Use of acyl phosphate esters in the modification of hemoglobin

A process for the preparation of acyl phosphate esters of the formula I or salts thereof: ##STR1## wherein R and R.sup.1 are the same or different and represent a linear or branched alkyl group having up to 4 carbon atoms, phenyl or benzyl; PA1 n is an integer; PA1 when n is 1, R.sup.2 represents the group ##STR2## wherein R.sup.4 is a linear or branched alkyl, alkenyl or alkynyl, a cyclic alkyl, a cyclic alkenyl, or aryl which may be substituted by alkyl, alkenyl, alkynyl, aryl, arylalkyl, or arylalkenyl, or PA1 when n is at least 2, R.sup.2 represents the group ##STR3## wherein R.sup.4 is as defined above. Novel compounds of the formula I wherein n is at least 2, and modified hemoglobin obtained using the novel compounds are also provided.

BACKGROUND OF THE INVENTION 
The present invention relates to a process for the preparation of acyl 
phosphate esters, novel acyl phosphate esters and their use in the 
preparation of modified proteins. 
Acyl phosphates (mixed anhydrides of a carboxylic acid and phosphoric acid) 
occur as intermediates in many biochemical processes. The acyl phosphates 
may function as activated carboxylic acids thus promoting the transfer of 
the acyl group to an acceptor. For example, in the formation of 
intramitochondrial acyl-coenzyme A in some organisms, acetate is activated 
by acetate kinase through reaction with ATP to produce acetyl phosphate 
prior to transfer of the acetyl group to coenzyme A. (Walsh, C. Enzymatic 
Reaction Mechanisms, W. H. Freeman Co.: New York, 1979 pp. 234-238). Amino 
acids are activated as aminoacyl adenylates prior to their incorporation 
into peptides and proteins on ribosomes (Ibid, pp. 241-248). 
The procedures reported for preparing acyl phosphate esters have many 
limitations. Phenyl acetyl phosphate has been prepared from phenyl 
phosphate and acetic anhydride (Jencks, W. P.; Carriuolo, J., J. Biol. 
Chem, 1959, 234, 1272, 1280; DiSabato, G., Jencks, W. P., J. Am Chem. Soc. 
1961, 83, 4400; Oestreich, C. H., Jones, M. M., Biochemistry 1966, 5, 
2926; Oestreich, C. H., Jones, M. M. Biochemistry 1967, 6, 1515; Briggs, 
P. J. et al, J. Chem. Soc. B. 1970, 1008) but the extension of this method 
to the use of alkyl phosphate in place of phenyl phosphate gives impure, 
uncharacterized products (Jencks, W. P. Carriuolo, J., J. Biol. Chem., 
1959, 234, 1272). Classical methods for the synthesis of 
aminoacyladenylates (coupling of . . . the N-protected amino acid with 
adenylic acid using dicyclohexylcarbodiimide) also gives impure products 
(Berg, P., J. Biol. Chem. 1958, 233, 608). 
Methyl acetyl phosphate has been prepared by reacting dimethyl acetyl 
phosphate with sodium iodide in acetone. (Kluger, R., Tsui, W. C., J. Org. 
Chem. 1980, 45, 2723 and Kluger, R., Tsui W. C., Biochem. and Cell Biol. 
1986, 64, 434). The synthesis of the dimethyl acetyl phosphate involves 
refluxing acetyl chloride and trimethyl phosphate for an extended period 
(Whetstone, R., U.S. Pat. No. 2,648,896; and Chem. Abstr. 1954, 48, 8250; 
and Kluger, R., Wasserstein, P., Biochemistry 1972, 11, 1544). However, 
the present inventors have found that this reaction may not be extended to 
more complex acid chlorides or to diacid chlorides, for example, succinyl 
chloride or fumaryl chloride. A potential alternative route for the 
preparation of dimethyl acetyl phosphate is to react an acyl halide with a 
dimethyl phosphate salt. Acetyl chloride has been reported to react with 
triethylammonium dimethyl phosphate to produce dimethyl acetyl phosphate 
but the material was isolated in an "impure and unstable state" (Avison, 
A.W.D., J. Chem. Soc. 1955, 732). A further limitation of this alternative 
route is that it must rigorously exclude water in order to be effective, 
since the diester is very reactive in water (Kluger, R., Wasserstein, P., 
Biochemistry 172, 11, 1544). 
Monoesters of acyl phosphates have been found to be stable in neutral 
aqueous solutions (Klimman, J. P., Samuel, D. Biochemistry, 1971, 10, 
2126) and have been reported to acetylate amino groups in sites which bind 
anions or proteins (Khger, R., Tsui W. C., J. Org. Chem., 1980, 45, 2723; 
Khger, R., Tsui W. C., Blochem. and Cell Biol. 1986, 64, 434, and Ueno, H. 
et al, Arch. Blochem. Biophys. 1986, 244, 795). Kern et al (Biochemistry, 
1985, 24, 1321) have shown that aminoacyladenylate selectively acylates 
amino residues of an aminoacyl t-RNA synthetase when it is produced by the 
enzyme from an amino acid which is not its normal substrate. Methyl acetyl 
phosphate hah been found to specifically acetylate amino groups 
exclusively in the region of the 2-3-diphosphoglycerate binding site in 
hemoglobin. (Ueno H., et al, Arch. Blochem. Biophys. 1986, 244, 795 and 
Ueno, H. et al, 1989, 26, 12344). 
SUMMARY OF THE INVENTION 
The present inventors have found that acyl phosphate esters can be prepared 
conveniently and in high yield by the reaction of a dialkyl or diphenyl 
phosphate and an acyl halide in the presence of an ether solvent. The 
dialkyl or diphenyl acyl phosphate esters obtained are readily converted 
to their corresponding salts by reaction with an alkali metal halide. The 
inventors have also found that the process is useful in producing novel 
acyl phosphate esters which can be used as cross-linking agents to produce 
modified proteins, for example modified hemoglobins. 
The present invention therefore provides a process for the preparation of 
acyl phosphate esters of the formula I or salts thereof 
##STR4## 
wherein 
R and R.sup.1 are the same or different and represent a linear or branched 
alkyl group having up to 4 carbon atoms, phenyl or benzyl; 
n is an integer; 
when n is 1, R.sup.2 represents the group 
##STR5## 
wherein R.sup.4 is a linear or branched alkyl, alkenyl or alkynyl, a 
cyclic alkyl, a cyclic alkenyl, or aryl which may be substituted by alkyl, 
alkenyl, alkynyl, aryl, arylalkyl or arylalkenyl, or 
when n is at least 2, R.sup.2 represents the group 
##STR6## 
wherein n is at least 2 and R.sup.4 is as defined above; which comprises 
(a) reacting a compound of the formula II 
##STR7## 
wherein R and R.sup.1 are as defined above and R.sup.3 is a counter ion, 
(i) with a compound of the formula III, 
##STR8## 
wherein R.sup.4 is as defined above and R.sup.5 is a leaving group which 
is generally known for esterification reactions, in the presence of a 
polar organic solvent, when a compound of the formula I wherein n is 1 is 
required, or, 
(ii) with a stoichiometric amount of a compound of the formula IV 
##STR9## 
wherein n is at least 2 and R.sup.4 and R.sup.5 are as defined above in 
the presence of a polar organic solvent, when a compound of the formula I 
wherein n is at least 2 is required, and, 
(b) if required, the acyl phosphate esters obtained are converted into the 
salts thereof. 
