U.S. patent application number 10/770224 was filed with the patent office on 2005-08-04 for polyoxyalkylene compound and method for making.
Invention is credited to Mohanty, Dillip K., Sharma, Ajit.
Application Number | 20050171002 10/770224 |
Document ID | / |
Family ID | 34808274 |
Filed Date | 2005-08-04 |
United States Patent
Application |
20050171002 |
Kind Code |
A1 |
Mohanty, Dillip K. ; et
al. |
August 4, 2005 |
Polyoxyalkylene compound and method for making
Abstract
A process for forming a conjugate of a polyoxyalkylene polymer
(such as polyethylene glycol) with a compound containing an amine
group(s) and/or a sulfide group(s) by reacting the compound with an
acrylate terminated polyoxyalkylene (such as polyethylene glycol
terminate at one end with acrlyate or methacrylate and terminated
at the other end with a methoxy group). The reaction is believed to
be a Michael addition. When the compound contains primary amine
groups (such as the surface primary amine groups of a PAMAM
dendrimer), it is usually desirable to convert the primary amine
groups to secondary amine groups before the reaction with the
acrylate terminated polyoxyalkylene.
Inventors: |
Mohanty, Dillip K.; (Mount
Pleasant, MI) ; Sharma, Ajit; (Mount Pleasant,
MI) |
Correspondence
Address: |
MCKELLAR IP LAW, PLLC
784 SOUTH POSEYVILLE ROAD
MIDLAND
MI
48640
US
|
Family ID: |
34808274 |
Appl. No.: |
10/770224 |
Filed: |
February 3, 2004 |
Current U.S.
Class: |
530/331 ;
514/21.9; 514/58; 530/300; 536/46 |
Current CPC
Class: |
C07C 251/24 20130101;
C07C 229/14 20130101; C07C 229/16 20130101 |
Class at
Publication: |
514/002 ;
514/058; 530/300; 536/046 |
International
Class: |
A61K 038/16; A61K
031/724 |
Claims
What is claimed is:
1. A compound corresponding to the formula: 32where R.sub.1 is an
organic radical where R.sub.2 is H or an organic radical where
R.sub.3 is H or an organic radical where R.sub.4 is a
polyoxyalkylene radical and where R.sub.5 is an organic radical or
H.
2. The compound of claim 1, wherein R.sub.1 is derived from a
dendrimer.
3. The compound of claim 1, wherein R.sub.1 is selected from the
group consisting of a group derived from a peptide or a
protein.
4. The compound of claim 1, wherein R.sub.4 is derived from
polyethylene glycol.
5. The compound of claim 2, wherein R.sub.2 is derived from a
cyclodextrin, wherein R.sub.3 is H, wherein R.sub.4 is derived from
polyethylene glycol and wherein R.sub.5 is CH.sub.3.
6. The compound of claim 3, wherein R.sub.2 is derived from a
cyclodextrin, wherein R.sub.3 is H, wherein R.sub.4 is derived from
polyethylene glycol and wherein R.sub.5 is CH.sub.3.
7. The compound of claim 3, wherein R.sub.2 is H, wherein R.sub.3
is H, wherein R.sub.4 is derived from polyethylene glycol and
wherein R.sub.5 is CH.sub.3.
8. A compound corresponding to the formula: 33where R.sub.6 is an
organic radical where R.sub.7 is H or an organic radical where
R.sub.8 is a polyoxyalkylene radical and where R.sub.9 is an
organic radical or H.
9. The compound of claim 8, wherein R.sub.6 is selected from the
group consisting of a group derived from a peptide or a
protein.
10. The compound of claim 8, wherein R.sub.6 is derived from
polyethylene glycol.
11. The compound of claim 9, wherein R.sub.7 is H, wherein R.sub.8
is derived from polyethylene glycol and wherein R.sub.9 is
CH.sub.3.
12. A method for preparing a compound corresponding to the formula:
34where R.sub.1 is an organic radical where R.sub.2 is H or an
organic radical where R.sub.3 is H or an organic radical where
R.sub.4 is a polyoxyalkylene radical and where R.sub.5 is an
organic radical or H, comprising the step of: reacting A with B,
wherein A is R.sub.1--N--R.sub.2 and wherein 35
13. The process of claim 12, wherein R.sub.1 is derived from a
dendrimer.
14. The process of claim 12, wherein R.sub.1 is selected from the
group consisting of a group derived from a peptide or a
protein.
15. The process of claim 12, wherein R.sub.4 is derived from
polyethylene glycol.
16. The process of claim 13, wherein R.sub.2 is derived from a
cyclodextrin, wherein R.sub.3 is H, wherein R.sub.4 is derived from
polyethylene glycol and wherein R.sub.5 is CH.sub.3.
17. The process of claim 14, wherein R.sub.2 is derived from a
cyclodextrin, wherein R.sub.3 is H, wherein R.sub.4 is derived from
polyethylene glycol and wherein R.sub.5 is CH.sub.3.
18. The process of claim 14, wherein R.sub.2 is H, wherein R.sub.3
is H, wherein R.sub.4 is derived from polyethylene glycol and
wherein R.sub.5 is CH.sub.3.
19. A method for preparing a compound corresponding to the formula:
36where R.sub.6 is an organic radical where R.sub.7 is H or an
organic radical where R.sub.8 is a polyoxyalkylene radical and
where R.sub.9 is an organic radical or H, comprising the step of:
reacting D with E, wherein D is R.sub.6--S and wherein E is 37
20. The compound of claim 19, wherein R.sub.6 is selected from the
group consisting of a group derived from a peptide or a
protein.
21. The compound of claim 19, wherein R.sub.8 is derived from
polyethylene glycol.
22. The compound of claim 20, wherein R.sub.7 is H, wherein R.sub.8
is derived from polyethylene glycol and wherein R.sub.9 is
CH.sub.3.
Description
BACKGROUND
[0001] The instant invention is in the field of chemical compounds
comprising polyoxyalkylene sub-structures such as polyethylene
glycol sub-structures. The instant invention also relates to
methods for producing chemical compounds comprising polyoxyalkylene
sub-structures.
[0002] Biologically active compounds comprising polyoxyalkylene
sub-structures can provide enhanced biocompatibility for the
compound, See, for example, U.S. Pat. No. 5,366,735 and U.S. Pat.
No. 6,280,745. A review of this subject by Zalipsky, in
Bioconjugate Chem., 1995, 6, p 150-165, identified polyethylene
glycol as one of the best biocompatible polymers to conjugate with
a biologically active compound (such as a drug, a protein, a
peptide or an enzyme) to produce a conjugate having improved
properties such as compatible solubility characteristics, reduced
toxicity and reduced immunogenicity.
