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Aminolyses of reactive monomers and polymers—II. N-hydroxyphthalimide esters
Ei.-opt'an Pohmer Journal. Vol. 15. pp. 603 to 605 © Pergamon Press Ltd 1979. Printed in Great Britain
0014-3057 79 0601-0603$02 (X) 0
A M I N O L Y S E S O F REACTIVE M O N O M E R S POLYMERS--II N-HYDROXYPHTHALIMIDE
AND
ESTERS
J. LABSK~" and J. KALAL Institute of Macromolecular Chemistry, Czechoslovak Academy of Sciences, 162 06 Prague 6, Czechoslovakia (Receited 17 October 1978)
Abstract--Some N-hydroxyphthalimide esters of N-methacrylated r,J-amino acids have been prepared and copolymerized with N-(2-hydroxypropyl)methacrylamide. The rates of aminolysis of monomers and copolymers have been compared.
INTRODUCTION
One of the possibilities for binding a biologically active c o m p o u n d to a soluble or insoluble polymer carrier consists in the formation of an amide bond between the polymer and the substrate. The vast array of information on the chemistry of peptides facilitates the choice of binding possibilities which may be applied in polymer chemistry. One of these possibilities is provided by activated esters, e.g. 4-nitrophenol esters already reported I-1,2]. Since 4-nitrophenol, released in the binding of the substrate to the polymer, may deactivate or exhibit toxic effects in some applications with sensitive biological materials, attempts have been made to find other activated esters. in this work, N-hydroxyphthalimide esters of methacryloylated to-amino acids were used in the preparation of soluble copolymers with N-(2-hydroxypropyl)methacrylamide. The polymers prepared may be of molecular weight higher t h a n for 4-nitrophenol esters: they react selectively with the amino group, their reactivity is higher than that for 4-nitrophenol esters, and at elevated temperature they may react also with the primary hydroxyl group. EXPERIMENTAL
Chemicals arm monomers
t,~-Amino acids and their derivatives have been denoted using a numerical system analogous to the letter code used for :t-amino acids. The figure indicates the number of carbon atoms of the amino acid; substitution at the amino group is expressed by a symbol for the respective acyl before the number, substitution of the hydrogen atom in the carboxylic group is expressed by a symbol for the respective alkyl behind the figure. Combination of two or more amino acids is expressed by combining the respective numerical symbols joined by a dash. Amino acids were commercial products or were obtained by the acid hydrolysis of the corresponding lactams (Fluka). Methacrylation proceeded under the conditions for the Schotten-Baumann reaction [1]; esterification of N-hydroxyphthalimide IFlukaJ was performed with dicyclohexylcarbodiimide. Physical properties of N-hydroxyphthalimide esters are given in Table 1. 603
N-Hydroxyphthalimide ester of 7-methacryloylaminoheptanoic acid (MA-7-ONHP): 20mmol of dicyclohexylcarbodiimide (4.1 g) and 21 mmol of N-hydroxyphthalimide (3.4 g) were added to 20 mmol (4.3 g) of 7-methacryloylaminoheptanoic acid in 100 ml chloroform with cooling to - 1 0 . The reaction mixture then stood for 24 hr al ambient temperature. The resulting dicyclohexyl urea was removed and the reaction mixture was shaken six times with 5", NaHCO 3 solution, until an orange-red colouration disappeared. The product was crystallized from ethanol-hexane mixture. Copolymers The copolymers were prepared by precipitation copolymerization in acetone at 60 (15 wt", of monomers: 5", of the monomer M2:0.1 wt"., of AIBN based on weight of monomers; 5 hr). N-(2-Hydroxypropyl)methacrylamide was prepared by a described method [3]. Molecular weights of copolymers were determined in DMF with Photo-Gonio Diffusometer Sofica 42.000. The characteristics of copolymers are given in Table 2. Aminolyses