In accordance with one embodiment of the invention, a process is provided 
for the preparation of acyl phosphate esters of the formula Ia or salts 
thereof: 
##STR10## 
wherein R and R.sup.1 are the same or different, and represent a linear or 
branched alkyl group having up to 4 carbon atoms, phenyl or benzyl; 
R.sup.4 is a linear or branched alkyl, alkenyl, or alkynyl, a cyclic 
alkyl, a cyclic alkenyl, or aryl which may be substituted by alkyl, 
alkenyl, alkynyl, aryl, arylalkyl or arylalkenyl which comprises reacting 
a compound of the formula II 
##STR11## 
wherein R and R.sup.1 are as defined above and R.sup.3 is a counter ion, 
with a compound of the formula III 
##STR12## 
wherein R.sup.4 is as defined above and R.sup.5 is a leaving group which 
is generally know for esterification reactions, in the presence of a polar 
organic solvent and if required, the compounds of the formula Ia obtained 
are converted into salts thereof. In accordance with a second embodiment 
of the invention, a process is provided for the preparation of acyl 
phosphate esters of the formula Ib or salts thereof; 
##STR13## 
wherein R and R.sup.1 are the same or different and represent a linear or 
branched alkyl group having up to 4 carbon atoms, benzyl or phenyl; n is 
an integer being at least 2, and R.sup.4 is a linear or branched alkyl, 
alkenyl or alkynyl, a cyclic alkyl, a cyclic alkenyl or aryl which may be 
substituted by alkyl, alkenyl, alkynyl, aryl, arylalkyl or arylalkenyl 
which comprises reacting a compound of the formula II 
##STR14## 
wherein R and R.sup.1 are as defined above and R.sup.3 is a counter ion, 
with a compound of the formula IV 
##STR15## 
wherein n is at least 2, R.sup.4 is a linear or branched alkyl, alkenyl, 
or leaving group which is generally known for esterification reactions, in 
the presence of a polar organic solvent, and if required, the compounds of 
the formula Ib obtained are converted into salts thereof. 
The present invention also provides novel acyl phosphate esters of the 
formula Ib or salts thereof 
##STR16## 
wherein R and R.sup.1 are the same or different and represent a linear or 
branched alkyl group having up to 4 carbon atoms, benzyl or phenyl, n is 
an integer being at least 2 and R.sup.4 represents a linear or branched 
alkyl, alkenyl, or alkynyl, a cyclic alkyl, a cyclic alkenyl or aryl which 
may be substituted by alkyl, alkenyl, alkynyl, aryl, arylalkyl or 
arylalkenyl. 
The invention also contemplates the use of a novel acyl phosphate ester of 
the formula Ib or salt thereof as defined above, as a cross-linking agent 
in the preparation of a modified hemoglobin. 
The invention further contemplates the modified hemoglobin obtained by 
cross-linking hemoglobin with a novel acyl phosphate ester of the formula 
Ib or a salt thereof as defined above. 
The invention still further contemplates a method of preparing a modified 
hemoglobin comprising: 
(a) cross-linking hemoglobin with an acyl phosphate ester of the formula Ib 
or a salt thereof as defined above; and, 
(b) purifying the resulting hemoglobin.

DETAILED DESCRIPTION OF THE INVENTION 
As mentioned hereinbefore, the present invention provides a process for the 
preparation of acyl phosphate esters of the formula I or salts thereof 
##STR17## 
wherein 
R and R.sup.1 are the same or different and represent a linear or branched 
alkyl group having up to 4 carbon atoms, phenyl or benzyl; 
n is an integer; 
when n is 1, R.sup.2 represents the group 
##STR18## 
wherein R.sup.4 is a linear or branched alkyl, alkenyl, or alkynyl, a 
cyclic alkyl, a cyclic alkenyl or aryl which may be substituted by alkyl, 
alkenyl, alkynyl, aryl, arylalkyl or arylalkenyl, or 
when n is at least 2 R.sup.2 represents the group 
##STR19## 
wherein n is at least 2 and R.sup.4 is as defined above; which comprises 
(a) reacting a compound of the formula II or a salt thereof 
##STR20## 
wherein R and R.sup.1 are as defined above and R.sup.3 is a counter ion, 
(i) with a compound of the formula III, 
##STR21## 
wherein R.sup.4 is as defined above and R.sup.5 is a leaving group which 
is generally known for esterification reactions in the presence of a polar 
organic solvent, when a compound of the formula I wherein n is 1 is 
required, or, 
(ii) with a stoichiometric amount of a compound of the formula IV 
##STR22## 
wherein n is at least 2 and R.sup.4 and R.sup.5 are as defined above, in 
the presence of a polar organic solvent, when a compound of the formula I 
wherein n is at least 2 is required, and, 
(b) if required, the acyl phosphate esters obtained are converted into 
salts thereof. 
In one embodiment of the invention a compound of the formula Ia 
##STR23## 
wherein R, R.sup.1 and R.sup.4 are as defined above, is prepared using 
reaction step (i) set out above. 
In accordance with a second embodiment of the invention a compound of the 
formula Ib 
##STR24## 
wherein n is at least 2 and R, R.sup.1 and R.sup.4 are as defined above, 
is prepared using reaction step (ii) set out above. 
The reaction using reaction step (ii) where n is 2 is generally represented 
by the scheme: 
##STR25## 
In the compounds of the Formula II, R and R.sup.1 may be the same or 
different and represent a linear or branched alkyl group having up to 4 
carbon atoms, phenyl or benzyl, preferably methyl, ethyl or benzyl. 
R.sup.3 in the compound of the Formula II is a counter ion and typically 
is an alkali metal ion such as sodium, lithium, potassium, preferably a 
sodium ion. 
In the compounds of the formula III and IV used in the process of the 
invention, R.sup.4 may be a linear or branched alkyl alkenyl, or alkynyl, 
a cyclic alkyl, a cyclic alkenyl, or aryl which may be substituted by 
alkyl, alkenyl, alkynyl, aryl, arylalkyl or arylalkenyl, preferably, a 
linear alkyl or alkenyl, phenyl, phenylalkyl, phenylalkenyl, 
diphenylalkyl, diphenylalkenyl or napthyl. R.sup.5 in the compounds of the 
formulae III and IV is a leaving group which is generally known for 
esterification reactions. Examples of suitable leaving groups are chloro-, 
bromo-, and iodo-. A general discussion of esterification reactions 
showing typical leaving groups may be found in Morrison, R. T. and R. N. 
Boyd, Organic Chemistry, 3d Ed. at pages 672-674. 