[0003] Polyethylene glycol (PEG) is a linear or branched
polyoxyalkylene terminated at the ends thereof with hydroxyl groups
and generally represented by the formula:
HO--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub- .2--OH. As discussed
by Henmanson in Chapter 15 of Bioconjugate Techniques (1966),
monomethoxy polyethylene glycol (mPEG) generally represented by the
formula:
CH.sub.3O--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--OH, is
usually used to prepare a polyethylene glycol conjugate with a
biologically active compound typically by way of a coupling
reaction between an amine group of the biologically active compound
and an amine receptive derivative (such as trichloro-s-triazine
activated mPEG) formed via the remaining terminal hydroxyl group of
the monomethoxy polyethylene glycol. An acrylate terminated PEG is
offered commercially by Shearwater Corporation (Huntsville, Ala.)
for vinyl polymerization or co-polymerization to produce graft
polymers or cross-linked materials with excellent properties for
biomaterial applications.
[0004] More recently, so called "second generation" PEGylation
chemistry has been developed to, for example, minimize problems of
diol impurity contamination of mPEG, to increase the molecular
weight of the polyoxyalkylene sub-structure and to increase
stability of the conjugate, see Roberts et al., Advanced Drug
Delivery Reviews 54 (2002) p459-476.
[0005] Dendrimers are hyperbranched, uniformly distributed
structures, having (at least ideally) definite molecular weight,
shape and nanometer size characteristics. Dendrimers were
discovered by Tomalia and co-workers at the Dow Chemical Company,
see Polym. J. 17 (1985) p 117-132. Dendrimers have been widely
studied as a drug delivery means, see for example Knusli et al., J.
Haematology, 82, 654 (1992). Dendrimers carrying the anti cancer
drug 5-fluorouracil have been PEGylated to reduce hemolytic
toxicity, drug leakage and macrophageal uptake while improving
stability and efficacy, see Bhadra et al., International Journal of
Pharmaceutics 257 (2003) p 111-124.
[0006] PAMAM dendrimers are the most common type of dendrimer and
are commercially available from Aldrich (Milwaukee, Wis.) in the
form of various "generations". PAMAM dendrimers are made by a
successive Michael addition synthesis scheme involving the reaction
of an acrylate group with an amine group. The so called "Generation
0" PAMAM dendrimer has the following formula: 1
[0007] The above Generation 0 PAMAM dendrimer has a molecular
weight of about 517 grams per mole. A Generation 1 PAMAM dendrimer
has a molecular weight of about 1,430 grams per mole and has eight
terminal primary amine groups. A Generation 2 PAMAM dendrimer has a
molecular weight of about 3,256 grams per mole and has sixteen
terminal primary amine groups. A Generation 10 PAMAM dendrimer has
a theoretical molecular weight of almost 935 kilograms per mole and
in theory has 4096 primary amine groups on the surface of the
dendrimer.
[0008] Despite the significant advances that have been made in the
field of methods for the PEGylation of biologically active
compounds (and more generally in the field of methods for the
conjugation of polyoxyalkylene sub-structures with biologically
active compounds), the existing methods generally require multiple
reactions and extensive purification of the product. It would be an
advance in this art if a process were discovered that required only
one reaction step and produced no by-products.
SUMMARY OF THE INVENTION
[0009] The method of the instant invention is a solution, at least
in part, to the above described problems of the prior art. The
instant invention provides a one step pegylation method that
ideally produces no by-products. In addition, the method of the
instant invention can be practiced at room temperature and under
conditions such as solvent compatibility that are mild relative to
maintenance of biological activity. In one embodiment, the instant
invention is applicable to biologically active compounds containing
an amine group. In another embodiment, the instant invention is
applicable to biologically active compounds containing a sulfide
group. The biologically active compound is reacted with an acrylate
terminated polyoxyalkylene (such as
H.sub.2C.dbd.CH--CO--O-PEG-O--CH.sub.3) in a one step process to
produce novel conjugates having many if not all of the benefits of
the prior art conjugates.
[0010] More specifically, the instant invention is a method for
preparing a compound corresponding to the formula: 2
[0011] where R.sub.1 is an organic radical
[0012] where R.sub.2 is H or an organic radical
[0013] where R.sub.3 is H or an organic radical
[0014] where R.sub.4 is a polyoxyalkylene radical
[0015] and where R.sub.5 is an organic radical or H, comprising the
step of: reacting A with B, wherein A is R.sub.1--N--R.sub.2 and
wherein 3
[0016] In another embodiment, the instant invention is a method for
preparing a compound corresponding to the formula: 4
[0017] where R.sub.6 is an organic radical
[0018] where R.sub.7 is H or an organic radical
[0019] where R.sub.8 is a polyoxyalkylene radical
[0020] and where R.sub.9 is an organic radical or H, comprising the
step of: reacting D with E, wherein D is R.sub.6--S and wherein
5
[0021] In addition, the invention is the compound of formula 1 and
the compound of formula 2 as described above.
DETAILED DESCRIPTION OF THE INVENTION
[0022] In general, the process of the instant invention can be
conducted at room temperature. Infrared (IR) spectra are obtained
using a thin film on a sodium chloride plate. Spectra are recorded
using a Nicolet 20DXB Fourier Transform (FT-IR) Spectrometer and
absorption is reported in wave number (cm.sup.-1). IR spectra cover
the range 1000-4000 cm.sup.-1.
[0023] Proton nuclear magnetic resonance spectra and carbon-13
nuclear magnetic resonance spectra are recorded for solutions in
appropriate solvents containing tetramethylsilane in case of
chloroform and methanol and 3(trimethylsilyl)propane Sulfonic acid
sodium salt (DSS) in case of Deuterium Oxide as internal standard
using a General Electric QE-300 NMR spectrometer. The NMR shifts
are reported in parts per million (.delta., PPM). The following
standard abbreviations were used in describing NMR data: s=singlet,
d=doublet, t=triplet, q=quartet, m=multiplet.
[0024] Mass Spectra are obtained by using a Hewlett-Packard Model
5995A gas Chromatograph/Mass Spectrometer with an ionizing
potential of 70 electron volts.
[0025] Starting Materials
[0026] 1. PAMAM Dendrimer (Generation=0)
[0027] Source: Aldrich Chemical; Structure: 6
[0028] The structure above may also be represented 7
[0029] Note: PAMAM Dendrimer is purchased in 20% solution in
methanol and used without further purification.