of monomers and copolymers The changes of concentrations of bound N-hydroxyphthalimide esters during aminolysis were followed using the spectrophotometer Cary 14. The concentrations of NHP esters and amines were 5 x 10-3M and 0.5M respectively: DMSO was used as solvent. The measurements were carried out in a 1 mm cell at 22 + 0.1 and at 295 nm. At this wavelength, there is the absorption maximum of NHP esters and also the largest difference between the absorptions of free and bound NHP. The amine solution was metered by precise syringes and the period between mixing and the beginning of measurement was 10see at most. To compare the rates of aminolysis, the time t t 2 (sec) corresponding to cleavage of the half of acylated N H P ester was employed. The end of reaction was always checked with ammonia. DISCUSSION
M o n o m e r s and copolymers containing N-hydroxyphthalimide esters were prepared, undergoing readily the aminolytic reaction with formation of amide linkage. As aminolysis by primary amines with the straight-chain alkyl groups is too rapid, sterically shielded amines were used for aminolysis. The generally higher reactivity of N H P esters follows from the comparison of rates of aminolysis from N H P esters
604
J. LABSKY and J. KALAL
Table 1. N-Hydroxyphthalimide esters of amino acids Compound
M.p. (C)
Yield E . [ l . m o l - l c m -t] ('!o) a b
MA-ONHP MA-2-ONHP MA-3-ONHP MA-4-ONHP MA-5-ONHP MA-6-ONHP MA-7-ONHP MA-12-ONHP MA-2-2-ONH P
121-123 162-163 122-123 91-93 83-85 85-86 87-88 88-90 178-180
78 55 38 35 26 40 60 20 30
1890 2100 2000 2060 1900 1880 1950 2010 2150
42,220 49,850 54,400 50,890 41,500 41,500 44,800 48,500 52,700
* Measured in DMSO at 25°; a 295nm; b 216nm. All elemental analyses are in good agreement with theory.
Table 3. Half-times (see) of aminolyses of hydroxyphthalimide esters in DMSO: A: tert-butylamine, B: tert-butylethylamine
MA-ONHP MA-2-ONHP MA-3-ONHP MA-4-ONHP MA-5-ONHP MA-6-ONHP MA-7-ONHP MA-12-ONHP MA-2-2-ONHP
Conversion (o,,)
MA-ONHP MA-2-ONHP MA-3-ONHP MA-4-ONHP MA-5-ONHP MA-6-ONHP MA-7-ONHP MA- 12-ONH P MA-2-2-ONH P
61 78 63 72 68 72 68 65 58
M2 Content (molO,,)
M~
2.3 1.48 1.3 2.6 2.7 2.5 1.2 1.8 2.0
125,000 242,000 113,000 180,000 57,000 82,000 69,000 98,000 78,000
with the corresponding monomers and polymers containing 4-nitrophenol [1] esters. This increase in the rate of aminolysis may be explained by the greater induction effect of two carbonyl groups in the vicinity of aminolytically cleavable linkage.
,_oo_o_ CO
Aminolysis of N-methacryloylphthalimide ONHP) proceeds thus:
I•H2CC ^u . ~ /
(MA-
R2NH
H~,, IOIe 111
--.-c,,c-c-o-,
125 8 8 12 15 20 25 30 8
,6P I R'.._C_N__CH
b
210 15 18 25 25 24 25 31 15
\
C--O--NHP
For the monomer MA-3-ONHP, it is possible to assume the cooperative effect of the amide bond which forms in the six-membered ring an intramolecular hydrogen bridge with the ester carbonyl. The carbon atom of the ester group carbonyl, in the sixmember ring thus created, is less accessible for the nucleophilic attack of amine with a bulky substituent for steric reasons than in the case of the five-membered ring. With increasing number of methylene groups between the amide bond and the aminolytically cleavable ester bond, the rate of aminolysis became steady (within the limits of experimental error) because the formation of intramolecular hydrogen bridges is less probable.
RzNH
/c
CH2 II NR 2
(~NHR2
The rates of aminolysis of copolymers with N H P esters are always less than the rates for corresponding monomeric esters. This fact may be explained by the shielding effect of the polymer chain preventing the bulky aikyl groups of the amines from approaching the ester bond. This mechanism has been proposed only on the basis of comparison with rates of aminolysis for 4-nitrophenol esters of analogous monomers, i.r.