For the reaction according to the invention with a compound of the formula 
III, (i.e. reaction step (i)) a compound of the formula II wherein R and 
R.sup.1 are methyl or benzyl and R.sup.3 is a sodium or lithium ion and a 
compound of the formula III wherein R.sup.5 is a chloro- group are 
preferred. Most preferably, the compound of the formula II is sodium 
dimethyl phosphate and in the compound of the formula II, R.sup.5 is a 
chloro- group. 
For the reaction according to the invention with a compound of the formula 
IV, (i.e. reaction step (ii)) a compound of the formula II wherein R and 
R.sup.1 are methyl and R.sup.3 is a sodium or lithium ion and a compound 
of the formula IV wherein n is 2 to 5, R.sup.4 is alkenyl, phenyl, 
phenylalkyl or diphenylalkenyl and R.sup.5 is a chloro- group are 
preferred. Most preferably, the compound of the formula II is sodium 
dimethyl phosphate and R.sup.5 is a chloro- group. A compound of the 
formula IV wherein n is 2 and R.sup.4 together with the two carbonyl 
groups is fumaryl, isophthalyl, terephthalyl, stilbene 3,3'-dicarboxylic 
acid or stilbene 4,4'-dicarboxylic acid and R.sup.5 is chloro- and a 
compound of the formula IV wherein n is 3 and R.sup.4 together with the 
three carbonyl groups is 1,3,5-benzene tricarboxylic acid are most 
preferred. 
The process according to the invention for the preparation of the compounds 
of the formula I is carried out in the presence of a polar organic 
solvent, in particular an ether solvent. Suitable ether solvents are 
diethyl ether, dioxane or tetrahydrofuran, preferably tetrahydrofuran. 
The reaction temperature for the process can be varied within a fairly wide 
range. In general the process can be carried out within a temperature 
range from -20.degree. C. to 75.degree. C. preferably from 20.degree. C. 
to 50.degree. C. most preferably 20.degree. C. to 25.degree. C. 
The compounds of the formulae II, III or IV used as starting materials in 
the process according to the invention are known from the literature or 
can be prepared by methods known from the literature. For example, sodium 
dimethyl phosphate may be prepared from trimethyl phosphate and sodium 
iodide in acetone. 
Depending on the temperature range, the reaction times are several hours to 
a few days. Typically, at temperatures in the range from 20.degree. C. to 
50.degree. C. the reaction times are between 1 and 72 hours, preferably 48 
hours. The reaction is generally carried out under normal pressure in a 
dry atmosphere. 
For the reaction of the compound of the formula II with the compound of the 
formula IV (i.e. reaction step (ii)) a stoichiometric amount of a compound 
of the formula II is added. For example, in the preparation of a compound 
of the formula I wherein n is 2, two equivalents of the compound of the 
formula II are reacted with the compound of the formula IV. 
If necessary, the products of the process may be purified by 
recrystallization from a suitable solvent or mixture of solvents or by 
column chromatography. The compounds of the formula I may be converted 
into their corresponding salts by reaction with an alkali metal halide, 
for example, sodium iodide or lithium iodide. Preferably, the compounds of 
the formula I are converted into their salts by reaction with a 
stoichiometric amount of sodium iodide in the presence of a solvent such 
as acetone. The conversion to the corresponding salts is generally carried 
out in the temperature range -20.degree. C. to 80.degree. C., preferably 
20.degree. C. to 25.degree. C. and the reaction times are between about 1 
to 12 hours, preferably 12 hours. The invention also provides novel acyl 
phosphate esters of the formula Ib or salts thereof 
##STR26## 
wherein n is an integer being at least 2, preferably 2 to 5, R and R.sup.1 
are the same or different and represent a linear or branched alkyl group 
having up to 4 carbon atoms, phenyl or benzyl, preferably methyl, ethyl or 
benzyl; and R.sup.4 represents a linear or branched alkyl, alkenyl or 
alkynyl, a cyclic alkyl, a cyclic alkenyl, or aryl which may be 
substituted by alkyl, alkenyl, alkynyl, aryl, arylalkyl or arylalkenyl, 
preferably a linear or branched alkyl or alkenyl, phenyl, benzyl, 
phenylalkyl, phenylalkenyl, diphenylalkyl, diphenylalkenyl or napthyl. 
Most preferably, in the compounds of the formula Ib or the salts thereof n 
is 2 to 3, R and R.sup.1 are the same and represent methyl; R.sup.4 
represents a linear alkenyl, phenyl, diphenylalkenyl, benzyl or napthyl. 
Specific examples of the compounds of the formula Ib of the present 
invention include: 
Fumaryl bis(methyl phosphates) represented by the formula set forth below: 
##STR27## 
Isophthalyl bis(methyl phosphates) represented by the formula set out 
below: 
##STR28## 
Terephthalyl bis(methyl phosphates) which is represented by the formula set 
forth below: 
##STR29## 
Stilbene 3,3'dicarboxylic acid bis(methyl phosphates) which is represented 
by the formula set forth below: 
##STR30## 
Stilbene 4,4 '-dicarboxylic acid bis(methyl phosphates) which is 
represented by the formula set forth below: 
##STR31## 
The compounds of the formula Ib may be present in the form of their salts. 
In general these are salts with alkali metal halides, for example sodium 
iodide and lithium iodide. The salts of the compounds of the formula Ib 
with sodium iodide are preferred. 
It will be appreciated that the radials R, R.sup.1, and R.sup.4 may carry 
one or more identical or different substituents. Examples of suitable 
substituents include linear or branched alkyl, halogen, cyano, nitro, 
alkylthio, alkoxy, amino and hydroxy. 
The compounds of the formula Ib and their salts are highly selective and 
react rapidly with amine nucleophiles. I t has been found that when these 
compounds associate with a protein they react with adjacent nucleophiles. 
By virtue of these properties, that is negative charge and electrophilic 
reactivity, the compounds of the formula Ib and their salts are suitable 
for use as site-directed reagents for protein modification. These 
compounds may also be combined with other selective electrophiles to 
provide reagents with further types of specificity. 
The compounds of the formula Ib and their salts are particularly useful as 
cross-linking agents in the preparation of a modified hemoglobin which can 
be used as a blood substitute. Particular compounds of the formula I or 
their salts, may be chosen as cross-linking agents based on calculations 
of their size relative to the known distances from cross-linking amino 
groups in the 2,3-diphosphoglycerate (hereinafter DPG) binding site of 
human hemoglobin. (Perutz, M. F., Nature (London), 1970, 228,726 and Ueno, 
H. et al., Arch. Blochem. Biophys. 1986, 244, p. 795; and Ueno, H. et al., 
J. Biol Chem. 1989, 26, 12344). Table I shows the calculated distances of 
the carboxyamide derivatives of the acyl phosphates resulting from the 
reaction of amino groups on the protein with fumaryl bis(methyl 
phosphate), isophthalyl bis(methyl phosphate); terephthalyl bis(methyl 
phosphate); stilbene 3,3'-dicarboxylic acid bis(methyl phosphate); and, 
stilbene 4,4'-dicarboxylic acid bis(methyl phosphate). 