[0030] 2. Poly Ethylene Glycol Methyl Ether Acrylate
[0031] Source: Aldrich Chemical Company 8
[0032] Used without further purification.
[0033] A PEG chain may be represented: 9
[0034] 3. Ethylene Diamine
[0035] Source: Aldrich Chemical Company
[0036] Structure: 10
[0037] Purification procedure: Ethylene diamine is distilled over
CaH.sub.2.
[0038] 4. Benzaldehyde
[0039] Source: Fisher Scientific
[0040] Structure: 11
[0041] Purification procedure: Benzaldehyde (about 75 mL) is placed
in a separatory funnel. It is washed with 10% NaCO.sub.3 until no
more CO.sub.2 evolved. Saturated NaCl solution is added to the
solution. The solution is then washed with a saturated solution of
Na.sub.2SO.sub.3 followed by washing with water. The organic layer
is collected and dried with MgSO.sub.4. The organic layer is
filtered and then distilled under vacuum.
[0042] 5. Acetophenone
[0043] Source: Fisher Scientific
[0044] Structure: 12
[0045] used without further purification.
[0046] 6. Propylamine
[0047] Source: Aldrich Chemical Company
[0048] Structure: 13
[0049] It is distilled over CaH.sub.2 before use.
[0050] 7. N-ethyl N-benzyl Amine
[0051] Source: Aldrich Chemical Company
[0052] Structure: 14
[0053] used without further purification.
[0054] 8. Methanol
[0055] Source: Burdick and Jackson
[0056] Structure: CH.sub.3OH
[0057] used without further purification.
[0058] 9. Molecular Sieve (Size 3A)
[0059] Source: EM Science
[0060] 1. Model Reaction
[0061] Molecular Weight: 1108 g/mole
[0062] Reaction Scheme: 15
[0063] Procedure: Ethylene diamine (0.5714 g, 0.9525*10.sup.-3
moles) and PEGMA (1 gm, 1.905*10.sup.-3) and 3 mL of methanol are
added to a clean and dry vial. The vial is capped and allowed to
shake for 2 hours. Methanol is removed under vacuum at room
temperature. The product is a sticky solid.
[0064] Spectral Data:
[0065] IR: cm.sup.-1 3514.3, 2873.2, 1723.8, 1656.3, 1462.3,
1449.6, 1407.5, 1344.2, 1293.6 1276.7, 1243.0, 1192.4, 1108.0
[0066] .sup.1H NMR: (.delta. ppm) in CD.sub.3OD 2.52(m), 2.69(s),
2.84(t), 3.35(s), 3.53(m), 3.62(d), 3.66(m), 4.70(t)
[0067] .sup.13C NMR: (a ppm) 33.33, 34.76, 45.67, 52.10, 53.92,
59.08, 62.17, 64.68, 70.05, 71.31, 72.91, 73.64
[0068] 2. PAMAM Dendrimer (Generation=0)--PEGMA (1/4 Equivalent)
Conjugate
[0069] Molecular weight: 1041 g/mole
[0070] Reaction Scheme: 16
[0071] Procedure: 1 mL of G.sub.0 solution (20% solution in
methanol) is taken in a clean dry vial. PEGMA (0.0254 g, 0.00019
moles) is added to the vial along with the 2 mL methanol. The vial
is capped tightly and allowed to shake for about 3 hours. The
methanol is removed after the reaction is over. The product is a
sticky solid.
[0072] Spectral Data
[0073] .sup.1H NMR: (.delta. ppm) in D.sub.2O 2.42(t), 2.60(s),
2.65(t), 2.70(t), 3.24(t), 3.28(s), 3.40(s), 3.62(m), 3.65(s),
3.80(m), 4.24(t).
[0074] .sup.13C NMR: (.delta. ppm) 32.59, 36.41, 36.65, 40.96,
41.13, 45.07, 46.97, 48.80, 49.06, 49.96, 58.01, 60.30, 69.41,
69.54, 69.63, 70.94, 71.69, 174.92, 175.03, 175.21, 175.30, 175.97,
180.62, 180.70
[0075] 3. PAMAM Dendrimer (Generation=0)--PEGMA (4 Equivalents)
Conjugate
[0076] Molecular weight: 2613 g/mole
[0077] Reaction Scheme: 17
[0078] Procedure: 1 mL of G.sub.0 solution (20% solution in
methanol) is taken in a clean dry vial. PEGMA (0.1016 g, 0.00076
moles) is added to the vial along with the 2 mL methanol. The vial
is capped tightly and allowed to shake for about 3 hours. The
methanol is removed after the reaction is over. The product is a
sticky solid.
[0079] Spectral Data:
[0080] .sup.1H NMR: (.delta. ppm) in D.sub.2O 2.42(t), 2.59(m),
2.70(t), 2.82(m), 3.29(t), 3.37(s), 3.62 (m), 3.69(s), 3.80(m),
4.30(d)
[0081] .sup.13C NMR: (.delta. ppm) 21.65, 33.90, 34.52, 34.90,
38.52, 39.21, 45.70, 47.05, 49.29, 49.29, 49.68, 50.82, 51.19,
51.35, 52.28, 53.26, 53.88, 54.01, 54.72, 54.90, 56.92, 60.55,
62.84, 66.43, 70.93, 71.95, 72.08, 72.17, 73.49, 74.23, 176.60,
176.70, 177.06, 177.51, 177.83, 180.58.
[0082] 4. PAMAM Dendrimer (Generation=0)--PEGMA (2
Equivalents)--Benzaldeh- yde (3 Equivalents)
[0083] Molecular weight: 1305 g/mol
[0084] Reaction Scheme: 18
[0085] Procedure: 2 mL of G.sub.0 and Benzaldehyde (0.24603 g,
0.00232107 moles) are placed in a clean dry vial, capped tightly
and allowed to shake for about 24 hours. PEGMA (0.40612 g, 0.000774
moles) is added thereafter. The vial is allowed to shake for about
100 hours. The methanol is removed under vacuum at room
temperature. The product is a sticky solid.