R'Cl =N. -- ,CH2
7
o
/ H 0 / ~) el~/\NHR2
el~)/xO-NHP O
e_ ~ c o ~
R-- ICI--N -- CH2 I t
H I¢/C\N R2
600 75 160 200 235 230 320 350 70
,6P . 16,e , I R-C''N/
,6,e 2 ~
450 35 125 140 165 160 170 165 28
The configuration of the five-membered ring also contributes to the high rate of aminolysis of monomer MA-2-ONHP; the inductive effect of the amide bond may also contribute.
NHR 2
Different rates of aminolysis by various amines may be explained by the different steric accessibilities of the amino groups and by the different basicities of the amines. A substantial increase in the rate of aminolysis with the next member of the homologous series (Table 3), i.e. MA-2-ONHP monomer, is obviously caused by the cooperative effect of the amide bond which leads to the formation of intermolecular hydrogen bridges. The aminolysis of monomer MA-2O N H P can be described thus:
Copolymer with HPMA* Amine A B
* N-(2-Hydroxypropyl)met hacrylamide.
Table 2. Copolymers of N-(2-hydroxypropyllmethacrylamide with NHP esters (M2) Monomer M2
Monomer Amine A B
Monomer type
Aminolyses of reactive monomers and polymers--II Spectra of the monomers MA-2-ONHP, MA-3O N H P and M A - 4 - O N H P in an inert solvent do not unambiguously reveal formation of intramolecular hydrogen bridges with the carbonyl of ester group. The formation of intermolecular hydrogen bridges with the carbonyl of the phthalimide group is not excluded.
605
REFERENCES
1. J. Labsk~, and J. Kfilal, Europ. Polym. J. 18, 167 11979). 2. J. Coupek, J. Labsk~, J. Kfilal, J. TurkovL and O. Valentov~i, Biochem. hiophy.s. Acta 481, 289 (1977). 3. J. Kope6ek and H. Ba~ilova~ Europ. Pol)'m. J. 9, 7 (1973).
0014-3057 79 0601-0603$02 (X) 0
A M I N O L Y S E S O F REACTIVE M O N O M E R S POLYMERS--II N-HYDROXYPHTHALIMIDE
AND
ESTERS
J. LABSK~" and J. KALAL Institute of Macromolecular Chemistry, Czechoslovak Academy of Sciences, 162 06 Prague 6, Czechoslovakia (Receited 17 October 1978)
Abstract--Some N-hydroxyphthalimide esters of N-methacrylated r,J-amino acids have been prepared and copolymerized with N-(2-hydroxypropyl)methacrylamide. The rates of aminolysis of monomers and copolymers have been compared.
INTRODUCTION
One of the possibilities for binding a biologically active c o m p o u n d to a soluble or insoluble polymer carrier consists in the formation of an amide bond between the polymer and the substrate. The vast array of information on the chemistry of peptides facilitates the choice of binding possibilities which may be applied in polymer chemistry. One of these possibilities is provided by activated esters, e.g. 4-nitrophenol esters already reported I-1,2]. Since 4-nitrophenol, released in the binding of the substrate to the polymer, may deactivate or exhibit toxic effects in some applications with sensitive biological materials, attempts have been made to find other activated esters. in this work, N-hydroxyphthalimide esters of methacryloylated to-amino acids were used in the preparation of soluble copolymers with N-(2-hydroxypropyl)methacrylamide. The polymers prepared may be of molecular weight higher t h a n for 4-nitrophenol esters: they react selectively with the amino group, their reactivity is higher than that for 4-nitrophenol esters, and at elevated temperature they may react also with the primary hydroxyl group. EXPERIMENTAL
Chemicals arm monomers