The compounds of formula Ib or the salts thereof may be reacted with 
liganded (oxy-, carboxy-, carbonmonoxy-, or derivatives) and unliganded 
(deoxy-) hemoglobin. The hemoglobin which may be cross-linked may be 
human, equine, porcine, ovine, bovine, simian or fish hemoglobin. 
The reaction with the compounds of the formula Ib or their salts and 
hemoglobin may occur at a temperature of from about 0.degree. C. to 
50.degree. C. preferably 35.degree. C. The pH of the reaction can vary 
from about 5.5 to about 10, preferably from about 5 to about 8, most 
preferably from about 6.8 to 7.5, with the reaction occurring in a buffer, 
typically 100 mM Bis-Tris buffer. The reaction time may vary but generally 
a sufficient degree of cross-linking occurs within 2 hours. The modified 
hemoglobin may then be separated from the unreacted hemoglobin and other 
impurities using techniques known in the literature. 
The hemoglobin modified using the above described reaction has been found 
to be cross-linked at the DPG binding site. In particular, it has been 
found that in the absence of DPG, the reaction of hemoglobin with fumaryl 
bis(methyl phosphate) produces material that is cross-linked between 
.beta. subunits (val-1 to lys-82) and between the same residues in a 
single .beta. subunit as well as crosslinked between .alpha. subunits 
between lys-99 in each subunit. In the presence of DPG, only the 
.alpha.-cross-link is formed. 
The compounds of the formula I and the salts thereof are highly specific 
for selected groups on the hemoglobin molecule resulting in a high yield 
of the desired modified hemoglobin product. 
The modified hemoglobin as in the present invention may be used as a blood 
substitute or blood plasma expander. The modified hemoglobin may be 
combined with a pharmaceutically acceptable carrier to prepare a 
pharmaceutical composition. Suitable pharmaceutically acceptable carriers 
include physiological saline, Ringer's solution, lactated Ringer's 
solution, Locke-Ringer's solution, Krebs-Ringer's solution, Hartmann's 
balanced saline and heparinized sodium-citrate-citric acid-dextrose 
solution. The modified hemoglobin may also be combined with other plasma 
substitutes and plasma expanders. Examples of plasma substitutes are 
poly(ethyleneoxide), polyvinylpyrrolidone, polyvinyl alcohol and ethylene 
oxidepolypropylene glycol condensates and examples of plasma expanders are 
linear polysaccharides, including dextrans, albumin, other plasma 
proteins, pectins, balanced fluid gelatin and hydroxyethyl starch. The 
modified hemoglobin and pharmaceutical compositions containing the 
modified hemoglobin may be administered using conventional methods. 
The following examples are further provided for illustrative purposes only 
and are in no way intended to limit the scope of the present invention. 
EXAMPLE 1 
a) Dimethyl acetyl phosphate. 
Dimethyl acetyl phosphate has previously been prepared by extended 
refluxing of a solution of acetyl chloride and trimethyl phosphate 
(Whetstone, R., U.S. Pat. No. 2,648,896, 1953; Chem. Abstr. 1954, 48, 
8250i; and Kluger, R., Wasserstein, P., Biochemistry 1972, 11, 1544). The 
reaction was accomplished much more rapidly by using acetyl bromide in 
place of acetyl chloride. However, the method could not be used in general 
since acid bromides are not accessible from more complex acid chlorides. 
The general method involves preparing acetyl dimethyl phosphate by 
dropwise addition of acetyl bromide (4 g, 32 mmol) to trimethyl phosphate 
(10 g, 71 mmol) at 50.degree. C. over a period of fifteen minutes. After 
an additional fifteen minutes, the reaction solution was distilled at 0.30 
torr through a 20 .times.1 m vacuum-jacketed column. An initial low 
boiling fraction of the excess trimethyl phosphate was followed by the 
product at 55.degree.-60.degree. C. Yield, 4.0 g, 75%. Analysis of 
product: proton NMR in CC.sub.4, relative to tetramethylsilane, .delta.2.2 
(3 H, d, J.sub.P-H =1.5 Hz), 3.75 (6 H, d, J.sub.P-H =11 Hz). The spectrum 
was identical to the .sup.1 H spectrum of dimethyl acetyl phosphate. 
(Avison, A. W. D., J. Chem. Soc. 1955, 732). 
b) Dimethyl acetyl phosphate. 
A suspension of sodium dimethyl phosphate (14.8 g, 0.1 mmol, from trimethyl 
phosphate and sodium iodide and acetyl chloride (7.8 g, 0.1 mmol) in dry 
tetrahydrofuran (80 mn) was stirred for two days at room temperature in a 
flask fitted with a drying tube. The reaction solution was filtered and 
the tetrahydrofuran was removed under reduced pressure. The resulting 
colorless liquid was Kugelrohr-distilled (Aldrich Kugelrohr apparatus, 
55.degree.-60.degree. C. 0.30 torr) to give 14.2 g (80%) of dimethyl 
acetyl phosphate. 
c) Methyl acetyl phosphate. Methyl acetyl phosphate was prepared from 
dimethyl acetyl phosphate (Kluger, R, Tsui W.-C., J. Org. Chem. 1980, 45, 
2723). A solution of sodium iodide (2.4 g, 16 mmol) in dry. acetone (15 
mL) was added to a solution of acetyl dimethyl phosphate (2 g, 16 mmol) in 
dry acetone (10 mL). The pale yellow solution stood overnight at room 
temperature. The precipitate was collected by filtration in a 
sintered-glass funnel, washed with dry acetone, followed by methylene 
chloride. The resulting white powder was dried under vacuum and 
recrystallized from hot isopropanol to give 2.2 g (80%) of the sodium salt 
of methyl acetyl phosphate. Analysis of product: .sup.1 H NMR in D.sub.2 
O, relative, to DSS, .delta.2.18 (3 H, J.sub.P-H =1.4 Hz), 3.67 (3 H, d, 
J.sub.P-H =11.6 Hz). The spectrum was identical to the previously reported 
.sup.1 H NMR spectrum of methyl acetyl phosphate. (Ibid, 2723). 