[0086] Spectral Data
[0087] IR: cm.sup.-1 1107.4, 1196.6, 1273.6, 1293.9, 1350.6,
1407.3, 1451.9, 1723.5, 2865.6
[0088] .sup.1H NMR: (.delta. ppm) in CD.sub.3OD
[0089] 2.30 (m), 2.45 (m), 2.60 (m), 2.70 (m), 2.95 (m), 3.34 (s),
3.62 (s), 4.20 (d), 7.40 (m), 7.75 (m), 7.795 (m), 8.30 (d)
[0090] .sup.13C NMR: (.delta. ppm) 33.50, 33.63, 33.83, 38.47,
39.11, 40.74, 41.02, 45.14, 50.41, 50.85, 51.76, 52.27, 53.65,
59.07, 60.99, 62.14, 64.76, 71.27, 71.30, 71.45, 72.87, 73.61,
128.78, 129.40, 129.78, 130.26, 131.40, 132.21, 137.01, 165.20,
173.86, 174.09, 174.63, 174.95
[0091] 5. PAMAM Dendrimer (Generation=0)--PEGMA (2
Equivalents)--Acetophen- one (3 Equivalents)
[0092] Molecular weight: 1347 g/mole
[0093] Procedure: 1 mL of G.sub.0 and Acetophenone (0.1395 g,
0.0021161 moles) are placed in a clean dry vial. 2 scoops of
molecular sieve were added to the vial. It was capped tightly and
allowed to shake for about 24 hours. PEGMA (0.4062 g, 0.000774
moles) was added thereafter and allowed to shake for about 100
hours. The methanol was removed under pump at room temperature. The
product is a sticky solid.
[0094] Spectral data
[0095] IR: cm.sup.-1 1036.2, 1107.4, 1139.8, 1196.6, 1251.3,
1273.6, 1295.9, 1348.6, 1407.3, 1447.3, 1597.8, 1634.3, 1658.6,
1723.5, 2865.6, 3514.1.
[0096] .sup.1H NMR: (.delta. ppm) in D.sub.2O 2.22(s), 2.43(t),
2.57(m), 2.65(s), 2.70(m), 2.80(d), 3.28(t), 3.30(m), 3.35(s),
3.38(s), 3.70(s), 3.63(m), 7.46(q), 7.55(t), 7.69(m), 7.98 (d)
[0097] .sup.13C NMR: (.delta. ppm) 17.65, 21.76, 28.42, 28.92,
32.90, 34.05, 35.21, 36.11, 41.12, 42.21, 42.62, 42.98, 46.12,
47.60, 49.61, 51.51, 52.61, 54.87, 57.03, 60.70, 63.00, 72.10,
72.23, 73.63, 74.37, 131.18, 131.47, 136.81, 139.00, 165.00,
177.50, 177.89, 177.80, 182.64.
[0098] 6. Model Reaction
[0099] a. Schiff Base Formation by the Reaction between Propylamine
and Acetophenone;
[0100] Molecular weight: 161 g/mol
[0101] Structure Scheme: 19
[0102] Procedure: Acetophenone (1.006 g, 0.00779 moles) and
Propylamine (2.510 g, 0.0425 moles) are added in a clean dry vial.
About 4 scoops of warm molecular sieve are added to the vial and
capped tightly. The vial is allowed to shake for about 96 hours.
The solution is collected using the solvent CH.sub.2Cl.sub.2 and
dried over MgSO.sub.4. The solution is filtered through celite and
dried under vacuum at room temperature.
[0103] Spectral data
[0104] IR: cm.sup.-1 (intensity) 1027.04, 1074.89, 1180.02,
1376.10, 1446.25, 1492.57, 1578.19, 1634.03, 2872.57, 2930.89,
2958.92, 3024.00, 3058.14, 3078.24.
[0105] MS: m/e (% of base) 162, 16, 160, 146, 132, 104, 91 (100%),
77
[0106] .sup.1H NMR: (.delta. ppm) in CDCl.sub.3 1.01 (t), 1.78 (q),
2.20 (s), 3.42 (t), 7.34 (m), 7.76 (m).
[0107] .sup.13C NMR (CD.sub.3OD): (.delta. ppm) 12.07, 15.31,
24.05, 29.05, 53.85, 54.99, 77.00, 125.61, 128.02, 129.10, 141.34,
164.67, 165.41, 168.22.
[0108] b. Reduction of Imine:
[0109] Molecular weight: 163 g/mole
[0110] Reaction Scheme 20
[0111] Procedure: Product (1.348 g, 0.008373 moles) of the above
reaction is placed in a clean dry vial. NaBH3CN (0.350 g, 0.00557
moles) was taken in a clean dry vial and about 1.5 mL of methanol
is added to that in order to make a clear solution. The solution is
added to the reaction vial and capped tightly. The vial is allowed
to shake for about 72 hours at room temperature. After 72 hours
about 6(N)HCl is added drop by drop until the pH is <2. Then the
solution is brought to a pH of >10 with 10% NaOH solution. The
opaque solution is then dried over MgSO.sub.4, filtered through
celite and dried over vacuum at room temperature.
[0112] Spectral Data
[0113] IR: cm.sup.-1 (intensity) 1027.53, 1071.61, 1286.90,
1370.64, 1450.87, 1492.02, 1601.13, 2871.74, 2931.94, 2959.54,
3025.48, 3062.21.
[0114] MS: m/e (% of base) 163, 162, 148, 134, 105 (100%), 77
[0115] .sup.1H NMR: (d ppm) in CD.sub.3OD 0.94 (t), 3.66 (d), 2.69
(m), 2.88 (m), 3.34 (s), 4.35 (q), 4.93 (s), 7.47 (m).
[0116] .sup.13C NMR: (.delta. ppm) in CD.sub.3OD 9.87, 18.30,
19.36, 58.14, 127.15, 129.05, 129.16, 136.54.
[0117] c. N-EthylBenzyl amine-PEGMEA Conjugates:
[0118] Structure: 21
[0119] Procedure: PEGMA (1 g, 0.00191 moles), N-ethyl
benzylamine(0.258 g, 0.00191 moles) and methanol are placed in a
clean dry vial. The vial is capped tightly and allowed to shake for
about 72 hours. The methanol is evaporated off under vacuum at room
temperature.
[0120] Spectral Data
[0121] .sup.1H NMR: (.delta. ppm) in CD.sub.3OD 1.03 (t), 2.48 (q),
2.77 (t), 3.34 (s), 3.57 (m), 3.82 (m), 4.19 (t), 4.28 (t), 7.23
(m), 7.29 (d).
[0122] .sup.13C NMR: (.delta. ppm) in CD.sub.3OD 11.96, 32.91,
33.05, 48.06, 49.57, 52.02, 58.80, 59.08, 62.18, 64.66, 64.81,
71.32, 71.51, 72.92, 73.64, 128.06, 129.21, 130.12, 140.15, 167.44,
174.10, 174.63.