t,~-Amino acids and their derivatives have been denoted using a numerical system analogous to the letter code used for :t-amino acids. The figure indicates the number of carbon atoms of the amino acid; substitution at the amino group is expressed by a symbol for the respective acyl before the number, substitution of the hydrogen atom in the carboxylic group is expressed by a symbol for the respective alkyl behind the figure. Combination of two or more amino acids is expressed by combining the respective numerical symbols joined by a dash. Amino acids were commercial products or were obtained by the acid hydrolysis of the corresponding lactams (Fluka). Methacrylation proceeded under the conditions for the Schotten-Baumann reaction [1]; esterification of N-hydroxyphthalimide IFlukaJ was performed with dicyclohexylcarbodiimide. Physical properties of N-hydroxyphthalimide esters are given in Table 1. 603
N-Hydroxyphthalimide ester of 7-methacryloylaminoheptanoic acid (MA-7-ONHP): 20mmol of dicyclohexylcarbodiimide (4.1 g) and 21 mmol of N-hydroxyphthalimide (3.4 g) were added to 20 mmol (4.3 g) of 7-methacryloylaminoheptanoic acid in 100 ml chloroform with cooling to - 1 0 . The reaction mixture then stood for 24 hr al ambient temperature. The resulting dicyclohexyl urea was removed and the reaction mixture was shaken six times with 5", NaHCO 3 solution, until an orange-red colouration disappeared. The product was crystallized from ethanol-hexane mixture. Copolymers The copolymers were prepared by precipitation copolymerization in acetone at 60 (15 wt", of monomers: 5", of the monomer M2:0.1 wt"., of AIBN based on weight of monomers; 5 hr). N-(2-Hydroxypropyl)methacrylamide was prepared by a described method [3]. Molecular weights of copolymers were determined in DMF with Photo-Gonio Diffusometer Sofica 42.000. The characteristics of copolymers are given in Table 2. Aminolyses of monomers and copolymers The changes of concentrations of bound N-hydroxyphthalimide esters during aminolysis were followed using the spectrophotometer Cary 14. The concentrations of NHP esters and amines were 5 x 10-3M and 0.5M respectively: DMSO was used as solvent. The measurements were carried out in a 1 mm cell at 22 + 0.1 and at 295 nm. At this wavelength, there is the absorption maximum of NHP esters and also the largest difference between the absorptions of free and bound NHP. The amine solution was metered by precise syringes and the period between mixing and the beginning of measurement was 10see at most. To compare the rates of aminolysis, the time t t 2 (sec) corresponding to cleavage of the half of acylated N H P ester was employed. The end of reaction was always checked with ammonia. DISCUSSION
M o n o m e r s and copolymers containing N-hydroxyphthalimide esters were prepared, undergoing readily the aminolytic reaction with formation of amide linkage. As aminolysis by primary amines with the straight-chain alkyl groups is too rapid, sterically shielded amines were used for aminolysis. The generally higher reactivity of N H P esters follows from the comparison of rates of aminolysis from N H P esters
604
J. LABSKY and J. KALAL
Table 1. N-Hydroxyphthalimide esters of amino acids Compound
M.p. (C)
Yield E . [ l . m o l - l c m -t] ('!o) a b
MA-ONHP MA-2-ONHP MA-3-ONHP MA-4-ONHP MA-5-ONHP MA-6-ONHP MA-7-ONHP MA-12-ONHP MA-2-2-ONH P
121-123 162-163 122-123 91-93 83-85 85-86 87-88 88-90 178-180
78 55 38 35 26 40 60 20 30
1890 2100 2000 2060 1900 1880 1950 2010 2150
42,220 49,850 54,400 50,890 41,500 41,500 44,800 48,500 52,700
* Measured in DMSO at 25°; a 295nm; b 216nm. All elemental analyses are in good agreement with theory.