EXAMPLE 2 
##STR32## 
(a) Fumaryl bis(dimethyl phosphate) (1) 
A suspension of sodium dimethyl phosphate (6.9 g, 47 mmol from trimethyl 
phosphate and sodium iodide in acetone) and fumaryl chloride (3.6 g, 24 
mmol) was stirred in dry tetrahydrofuran (60 mL, dried by distillation 
from sodium benzophenone ketyl) under nitrogen at room temperature for two 
days. The solution was then filtered through a sintered glass funnel and 
the solvent was removed, leaving the product as a solid. Recrystallization 
from benzene and ether yielded the product as white flakes (4.7 g, 61%, mp 
76.degree.-77.degree. C.). Analysis of product: IR (KBr)-C.dbd.0 1743 
cm.sup.-1 ; .sup.1 H NMR (CDC.sub.3) .delta.6.93 (H--C.dbd.C, 2 H, s), 
4.05 (--OCH.sub.3, 12 H, d, J.sub.P-H =11.6 Hz). .sup.13 C NMR 
(CDC.sub.3): .delta.158.12 (d, J.sub.P-C =7.9 HZ), 134.39 (d, J.sub.P-C 
=9.4 Hz), 55.39 (d, J.sub.P-C =5.9 HZ); .sup.31 P NMR (CHC.sub.3) 
.delta.-15.4 (hept, J.sub.P-H =11.6 Hz). In the analysis of the product, 
proton NMR spectra were recorded on a Varian T-60 (60M Hz) spectrometer or 
a Varian Gemini (200M Hz) spectometer. Phosphorous spectra were recorded 
on a Varian XL-200 spectrometer. .sup.13 C NMR spectra were recorded on 
the Varian Gemini spectrometer. Infrared spectra were recorded on a 
Nicolet SDX FTIR spectrometer. 
The reaction was repeated with the addition of 0.01 and 0.1 equivalents of 
18-crown-6 (in order to increase the extent of dissolution of sodium 
phosphate). The yield was lower in both cases than when the crown ether 
was absent. 
(b) Fumaryl bis(sodium methyl phosphate) (1a) 
A solution of sodium iodide (0.9 g, 6 mmol) in dry acetone (6 mL) was added 
to an acetone (6 mL) solution of fumaryl bis(dimethyl phosphate) (1 g, 3 
mmol) in a 25 mL flask. The solution was shaken and the flask was left for 
twelve hours, during which time the product precipitate was a pale yellow 
powder. Filtration, followed by washings with dry acetone and methylene 
chloride resulted in an off-white powder that was dried under vacuum. One 
gram of the material was recrystallized by dissolving in 20 mL methanol. 
Then 40 mL 1:1 ethanol: isopropanol was added and the solution stood for 
30 min. The resulting crystals were collected and dried in vacuo 
(mp&gt;200.degree. C. 93% yield) Analysis of product: IR (KBr): C.dbd.O 1714 
cm.sup.-1. .sup.1 H NMR (D.sub.2 O) .delta.6.85 (H--C.dbd.C,2 H, d, J=2 
Hz), 3.65 (--OCH.sub.3, 6 H, d, J.sub.P-H =12 Hz). .sup.13 C NMR (D.sub.2 
O): .delta.163.0 (d, J.sub.P-C =6.4 Hz), Anal. 135.6 (d, J.sub.P-C =7.6 
Hz), 54.76 (d, J.sub.P-C =6.4 Hz). Anal. (C.sub.6 H.sub.8 O.sub.10 P.sub.2 
Na.sub.2) C,H,P. 
The product was identified as a symmetrical monomethyl phosphate by 
analysis of proton-coupled .sup.31 P NMR spectra and proton NMR spectra. 
The proton-coupled .sup.31 P NMR spectra of the bis(dimethyl phosphates) 
consists of a single phosphorous signal which is a septet due to coupling 
of two equivalent phosphorus nuclei to equivalent sets of six protons 
(from the two methyl groups). Cleavage of one methyl group from each end 
converts the material to one whose phosphorus NMR signal is a quartet. 
Integration of the signal of the methoxy protons in the proton NMR 
spectrum, relative to that of the remaining protons in the molecule, shows 
that cleavage of half of the ester groups has occurred. 
EXAMPLE 3 
##STR33## 
(a) Isophthalyl bis(dimethyl phosphate) (2) 
isophthalyl bis(dimethyl phosphate) was prepared from isophthalyl 
dichloride (4.83 g, 23 mmol) and sodium dimethyl phosphate (6.9 g, 47 
mmol) in dry tetrahydrofuran (50 mL) as set forth in Example 1, to produce 
a colorless oil in 83% yield. Analysis of product: IR (film) C.dbd.O 1754 
cm.sup.-1 ; .sup.1 H NMR (CDC.sub.3) .delta. 8.72 (1 H, t, J=1.7 Hz), 8.35 
(2 H, dd, J=1.7, 7.8 Hz), 7.68 (1 H, t, J=7.8 Hz), 4.02 (12 H, d, 
J.sub.P-C =11.7 Hz). .sup.13 C NMR (CDC.sub.3) .delta.160.60 (d, J.sub.P-C 
=7.0 Hz), 137.39 (d, J.sub.P-C =11.5 Hz), 136.94, 133.43, 130.39, 56.23 
(d, J.sub.P-C =4.8 Hz). 
(b) Isophthalyl bis(sodium methyl phosphate)(2a) 
Isophthalyl bis(sodium methyl phosphate) was prepared in 95% yield from 
isophthalyl bis(dimethyl phosphate) (1.82 g, 4.8 mmol) and sodium iodide 
(1.44 g, 9.6 mmol) as set forth in Example 1. Analysis of product: 
mp&gt;200.degree. C.; IR (KBr) C.dbd.O 1720 cm.sup.-1 ; .sup.1 H NMR (D.sub.2 
O) .delta.8.52 (1 H, t, J=1.8 Hz), 8.17 (2 H, dd, J=1.8, 7.9 Hz), 7.52 (1 
H, t, J=7.9 Hz), 3.56 (6 H, d, J.sub.P-H =11.4 Hz); .sup.13 C NMR (D.sub.2 
O) .delta.166.40 (d, J.sub.P-C =8.1 Hz), 138.72, 137.95 (d, J.sub.P-C 
=10.0 Hz), 134.89, 132.66, 56.91 (d, J.sub.P-C =6.2 Hz). The product was 
identified as a symmetrical monomethyl phosphate by analysis of 
proton-coupled .sup.- P NMR spectra and proton NMR spectra as set forth in 
Example 1. 
EXAMPLE 4 
##STR34## 
(a) Terephthalyl bis(dimethyl phosphate) (3) 
Terephthalyl bis(dimethyl phosphate) was prepared from terephthalyl 
dichloride (4.83 g, 23 mmol) and sodium dimethyl phosphate (6.9 g, 47 
mmol) in dry tetrahydrofuran (50 mL) as set forth in Example 1, to give a 
white solid. Recrystallization from benzene with addition of ether gave 
crystals: mp 81.degree.-82.degree. C. in 91% yield. Analysis of product: 
IR (KBr) C.dbd.O 1743 cm.sup.-l. .sup.1 H NMR (CDC.sub.3) .delta.160.00 
(d, J.sub.P-C =7.9 Hz), 133.10 (d, J.sub.P-C =8.6 Hz), 130.90, 55.48 (d, 
J.sub.P-C =4.7 Hz). 
(b) Terephthalyl bis(sodium methyl phosphate) (3a) 
Terephthalyl bis(sodium methyl phosphate) was prepared in 95% yield from 
terephthalyl bis(dimethyl phosphate) (1 g, 2.6 mmol) and sodium iodide 
(0.78 g, 5.2 mmol) in acetone as set forth in Example 1. Analysis of 
Product: mp&gt;200.degree. C.; IR (KSr) C.dbd.O 1715 cm.sup.-1 ; .sup.1 H NMR 
(D.sub.2 O) .delta.7.93 (4 H, s) , 3.56 (6 H, d, J.sub.P-H =11.5 Hz); 
.sup.13 C NMR (D.sub.2 O)!.delta.166.36 (d, J.sub.P-C =8.1 Hz), 136.63 (d, 
J.sub.P-C =7.0 Hz), 133.35, 56.72 (d, J.sub.P-C =2.6 Hz). Anal. 