[0123] 7. Pegylation of Reduced PAMAM Dendrimer
(Generation=0)--Benzaldehy- de (4 Equivalents) Conjugates:
[0124] Molecular weight: 2977 g/mole
[0125] Reaction Scheme: 222324
[0126] Procedure: 2 mL (7.737.times.10.sup.-4 moles) of G.sub.0 and
benzaldehyde (0.328 g, 3.095.times.10.sup.-3 moles) are placed in a
clean dry vial. About 2.5 mL methanol is added to the reaction
vial, capped tightly and allowed to shake for about 24 hours.
Sodium cyanohydridoborate (0.129 g, 2.058.times.10.sup.-3 moles) is
taken in a clean dry vial and a clean solution with minimum volume
(about 1 mL) of methanol is made. The solution is added to the
reaction vial and capped tightly. The vial is allowed to shake for
24 hours more. Thereafter PEGMA (0.8124 g, 1.547.times.10.sup.-3
moles) is added and the vial is shaken for another 60 hours. Then
concentrated HCl (about 6 N) is added drop by drop until the pH is
<2. The acidic solution is kept for about 2 hours and then is
brought up to a pH of >10 by the drop by drop addition of 10%
NaOH solution and then dried over MgSO.sub.4 and filtered through
celite. The methanol is then evaporated under vacuum at room
temperature.
[0127] Spectral Data:
[0128] A. G.sub.0+4Benzaldehyde
[0129] .sup.1H NMR: (.delta. ppm) in CD.sub.3OD 2.23 (t), 2.35 (s),
2.59 (t), 3.34 (s), 3.48 (t), 3.66 (t), 4.92 (s), 5.46 (s), 7.40
(m), 7.53 (t), 7.60 (t), 7.63 (t), 7.65 (t), 7.73 (m), 7.84 (m),
7.86 (t), 8.25 (s), 10.00 (s).
[0130] .sup.13C NMR: (.delta. ppm) in CD.sub.3OD 32.56, 34.24,
35.93, 39.10, 40.94, 42.80, 50.93, 51.784, 52.65, 53.58, 59.06,
59.11, 60.86, 61.05, 62.67, 62.73, 128.28, 128.62, 128.92, 129.02,
129.50, 129.59, 130.39, 130.72, 130.73, 130.95, 131.05, 131.15,
131.55, 131.63, 131.71, 133.08, 133.18, 133.28, 134.35, 134.45,
136.83, 136.93, 137.07, 163.94, 166.04, 174.66, 192.77, 192.83,
192.90, 195.09, 195.15, 195.21.
[0131] B. G.sub.0+4Benzaldehyde+NaBH.sub.3CN
[0132] .sup.1H NMR: (.delta. ppm) in CD.sub.3OD 2.54 (m), 2.53 (m),
2.73 (m), 2.86 (m), 3.34 (s), 3.38 (s), 3.59 (s), 3.76 (s), 4.622
(s), 4.89 (s), 5.01 (t), 5.11 (m), 7.34 (m), 7.54 (m), 7.76 (m),
8.35 (m)
[0133] .sup.13C NMR: (.delta. ppm) in CD.sub.3OD 34.67, 38.41,
39.85, 42.01, 42.96, 47.43, 51.23, 52.33, 53.71, 54.15, 57.10,
59.42, 65.19, 116.96, 120.39, 127.92, 128.13, 128.51, 128.76,
128.89, 129.26, 129.43, 129.66, 129.87, 135.37, 136.62, 139.00,
140.63, 142.63, 174.51, 174.60, 175.02, 175.09, 175.16.
[0134] C. G.sub.0+4Benzaldehyde+NaBH.sub.3CN+PEGMEA
[0135] IR: cm.sup.-1 (intensity) 11091, 1198.77, 1249.93, 1293.76,
1350.91, 1452.86, 1542.64, 1564.72, 1630.09, 1658.20, 1736.78,
2871.20.
[0136] .sup.1H NMR: (.delta. ppm) in DMSO-d.sub.6 2.15 (s), 2.42
(t), 2.49 (m), 2.58 (s), 2.60 (d), 3.08 (d), 3.15 (s), 3.22 (s),
3.41 (t), 3.49 (s), 3.53 (s), 4.47 (d), 4.66 (t), 7.21 (m), 7.29
(m), 7.41 (m), 7.54 (m), 7.88 (m).
[0137] .sup.13C NMR: (.delta. ppm) in DMSO-d.sub.6 31.86, 32.04,
33.21, 36.48, 48, 61, 48.86, 49.79, 51.25, 52.30, 57.57, 58.08,
60.20, 62.87, 69.60, 69.80, 71.30, 72.35, 126.43, 126.63, 126.81,
127.31, 127.51, 128.70, 128.17, 128.51, 128.71, 139.32, 142.57,
171.21, 172.52.
[0138] 8. Pegylation of Reduced PAMAM Dendrimer
(Generation=0)--Acetopheno- ne (4 Equivalents) conjugates:
[0139] Molecular weight: 3033 g/mole
[0140] Reaction Scheme: 252627
[0141] Procedure: 2 mL (7.737.times.10.sup.-4 moles) of G.sub.0 and
acetophenone (0.372 g, 3.095.times.10.sup.-3 moles) are placed in a
clean dry vial. About 2.5 mL methanol is added to the reaction vial
along with 3 scoops of molecular sieve (3A), capped tightly and
allowed to shake for about 24 hours. Sodium cyanohydridoborate
(0.129 g, 2.058.times.10.sup.-3 moles) is taken in a clean dry vial
and a clean solution with minimum volume (about 1 mL) of methanol
is made. The solution is added to the reaction vial and capped
tightly. The vial is allowed shake for 72 hours more. Thereafter
PEGMA (0.8124 g, 1.547.times.10.sup.-3 moles) is added and the vial
is allowed to shake for another 120 hours. The solution turns to a
pink color. Then concentrated HCl (about 6 N) is added drop by drop
until the pH is <2. The acidic solution is kept for about 2
hours and then is brought up to a pH of >10 by the drop by drop
addition of 10% NaOH solution, dried over MgSO.sub.4 and filtered
through celite. The methanol is then evaporated under vacuum at
room temperature.
[0142] Spectral Data:
[0143] A. G.sub.0+4 Acetophenone:
[0144] .sup.1H NMR: (.delta. ppm) in CDCl3 2.20 (m), 2.65 (d), 2.57
(t), 2.62 (S), 2.72 (t), 3.22 (m), 3.34 (m), 3.49 (d), 3.55 (t),
7.07 (m), 7.27 (s), 7.35 (m), 7.47 (t), 7.59 (t), 7.72 (m), 7.98
(d).
