Pd/C and hydrogenated at 
20 psi on a Parr apparatus at room temperature for 16.5 hours. (The 
warming is necessary, with cooling before adding the catalyst. On 
occasion, the starting material will crystallize upon cooling.) The 
catalyst was filtered off and the filtrate evaporated in vacuo to give a 
white solid weighing 1.98 g (82% yield). The structure was confirmed as 
that of the title compound by IR, UV/VIS and TLC as follows: 
IR (KBr): 2880 (s), 1720 (w, C.dbd.O), 1470, 1365, 1345. 1285, 1115 (vs), 
970, 845 (note that the peak at 1540 is no longer present). 
UV/VIS: (CH.sub.3 OH) 316 (0.068), 224 (0.815); (CH.sub.2 Cl.sub.2) 314 
(0.247), 238 (1.09), 
TLC (4/1 C.sub.2 H.sub.5 OH/25% concentrated aqueous NH.sub.3): R.sub.f 
0.6-0.7, one spot, gives blue fluorescence (the nitro starting material 
does not fluoresce); the product gives a weak reaction with ninhydrin. 
EXAMPLE 3 
A. Preparation of 
.omega.-Methoxy-.alpha.-(p-nitrophenylaminocarbonyl)-polyethylene Glycol 
This compound was prepared as a precursor to 
.omega.-methoxy-.alpha.-(m-aminophenylaminocarbonyl)polyethylene glycol, 
whose preparation is described in part B which follows. 
.omega.-Methoxy-.alpha.-hydroxy polyethylene glycol with an average 
molecular weight of approximately 5000 (0.90 g, 0.018 mMol) was dissolved 
in 25 mL of dry CH.sub.2 Cl.sub.2 with two drops of triethylamine, 
p-Nitrophenylisocyanate (30 mg, 0.18 mMol) was then added and the mixture 
refluxed overnight. After 26 hours the solvent was removed in vacuo to 
give a yellow waxy solid weighing 0.78 g, whose structure was verified as 
that of the title compound by IR as follows: 
IR (KBr): 1730 (carbamate), 1600, 1510 (sh), plus the usual absorptions 
associated with polyethylene glycol (1468, 1360, 1245, 1280, 1240, 1110, 
965, 845). Absorption at 2270 was not observed, indicating that no 
unreacted p-nitrophenyl isocyanate remained. 
Preparation of 
.omega.-Methoxy-.alpha.-(p-aminophenylaminocarbonyl)-polyethylene Glycol. 
The product of part A of this Example (0.78 g) was dissolved in 100 mL of a 
warm 1:1 mixture of methanol and tetrahydrofuran, and hydrogenated at 1 
atmosphere with 10% Pd/C for 65 hours. The catalyst was filtered off and 
the solvent removed in vacuo to give a pale brown solid weighing 0.72 g, 
whose structure was confirmed as that of the title compound by TLC and IR 
as follows: 
TLC (90/10 CH.sub.2 Cl.sub.2 /CH.sub.3 OH): R.sub.f 0.4 (faint), 0.23-0.3 
(major) 0.18 (minor). All three spots were ninhydrin positive. 
IR (KBr): 1720, 1525 (w) plus the usual absorptions associated with 
polyethylene glycol (see part A) Note: (1) The peak at 1600 cm.sup.-1 in 
the starting material (1595 in p-nitrophenylisocyanate), assigned to 
NO.sub.2 is gone: and (2) C.dbd.O shows a shift from 1730 to 1720 
cm.sup.-1 similar to examples tabulated by D. Dolphin and A. Wiese, 
Tabulation of Infrared Spectral Data, Wiley & Sons (1977). 
The foregoing is offered primarily for purposes of illustration. It will be 
readily apparent to those skilled in the art that variations and 
modifications of the structures and procedures described herein may be 
made without departing from the spirit and scope of the invention.