(C.sub.10,H.sub.10,O.sub.10,P.sub.2,Na.sub.2) CHP. The product was 
identified as a symmetrical monomethyl phosphate by analysis of 
proton-coupled .sup.31 P NMR spectra and proton NMR spectra as set forth 
in Example 1. 
EXAMPLE 5 
##STR35## 
(a) Stilbene 3,3'-dicarboxylic acid 
Stilbene 3,3'-dicarboxylic acid was prepared by the method of Toland J., et 
al. J. Am Chem. Soc. 1953, 75, p. 2263. 
(b) Stilbene 3,3'-dichloroformate: 
Stilbene 3,3'-dicarboxylic acid (4.9 g, 18 mmol), thionyl chloride (50 mL) 
and a catalytic amount of dry dimethyl formamide (10 drops) were refluxed 
for twelve hours. Excess thionyl chloride (20 mL) was distilled off and 
the product crystallized as yellow needles. The crystals (2.53 g, 8.3 
mmol, 46%) were collected by filtration, washed sparingly with ether, and 
pumped dry. Recrystallization from toluene gave pure material (mp 
179.degree.-181.degree. C.). .sup.1 H NMR (CDC.sub.3) .delta.3.68 (16 H, 
s), 7.07 (2 H, s), 7.20-7.51 (H, m). 
(c) Stilbene 3,3'-dicarboxylic acid bis(dimethyl phosphate) (4) 
Stilbene 3,3 '-dichloroformate (2.4 g, 7.9 mmol) and sodium dimethyl 
phosphate (2.3 g, 15.8 mmol) were stirred at room temperature in dry 
tetrahydrofuran for 48 hours under nitrogen. The reaction mixture was 
filtered and solvent evaporated to give a solid. This was recrystallized 
from benzene/ether to give pure material (2.62 g, 5.4 mmol, 68% yield). 
Analysis of product: .sup.1 H NMR (CDC.sub.3) d 8.19 (1 H, t, J =1.6 Hz), 
7.99 (1 H, dr, J=1.6, 7.7 Hz), 7.95 (1 H, dt, J =1.6 Hz, 7.7,), 7.52 (1 H, 
t, J=7.7 Hz), 4.02 (12 H, d, J =11.7 Hz); .sup.13 C NMR (CDC.sub.3) 
.delta.161.00 (d, J.sub.P-C =8.3 Hz), 137.60, 32.50, 130.00, 129.33, 
128.91, 128.74, 128.56 (d, J.sub.P-C =8.5 Hz), 55.35 (d, J.sub.P- C =5.4 
Hz). Thin Layer Chromatography:R.sub.f =0.29 (Silica plates, 1:1 
dichloromethane: ethyl acetate). 
(d) Stilbene 3,3'-dicarboxylic acid bis(sodium methyl phosphate) (4a) 
Stilbene 3,3'-dicarboxylic acid bis(sodium methyl phosphate) was prepared 
from stilbene 3,3'-dicarboxylic acid bis(dimethyl phosphate) (3.52 g, 7.3 
mmol) and sodium iodide (2.19 g, 14.6 mmol) in acetone as above in 93% 
yield. Analysis of product: mp&gt;200.degree. C., IR (KBr) C.dbd.O 1714 
cm.sup.-1 ; .sup.1 H NMR (D.sub.2 O) .delta.7.51 (2H, d, J - 7.9 Hz), 7.47 
(2 H, s) 7.22 (2 H, d, J=7.9 Hz), 7.10 (12 H, t, J=7.9 Hz), 6.56 (s, 2 H), 
3.62 (6 H, d, J=11.4 Hz); .sup.13 C NMR (D.sub.2 O) .delta.167.00 (d, 
J.sub.P-C =8.3 Hz), 140.01, 134.88, 132.30, 132.08, 131.94, 131.76, 130.05 
(d, J.sub.J-C =8.1 Hz), 56.95. Thin Layer Chromatography:R.sub.f =0.39 
(Silica plates, ethanol.) The product was identified as a symmetrical 
monomethyl phosphate by analysis of proton-coupled .sup.31 P NMR spectra 
and proton NMR spectra as set forth in Example 1. 
EXAMPLE 6 
##STR36## 
(a) Stilbene 4,4 '-dicarboxylic acid 
Stilbene 4,4 '-dicarboxylic acid was prepared by the method of Toland J., 
et al, J. Am. Chem. Soc., 1953, 75, p.2263. 
(b) Stilbene 4,4'-dichloroformate. 
A suspension of stilbene 4,4'dicarboxylic acid (5 g, 18.6 mmol), thionyl 
chloride (60 mL) and a catalytic amount of dry dimethylformamide (0.25 mL) 
were refluxed for 24 hours. The dicarboxylic acid did not dissolve, but 
was converted directly into the diacid chloride which was also largely 
insoluble in the reaction medium. The reaction solution was refrigerated 
for 12 hours. The yellow crystalline product was collected by filtration, 
washed sparingly with acetone and pumped to dryness (5.2 g, 17 mmol, 92% 
yield). Analysis of product: mp 238.degree. C. .sup.1 H NMR (dimethyl 
sulfoxide-d.sub.6) .delta.7.95 (4 H, d, J=10 Hz), 7.73 (4H, d, J=10 Hz), 
7.48 (2 H, s). 
(c) Stilbene 4,4'-dicarboxylic acid bis(dimethyl phosphate) (5) 
Stilbene 4,4'-dichloroformate (2.6 g, 8.5 mmol) and sodium dimethyl 
phosphate (3.15 g, 21.3 mmol) were stirred in dry tetrahydrofuran at 
50.degree. C. under nitrogen for 48 hours. The reaction mixture was 
filtered through a sintered glass funnel and the solvent removed by 
evaporation to give the crude 15 product. Recrystallization from 
tetrahydrofuran/ether gave pure material (1.19 g, 23%). Analysis of 
product: mp 176.degree.-177.degree. C. .sup.1 H NMR (CDC.sub.3).delta.8.08 
(4 H, d, J=8.5 Hz), 7.65 (4 H, d, J=8.5 Hz), 7.29 (2 H, s), 4.03 (12 H, d, 
J=11.7. Hz). .sup.13 C 160.5 (d, J.sub.P-C =8.1 Hz), 142.5, 131.1, 130.5, 
127.1, 127.0. 55.2 (d, J.sub.P-C 5.9 Hz). Thin layer 
chromatography:R.sub.f =0.27 (Silica plates, 1: 1 dichloromethane/ethyl 
acetate). 
(d) Stilbene 4,4'-dicarboxylic acid bis(sodium methyl phosphate) (5a). 