[0145] .sup.13C NMR: (.delta. ppm) in CDCl.sub.3 16.19, 16.27,
34.03, 34.13, 34.31, 50.36, 50.68, 50.80, 51.38, 51.54, 51.69,
76.58, 77.42, 125.84, 126.56, 128.17, 129.62, 133.10, 140.83,
167.14, 167.32, 172.68, 172.77, 173.01.
[0146] B. G.sub.0+4 Acetophenone+NaBH.sub.3CN
[0147] .sup.1H NMR: (.delta. ppm) in CD.sub.3OD 1.37 (m), 1.43 (d),
2.36 (m), 2.51 (t), 2.60 (s), 2.72 (m), 2.85 (t), 3.27 (m),
3.30(m), 3.37 (s), 3.78 (m), 7.22 (m), 7.31(m), 7.47 (m), 7.49 (d),
7.52 (d), 7.58 (d), 7.98 (d), 8.01 (t).
[0148] .sup.13C NMR: (.delta. ppm) in CD.sub.3OD 23.99, 25.62,
34.57, 39.96, 41.10, 42.918, 47.66, 49.85, 51.19, 52.29, 59.12,
70.81, 126.42, 127.77, 128.05, 128.15, 129.24, 129.56, 145.93,
147.80, 174.99, 175.21.
[0149] C. G.sub.0+4 Acetophenone+NaBH.sub.3CN+PEGMEA
[0150] IR: cm.sup.-1 (intensity) 1101.38, 1199.40, 1249.53,
1288.77, 1350.85, 1452.80, 1580.67, 1630.78, 1657.81, 1736.70,
2873.04.
[0151] .sup.1H NMR: (.delta. ppm) in DMSO-d.sub.6 1.18 (d), 1.28
(d), 2.15 (s), 2.36 (t), 2.49 (t), 2.56 (s), 2.65 (t), 3.03 (t),
3.14 (d), 3.21 (s), 3.40 (t), 3.48 (s), 3.54 (m), 4.22 (q), 4.68
(t), 7.64 (d), 7.27 (m), 7.87 (m), 8.07 (m).
[0152] .sup.13C NMR: (.delta. ppm) in DMSO-d.sub.6 24.64, 32.11,
33.33, 36.72, 46.76, 48.62, 48.98, 49.98, 49.98, 51.29, 52.34,
57.42, 58.12, 60.22, 69.64, 69.84, 71.34, 72.38, 125.35, 126.54,
127.48, 128.03, 128.23, 146.24, 171.35, 171.43, 172.55.
[0153] Analysis of spectral data especially the .sup.1H NMR
analysis of the pegylated PAMAM dendrimer revealed the fact that
the double bond of PEGMA is allowed to react with the terminal
amine groups of PAMAM dendrimer qualitatively. However, it was
observed that the desired product, 3, is not apparently formed.
Detailed analysis of NMR spectra suggested that although 3 forms
first, it is rapidly converted into 15 in the presence of solvent
methanol. A considerable amount of compound 15 gets converted back
to 3 during the removal of methanol at the completion of the
reaction. Moreover, both 3 and 15 are unstable in presence of water
and are hydrolyzed rapidly to 16. The mechanism is believed to be a
series of substitution reactions described below. The intra
molecular hydrogen bond appears to be playing an important role to
facilitate above described reactions. 2829
[0154] An examination of the above scheme clearly demonstrates the
proposition that the hydrogen atom on the nitrogen atom plays a
pivotal role in the production of 15 and 17, instead of the desired
product 3. Furthermore, it is suspected that the dendrimer molecule
under investigation can speed up trans-esterification and
hydrolysis processes readily. It is also suspected that
trans-esterification and hydrolysis processes occurs for quarter as
well as per pegylation. It is observed from the NMR spectrum that
the intensity ratio of the peaks corresponding to the methoxy
groups (OCH.sub.3) of methanol and compound 16 was changed with the
time upon the addition of the water. When water is added to the
pegylated PAMAM dendrimer, the changes of the intensity ratio of
the absorbance correspond to methoxy groups of methanol and
compound 16 can be monitored by .sup.1H NMR spectrum. The ratio Vs
time is noted as illustrated in Table 1 below. It is suspected from
the data that rates of hydrolysis follow the same trend.
1TABLE 1 Time(min) Ratio 1/Rato 7 0.48 2.1 19 0.47 2.13 22 0.51
1.97 27 0.65 1.54 31 0.41 2.44 35 0.57 1.74 39 0.66 1.51 44 0.53
1.87 48 0.67 1.5 52 0.76 1.3 56 0.69 1.44 61 0.71 1.41 67 0.71 1.4
6870 1.49 0.67
[0155] In order to circumvent the undesired reactions after
pegylation, it was decided to block three of the four amine groups
of PAMAM Dendrimer (Generation=0) to remove all the hydrogen
attached to three primary residual nitrogen atoms. The remaining
primary residual nitrogen atom can then be pegylated without any
unwanted product. Benzaldehyde and acetophenone were used for above
purpose (and almost any aldehyde or ketone can be used for this
purpose and an aldehyde substituted cyclodextrin is believed to be
especially useful in this respect). As carbonyl group of
benzaldehyde and acetophenone reacts with primary residual amine
groups of dendrimer (Generation=0) to form an imine. Reactions were
carried out using little excess of such reagents. Analysis of the
product by NMR indicated that the expected product 5 was formed
along with the other side products. Blocking only three residual
amine groups out of four was not achieved successfully and was
complicated due to distribution of benzaldehyde and acetophenone
molecules as well, since side products 9, 10 and others formed
along with the expected product 5. 3031
[0156] Model reactions 6.a and 6.b show that formation of Schiff
base followed by reduction of imine functional group can be
achieved. The presence of absorbance at 1642 cm.sup.-1 confirm the
formation of imine. On the other hand the absence of that
absorbance in IR spectrum after reduction proved that imine
functional group can be reduced. Furthermore, the secondary amine
can be pegylated and form the desired product. All the four primary
amine groups of dendrimer molecules (Generation=0) can be converted
to imine groups by reacting with carbonyl groups of, for example,
benzaldehyde as well as acetophenone.
[0157] The synthesis routes are shown in scheme 7 and 8 to remove
the hydrogen atoms attached to residual primary amine groups. An
examination of the spectra reveals the presence of absorbance
corresponding to imine functional groups. Analysis of the products
by IR and NMR indicates that all amine groups were converted to
imines. The imine groups of products 9 and 12 can be reduced by
selective reducing agent as dendrimer molecule contains acid amide
groups which are very sensitive to reducing agents. Sodium
cyanohydridoborate hydride is used for this specific purpose.