Table 3. Half-times (see) of aminolyses of hydroxyphthalimide esters in DMSO: A: tert-butylamine, B: tert-butylethylamine
MA-ONHP MA-2-ONHP MA-3-ONHP MA-4-ONHP MA-5-ONHP MA-6-ONHP MA-7-ONHP MA-12-ONHP MA-2-2-ONHP
Conversion (o,,)
MA-ONHP MA-2-ONHP MA-3-ONHP MA-4-ONHP MA-5-ONHP MA-6-ONHP MA-7-ONHP MA- 12-ONH P MA-2-2-ONH P
61 78 63 72 68 72 68 65 58
M2 Content (molO,,)
M~
2.3 1.48 1.3 2.6 2.7 2.5 1.2 1.8 2.0
125,000 242,000 113,000 180,000 57,000 82,000 69,000 98,000 78,000
with the corresponding monomers and polymers containing 4-nitrophenol [1] esters. This increase in the rate of aminolysis may be explained by the greater induction effect of two carbonyl groups in the vicinity of aminolytically cleavable linkage.
,_oo_o_ CO
Aminolysis of N-methacryloylphthalimide ONHP) proceeds thus:
I•H2CC ^u . ~ /
(MA-
R2NH
H~,, IOIe 111
--.-c,,c-c-o-,
125 8 8 12 15 20 25 30 8
,6P I R'.._C_N__CH
b
210 15 18 25 25 24 25 31 15
\
C--O--NHP
For the monomer MA-3-ONHP, it is possible to assume the cooperative effect of the amide bond which forms in the six-membered ring an intramolecular hydrogen bridge with the ester carbonyl. The carbon atom of the ester group carbonyl, in the sixmember ring thus created, is less accessible for the nucleophilic attack of amine with a bulky substituent for steric reasons than in the case of the five-membered ring. With increasing number of methylene groups between the amide bond and the aminolytically cleavable ester bond, the rate of aminolysis became steady (within the limits of experimental error) because the formation of intramolecular hydrogen bridges is less probable.
RzNH
/c
CH2 II NR 2
(~NHR2
The rates of aminolysis of copolymers with N H P esters are always less than the rates for corresponding monomeric esters. This fact may be explained by the shielding effect of the polymer chain preventing the bulky aikyl groups of the amines from approaching the ester bond. This mechanism has been proposed only on the basis of comparison with rates of aminolysis for 4-nitrophenol esters of analogous monomers, i.r.
R'Cl =N. -- ,CH2
7
o
/ H 0 / ~) el~/\NHR2
el~)/xO-NHP O
e_ ~ c o ~
R-- ICI--N -- CH2 I t
H I¢/C\N R2
600 75 160 200 235 230 320 350 70
,6P . 16,e , I R-C''N/
,6,e 2 ~
450 35 125 140 165 160 170 165 28
The configuration of the five-membered ring also contributes to the high rate of aminolysis of monomer MA-2-ONHP; the inductive effect of the amide bond may also contribute.
NHR 2
Different rates of aminolysis by various amines may be explained by the different steric accessibilities of the amino groups and by the different basicities of the amines. A substantial increase in the rate of aminolysis with the next member of the homologous series (Table 3), i.e. MA-2-ONHP monomer, is obviously caused by the cooperative effect of the amide bond which leads to the formation of intermolecular hydrogen bridges. The aminolysis of monomer MA-2O N H P can be described thus:
Copolymer with HPMA* Amine A B
* N-(2-Hydroxypropyl)met hacrylamide.
Table 2. Copolymers of N-(2-hydroxypropyllmethacrylamide with NHP esters (M2) Monomer M2
Monomer Amine A B
Monomer type
Aminolyses of reactive monomers and polymers--II Spectra of the monomers MA-2-ONHP, MA-3O N H P and M A - 4 - O N H P in an inert solvent do not unambiguously reveal formation of intramolecular hydrogen bridges with the carbonyl of ester group. The formation of intermolecular hydrogen bridges with the carbonyl of the phthalimide group is not excluded.
605
REFERENCES
1. J. Labsk~, and J. Kfilal, Europ. Polym. J. 18, 167 11979). 2. J. Coupek, J. Labsk~, J. Kfilal, J. TurkovL and O. Valentov~i, Biochem. hiophy.s. Acta 481, 289 (1977). 3. J. Kope6ek and H. Ba~ilova~ Europ. Pol)'m. J. 9, 7 (1973).