Stilbene 4,4 '-dicarboxylic acid bis(sodium methyl phosphate) was prepared 
from stilbene 4,4'-dicarboxylic acid bis(dimethyl phosphate) and sodium 
iodide in acetone as set forth in Example 1 Analysis of product: 
mp&gt;200.degree. C. IR (KBr) C.dbd.O 1715 cm.sup.-1 ; .sup.1 H NMR (D.sub.2 
O) .delta.7.88 (4 H, d, J=8.1 Hz), 7.27 (4 H, d, J =8.1 HZ), 6.77 (2 H, 
s), 3.80 (6 H, d, J=11.3 Hz); .sup.13 C NMR (D.sub.2 O) .delta.166.77 (d, 
J.sub.P-C =8.3 Hz), 144.8, 133.5, 132.7, 1308 (d, J.sub.P-C =8.1 Hz), 
129.7, 56.6. .sup.- P NMR .delta.-2.38 (from H.sub.3 PO.sub.4). The 
product was identified as a symmetrical monomethyl phosphate by analysis 
of proton coupled .sup.31 P NMR spectra and proton NMR spectra as set 
forth in Example 1. 
EXAMPLE 7 
Benzene 1,3,5-tricarboxylic acid tris(sodium methyl phosphate) was prepared 
from benzene 1,3,5-tricarboxylic acid tris(dimethyl phosphate), (prepared 
from sodium dimethyl phosphate and 1,3,5-benzene tricarbonyl trichloride) 
and sodium iodide as set forth in Example 3 by proportionally increasing 
the amount of the compounds used in the process. 
EXAMPLE 8 
The solution stability of the bis(methyl acyl phosphates) was examined. The 
hydrolysis of fumaryl bis(sodium methyl phosphate), stilbene 
3,3'dicarboxylic acid bis(sodium methyl phosphate) and stilbene 
4,4'dicarboxylic acid bis(sodium methyl phosphate) in 0.100M tris-HCL 
buffer, pH 7.4, 37.degree. C., were followed by integrating signals for 
the reactant and the phosphorus-containing product, methyl phosphate, in 
the .sup.31 P NMR spectrum. Production of methyl phosphate was observed as 
an indication of hydrolysis or of acylation of the buffer. The half life 
of the fumaryl compound, was 36 hours under these conditions. The half 
life of the 3,3'stilbene derivative, was 100 hours. The reaction of the 
4,4'stilbene derivative was followed for nine hours and there was no 
change observed in the spectrum over this time. This indicates that the 
reagents are sufficiently stable in solution and that they do not react 
rapidly with buffer. This is in agreement with earlier reports of the 
reactivity of acyl phosphate esters (Disabato, G.; Jencks, W. P., J. Am. 
Chem. Soc. 1961, 83, 4393, 4400). 
EXAMPLE 9 
Cross-linking of Hemoglobin 
Solutions (2 mL) of 4,4'stilbene dicarboxylic acid bis(methyl phosphate) at 
concentrations of 0.3 mM, 3.0 mM, 30 mM, and 300 mM were prepared in 
0.100M tris-HCL buffer pH 7.4. A control solution containing only the 
buffer was prepared. To each of these freshly prepared solutions, 40 mg of 
human hemoglobin (Sigma Chemical Company) was added so that the resulting 
solution was 0.3 mM in hemoglobin. The reaction mixtures were than stirred 
vigorously and incubated at 37.degree. C. After four hours the solutions 
were dialyzed against 0.100M sodium phosphate buffer, pH 7.0, for 20 hours 
at 4.degree. C. to remove the unreacted acyl phosphates. The samples were 
then lyophilized and analyzed by SDS-polyacrylamide gel electrophoresis to 
assess the extent of intersubunit cross-linking (Weber, K; Osborn, M.; J. 
Biol. Chem. 1969, 244, 4406). Prior to electrophoresis, the hemoglobin 
samples, cross-linked bovine hemoglobin standard (Sigma Dalton Mark VII-L 
doubly crystallized, dialyzed, lyophilized), and molecular weight 
standards were denatured by boiling for 15 min. in 0.11M sodium phosphate 
buffer, pH 7.0, which contained 1% sodium dodecyl sulfate, 1% 
2-mercaptoethanol, 36% urea, and 0.015% bromphenol blue. The final protein 
concentrations were 2 mg/mL and 10 to 20 .mu.L aliquots were loaded on the 
gel. The process was conducted using a Bio-Rad Mini-Protean II dual slab 
cell apparatus. The extent of cross-linking of the hemoglobin could be 
estimated by visual comparison of the resolved electrophoretic bands after 
fixation followed by staining with Coomassie Brilliant Blue R. 
The above procedure was repeated with solutions of fumaryl bis(methyl 
phosphate) at concentrations of 0.3 mM, 3.0 mM and 30 mM; 3,3'stilbene 
dicarboxylic acid bis(methyl phosphate) at a concentration of 30 mM and 
methyl acetyl phosphate at a concentration of 0.6M. 
The SDS gel electrophoresis indicated that the reaction of hemoglobin with 
stilbene 3,3'dicarboxylic acid bis(methyl phosphate), stilbene 
4,4'-dicarboxylic acid bis(methyl phosphate), and with fumaryl bis(methyl 
phosphate) produced dimeric and tetrameric species. The lanes containing 
the hemoglobin modified with these reagents showed bands which correspond 
to the dimer (32,000), trimer (48,000) and tetramet (64,000) (compared 
with the standard cross-linked bovine hemoglobin). As well, there were 
trace bands of higher molecular weight. Bands for unreacted hemoglobin 
indicated that the material is fully dissociated into monomers. Control 
experiments with hemoglobin which had been reacted with methyl acetyl 
phosphate and with stilbene 3,3'-dicarboxylic acid gave materials which 
are monomeric according to the gel patterns. These results indicate that 
cross-linking reactions are taking place only in the presence of the 
difunctional acyl phosphate esters. 
EXAMPLE 10 
Characterization of Cross-linked Hemoglobin 
The effect of pH, type of buffer, and ligand state of the cross-linked 
hemoglobin was examined for the reaction of fumaryl bis(methyl phosphate) 
(FMP) with hemoglobin. No significant differences were observed in amounts 
or varieties of reaction products when reacting carbonmonoxyhemoglobin 
(COHb) with FMP in bisTris compared to HEPES 
[4-(2-hydroxy-ethyl)-1-piperazine 
ethanesulphonic acid]at pH 7.5, 35.degree., for 2 hours. Over the range of 
pH from 6.8 to 7.5, the greatest reaction between COHb and FMP was 
obtained at the pH of 7.2 in bisTris. The reaction at pH 7.2, . 35.degree. 
with 1 mM COHb and 2 mM FMP resulted in the conversion of about 75% of the 
hemoglobin to fumaryl derivatives in 2 hours with little further change by 
3 hours. The major modified component was approximately 35% of the total 
hemoglobin by 10 minutes and did not change appreciably thereafter. A 
second peak of reacted material observed by anion exchange chromatography 
increased over 3 hours from 14% at 10 minutes to about 30% by 3 hours. 