[0158] An examination of .sup.1H and .sup.13C NMR spectra as well
as IR spectra showed that the absence of the absorbance due to
imine group. The absorbance of the reduced products 10 and 13 are
identified. The hydrogen atoms of compounds 10 and 13 associated
with four nitrogen atoms are pegylated in the following step. NMR
analysis of the products indicates that compounds 10 as well as 13
were pegylated. The examination of spectral data showed the
formation of alcohol of the corresponding aldehyde as side product
as excess aldehyde was used for those reactions. In addition, Boron
complex compound was formed during the reduction step. Most of the
absorbance of the final product in .sup.1H and .sup.13C NMR spectra
is identified with the help of .sup.1H--.sup.1H and
.sup.1H--.sup.13C correlation spectra.
[0159] It is noticed that reaction schemes 7 as well as 8 can not
apparently be accomplished step by step. It is thought necessary
that reduced compounds 10 and 13 to be worked up in order to remove
unwanted Boron complex compounds after reduction of the imine.
[0160] It is observed that reduced product can not be dissolved
further in low boiling point solvent such as methanol once solvent
was removed after working up. This is apparently because of the
formation of intra-molecular H-bonding after the solvent is removed
at room temperature under vacuum. This kind of intra-molecular
H-bond makes reduced compounds 10 and 13 reluctant to form further
inter-molecular H-bonding with solvent. For the sake of simplicity
in isolating the product, a low boiling solvent is probably the
best choice for this reaction.
[0161] Therefore, a modified procedure is developed in order to
avoid such solubility problem. The problem associated with
solubility is avoided by pegylating compounds 10 and 13 before work
up. The analysis of .sup.1H NMR indicated that double bonds of
PEGMEA are reacted with secondary amine groups of compounds 10 and
13.
[0162] The reactions are carried and dendrimers are pegylated first
without working it up. Spectral data showed that dendrimer is
successfully pegylated. It can be problematic to work up the
pegylated products 11 and 14 as cleavage or hydrolysis could have
been possible during work up. However, examination of the .sup.1H,
.sup.13C NMR spectra showed that the PEG molecules remain unchanged
after treatment with strong acid and base. It is observed that
delicate ether and carbonyl moieties of pegylated products 11 and
14 are neither cleaved nor hydrolyzed.
[0163] Gel electrophoresis of compound 11 and 14 along with
different generation of dendrimer (ladder) are carried out to
confirm the formation of compounds 11 and 14. The migration of 10
and 11 is slower than that of 13 and 14 and is explained on the
basis of steric effects.
[0164] Aldehyde substituted beta cyclodextrins are especially
useful in the instant invention to block primary amines. For
example, an amine terminated polyethylene glycol can be reacted
with an aldehyde substituted beta cyclodextrin at room temperature
in aqueous sodium cyanohydridoborate to couple the cyclodextrine to
the polyethylene glycol via a nitrogen atom to form a pegylated
cyclodextrine adduct. Then, the pegylated cyclodextrine adduct can
be reacted with the polyoxyalkylene acrylate to form a cyclodextrin
adduct that is further pegylated.
[0165] Chitosan can be reacted with an aldehyde substituted beta
cyclodextrin at room temperature in aqueous sodium
cyanohydridoborate to block the primary amines of the chitosan
followed by reaction with the polyoxyalkylene acrylate to form a
pegylated and beta cyclodextrin substituted chitosan. Peptides,
polypeptides and proteins containing primary amines can be reacted
with an aldehyde substituted beta cyclodextrin at room temperature
in aqueous sodium cyanohydridoborate to block the primary amines of
the peptide, polypeptide or protein followed by reaction with the
polyoxyalkylene acrylate to form a pegylated and beta cyclodextrin
substituted peptide, polypeptide or protein.
[0166] As a specific example, the following scheme can be used in
the instant invention to first convert the primary amines of
poly-L-arginine to secondary amines by the addition of a
cyclodextrin to the amine group and then pegylation with a
polyethylene glycol acrylate. A 3 necked, 25-mL, round-bottomed,
flask is fitted with nitrogen inlet, a condenser with drying tube,
a rubber septum, and a magnetic stir bar. The following ingredients
are added to the flask: poly-L-arginine hydrochloride (5 mg, 0.663
pmol), beta cyclodextrin monoaldehyde (58.0 mg, 51.2 pmol), 2-mL of
deionized water and 1-mL sodium hydroxide solution (0.2586M).
Immediately after addition of the base, sodium cyanoborohydride
(8.6 mg, 0.137 mmol) is added. The reaction mixture is stirred for
72 hours at room temperature. Half the reaction mixture (1.5-mL) is
then removed and precipitated in 5-mL acetone for analysis to
confirm the desired reaction. The remainder of the reaction mixture
is mixed with 1-mL of poly(ethylene glycol)methyl ether acrylate
aqueous solution (0.0286M). The reaction mixture is then stirred
for an additional 72 hours at room temperature and then
precipitated in 10-mL of acetone and centrifuged to yield a white
solid that is dried under vacuum overnight. Analysis of the white
solid confirms the desired formation of a pegylated beta
cyclodextrin-poly-L-arginine conjugate.
[0167] As a specific further example, the following scheme can be
used in the instant invention to first convert the primary amines
of poly-L-lysine to secondary amines by the addition of a
cyclodextrin to the amine group and then pegylation with a
polyethylene glycol acrylate. A 3 necked, 25-mL, round-bottomed,
flask is fitted with a nitrogen inlet, a condenser with drying
tube, a rubber septum and a magnetic stir bar. The following
ingredients are added to the flask: poly-L-lysine hydrochloride (8
mg, 0.5 .mu.mol), beta cyclodextrin monoaldehyde (55.0 mg, 48.5
pmol), 2-mL of deionized water and 1-mL sodium hydroxide solution
(0.0485M). Immediately after addition of the base, sodium
cyanoborohydride (8.1 mg, 0.129 mmol) is added. The reaction
mixture is then stirred for 72 hours at room temperature. Half the
reaction mixture (1.5-mL) is then removed and precipitated in 5-mL
acetone for analysis to confirm the production of the desired
product. To the remainder of the reaction mixture, 0.5-mL
poly(ethylene glycol)methyl ether acrylate aqueous solution
(0.0502M) is added. The reaction mixture is then stirred for an
additional 72 hours at room temperature and then precipitated in
10-mL of acetone and centrifuged to yield a white sold that was
dried under vacuum overnight. Analysis of the white solid confirms
the desired formation of a pegylated beta
cyclodextrin-poly-L-lysine conjugate.
[0168] As a specific additional example, the following scheme can
be used in the instant invention to first convert the primary
amines of Chitosan to secondary amines by the addition of a
cyclodextrin to the amine group and then pegylation with a
polyethylene glycol acrylate. A 3 necked, 25-mL, round-bottomed,
flask is fitted with a nitrogen inlet, a condenser with drying
tube, a rubber septum and a magnetic stir bar. The following
ingredients are added to the flask: low molecular weight chitosan
(50 mg, 0.185 mmol) dissolved in 15-mL of 0.1M hydrochloric acid,
2.00 g of beta-glucero-phosphate dissolved in 4-mL of deionized
water, beta cyclodextrin monoaldehyde (421 mg, 0.370 mmol) and
sodium cyanoborohydride (33 mg, 0.525 mmol). The reaction mixture
is then stirred for 72 hours at room temperature. 14-mL of the
reaction mixture is removed for analysis to confirm the production
of the desired product. To the remainder of the reaction mixture,
4.50-mL poly(ethylene glycol)methyl ether acrylate aqueous solution
(0.0220M) is added. The reaction mixture is then stirred for and
additional 72 hours at room temperature. The resulting solution is
then lyophilized to yield a white fibrous solid. The solid is then
washed with acetone using a soxhlet and dried under vacuum
overnight. Analysis of the solid confirms the formation of the
desired pegylated beta cyclodextrin-chitosan conjugate.
[0169] As a yet further specific additional example, the following
scheme can be used in the instant invention to pegylate
glutathione. Glutathione (0.153 g, 0.0005 mole), poly (ethylene
glycol)methyl ether acrylate (0.225 g, 0.0005 mole) are dissolved
in 4 mL of a buffer solution (pH=5.8) in a screw cap vial. The
clear aqueous solution is allowed to mix on a tabletop shaker for 3
h at room temperature. The entire reaction mixture is lypholized to
produce the desired pegylated glutathione.
[0170] The following scheme can be used in the instant invention
for the pegylation of proteins: (a) the protein is dissolved or
dispersed in 0.1M bicarbonate buffer, pH 9.1 (the concentration of
proteins as a rule can be measured from their extinction
coefficients at 280 nm); (b) mPEG acrylate solutions are prepared
at various concentrations in 0.1M bicarbonate buffer, pH 9.1; (c) a
known volume of the protein solution is mixed with the mPEG
solution in various vials to yield various amino/mPEG ratios; (d)
samples are incubated under defined temperatures and times with
appropriate control tubes; and (e) after reaction, the reaction
mixture is subjected to native gel electrophoresis in 10%
polyacrylamide gels (protein staining, as a rule, is performed with
Coomassie Blue).
[0171] As a final additional specific example, the following scheme
can be used in the instant invention to first convert the primary
amines of a PAMAM dendrimer to secondary amines by the addition of
a cyclodextrin to the amine group and then pegylation with a
polyethylene glycol acrylate. A 3 necked, 25-mL, round-bottomed,
flask is fitted with a nitrogen inlet, a condenser with drying
tube, a rubber septum and a magnetic stir bar. The following
ingredients are added to the flask: PAMAM generation 0 dendrimer
(160 mg, 0.310 mmol) dissolved in 1-mL deionized water, beta
cyclodextrin monoaldehyde (1.5010 g, 1.32 mmol) dissolved in 15-mL
of deionized water and sodium cyanoborohydride (229.6 mg, 3.65
mmol). The reaction mixture is then stirred for 72 hours at room
temperature. 6-mL of the reaction mixture is then removed and
precipitated in methanol to confirm the production of the desired
product. To the remainder of the reaction mixture, 9-mL
poly(ethylene glycol) methyl ether acrylate aqueous solution
(0.0880M) are added. The reaction mixture is then stirred for an
additional 72 hours at room temperature and then precipitated in
10-mL of acetone and centrifuged to yield a white sold that was
dried under vacuum overnight. Analysis confirms the desired
production of pegylated beta cyclodextrin PAMAM dendrimer
conjugate.
[0172] Thus, it should be appreciated that in the instant invention
any compound containing an amine group can be reacted with the
polyoxyalkylene acrylate to form a conjugate comprising a
polyoxyalkylene sub-structure. Furthermore, when the amine group is
a primary amine, then it may be necessary (such as in the case of a
PAMAM dendrimer) as a preliminary step to "block" the primary
amine(s), as discussed above in detail, by reaction of such primary
amine(s) with an aldehyde or ketone followed by conversion of the
resulting imine to a secondary amine. Many drug compounds contain
amine group(s) and it should be understood that the instant
invention is an excellent means of converting such drugs to a
polyoxyalkylene conjugate of the drug.
[0173] The term "polyoxyalkylene" is defined in the above
referenced U.S. Pat. No. 6,280,745, herein fully incorporated by
reference, and includes polyethylene glycol, polypropylene glycol,
as well as block and random polyethylene glycol/polypropylene
glycol co-polymers. Although acrylate terminated polyethylene
glycols are commercially available, acrylate terminated
polyethylene glycol can be prepared, for example, by reacting a
monomethoxy polyethylene glycol with acryloyl chloride or, for
example, with methacroloyl chloride.
[0174] The molecular weight of the polyoxyalkylene sub-structure of
the instant invention can be tailored so that the conjugate has
desired properties such as solubility characteristics that are more
compatible with the biologic system. In many cases, the preferred
molecular weight of the polyoxyalkylene sub-structure of the
instant invention will be in the range of from about 500 to about
5000 grams per mole.
[0175] In addition to reactions with amines, the acrylate
terminated polyoxylakylene of the instant invention also can be
reacted with a terminal sulfur (sulfide) group(s) of a biologically
active compound to produce novel compounds. For example an aqueous
buffered (pH=5.8) solution of glutathione can be pegylated at room
temperature by a two hour reaction with the acrylate terminated
polyethylene glycol of the instant invention. Polycysteine can be
similarly pegylated.
[0176] The process of the instant invention produces novel
compounds that, as expected, maintain their biological activity.
For example, bovine erythrocyte carbonic anhydrase (CAB) pegylated
with mPEG acrylate at room temperature in a pH 9.1 aqueous buffer
(mole ratio of CAB to mPEG acrylate of 1:8; 1:2 and 8:1) maintains
its biological activity. As a further example, hen egg white
lysozyme (HEWL) pegylated with mPEG acrylate at room temperature in
a pH 9.1 aqueous buffer (mole ratio of HEWL to mPEG acrylate of 1:2
and 8:1) also maintains its biological activity.
* * * * *