From these results the reaction conditions were standardized to 1 mM Hb, 2 
mM FMP, pH 7.2 in 100 mM bisTris, at 35.degree. C. for 2 hours. 
Structural characterization of the major and some minor modified 
hemoglobins that result form reacting deoxyHb and COHb with FMP were made 
by isolating single hemoglobin components wherever possible. The initial 
separation was made on a preparative size Synchropak AX300 anion exchange 
column. Further purification was obtained by rechromatography of anion 
exchange zones containing mixtures of hemoglobins on a preparative size 
Synchropak CM300 cation exchange column. Zones from cation exchange 
rechromatography were then subjected to globin chain separation using 
Vydak C-4 large pore reverse phase columns. In some cases, this was 
sufficient for identification of the unmodified globin chains. In most 
cases the globin chain was isolated, treated by oxidations or 
aminoethylation to stabilize the zysteinyl residues, hydrolyzed with 
trypsin and glu-C proteonase and the resultant peptides were separated and 
analyzed for amino acid composition. Table II lists the components 
isolated and the structural modifications identified for the FMP treated 
hemoglobin. 
The number, chromatographic elution positions, and amounts of products of 
hemoglobin reacted with FMP varies with the ligand state of the hemoglobin 
and with the presence or absence of 2,3-DPG (2,3, diphosphoglycerate). 
FIG. 1 is an artion exchange HPLC chromatogram of the reaction mixture 
after treatment of deoxyhemoglobin with FMP. FIG. 2 is the same using 
COHb. Zone 1 in each case is the same and represents unreacted Hb A. The 
second major zone for the COHb is comprised primarily of one of the 
components found for the third zone for the deoxyHb reaction mixtures. The 
third major zone in the CohB appears to chromatograph like the last or 
fifth zone of the deoxyHb mixtures. FIGS. 3 is an artion exchange HPLC 
chromatogram of the reaction products resulting from deoxyHb treated with 
FMP in the presence of 2,3-DPG. The elution conditions are slightly 
different from those used in chromatograms shown in FIGS. 1 and 2. A 
control of deoxyHb reacted with FMP in the absence of 2,3-DPG using the 
same chromatographic conditions is shown in FIG. 4. The first zone on each 
is unreacted Hb A. The second zone in FIG. 3 is not found in either of the 
other two conditions and contains a modified hemoglobin with a fumaryl 
bridge between the two .alpha.-99 lysyl residues. This is the same as the 
major reaction product found in the product DBBF obtained from Baxter 
Travenol which is prepared using the bis(3,5-dibromosalicyl) fumarate 
cross-linker. The third zone in FIG. 3 appears to be a mixture of products 
containing modifications of both alpha and beta chains but without 
significant amounts of cross-linked hemoglobins. 
The results indicate that the cross-linking site of reaction of human 
hemoglobin with fumaryl bis(methyl phosphate) is the highly cationic site 
which binds 2,3-diphosphoglycerate. In the absence of 
2,3-diphosphoglycerate, reaction with fumaryl 15 bis(methyl phosphate) 
produces material that is cross-linked between .beta. subunits (val-1 to 
lys-82) and between the same residues in a single .beta. subunit as well 
as a cross-link between .alpha. subunits between lys-99 in each subunit. 
In the presence of 2,3-diphosphoglycerate, only the .beta. cross-link is 
formed. 
EXAMPLE 11 
Isophthalyl bis(methyl phosphate) or 3,3'-stilbene bis(methyl phosphate) 
were reacted with hemoglobin. Modified hemoglobin components were purified 
on the preparative size Synchropak AX300 and Synchropak CM300 columns 
using the HPLC system. .beta..sub.1 1-.beta..sub.2 82 and .beta..sub.1 
82-.beta..sub.2 82 stilbene hemoglobins and .beta..sub.1 1-.beta.82 
cross-linked and .beta..sub.1 1-.beta..sub.2 82 uncross-linked isophthalyl 
hemoglobins were isolated. 
TABLE I 
Estimated distance between amino groups that can be crosslinked with 
monoesters of the compounds of the formula Ib, as calculated from the span 
of the diamide derived from the dicarboxylic acid. 
______________________________________ 
Compounds of the Formula Ib 
Span of Amine (A.degree.) 
______________________________________ 
Fumaryl 6.1 
Isophthalyl 7.2 
Terephthalyl 7.4 
3,3"-Stilbene 13.7 
4,4"-Stilbene 13.9 
______________________________________ 
TABLE II 
______________________________________ 
STRUCTURE OF GLOBIN CHAINS FROM 
PUMARYL BIS (METHYL PHOPHATE) 
MODIFIED HEMOGLOBINS 
Anion 
Zone # Cation Zone # 
Chain Zone # 
Chain and Modificat 
______________________________________ 
A. Hemoglobins From FMP Treated Deoxyhemoglobin 
AX-1 CM-1 I .beta.-unmodified 
" " II .alpha.-unmodified 
AX-2 CM-1 I .alpha.-unmodified 
" " II .beta..sub.1 1-.beta..sub.2 82 
AX-3b CM-1 I .beta.-unmodified 
" " II .alpha.-unmodified 
" " III .beta..sub.1 1-.beta..sub.2 82 
" " IV .beta..sub.1 1-.beta..sub.2 82 
" " V .alpha.-modification? 
" " VI .alpha..sub.1 99-.alpha..sub.2 99 
AX-3b CM-2 I .alpha.-unmodified 
" " II .beta..sub.1 82-.beta..sub.2 82 
AX-3b CM-3 I .beta.-unmodified 
" " II .alpha..sub.1 1-Fumarate? 
" " III .beta..sub.1 82-.beta..sub.2 82 
AX-3c CM-1 I .beta..sub.1 1-Fumarate? 
" " II .alpha.-unmodified 
AX-3c CM-2 I .beta.-unmodified 
" " II .beta.-modification? 
" " III .alpha.-unmodified 
" " IV .alpha.-modification? 
" " V .alpha..sub.1 99-.alpha..sub.2 99 
AX-4 CM-1 I .alpha.-unmodified 
" " II .beta..sub.1 1-.beta..sub.1 82 
AX-5 CM-1 I .beta..sub.1 82-Fumarate 
" " II .alpha.-unmodified 
B. Hemoglobins From FMP Treated Deoxyhemoglobin 
with 2,3-DPG 
AX-1 CM-1 I .beta.-unmodified 
" " II .alpha.-unmodified 
AX-2 CM-1 I .beta.-unmodified 
" " II .alpha..sub.1 99-.alpha..sub.2 99 
AX-3 CM-1 I .beta.-modified? 
" " II .alpha.-modified? 
C. Hemoglobins From FMP Treated 
Carbonmonoxyhemoglobin 
AX-1 CM-1 I .beta.-unmodified 
" " II .alpha.-unmodified 
AX-2 CM-1 I .alpha.-unmodified 
" " II .beta..sub.1 82-.beta..sub.2 82 
______________________________________