Competitive Reactions During Amine Addition to cis-Aconityl Anhydride
473
Synthesis of cis-Aconityl Glycyl Amide (19) Under Interfacial
Conditions
since an analogous strategy has been used in conjugation
protocols (e.g. (3)→(4), Scheme 1). Unfortunately to date,
attempts to prepare active esters (e.g. pentachloro phenol,
mixed anhydrides, acid chlorides, N-hydroxysuccinimide
esters) produced complex product mixtures (1H NMR).
These reactions were typically deeply red-brown coloured
solutions with several products observed by thin layer
To a 10 mL single neck round bottom flask with a magnetic stirrer was
added cis-aconityl anhydride (2) (1.20 g, 7.7 mmol, 1.2 equiv.) and
reagent grade acetonitrile (5.0 mL). Glycine (0.49 g, 6.5 mmol) was
added to this solution and the suspension stirred for 36 h at ambient
temperature. The solid was isolated by vacuum filtration and washed
with acetonitrile to give the desired product (19) (1.32 g, yield 91%) as
an off-white coloured solid after drying in vacuum. 1H NMR: (in D2O,
Na2CO3, 250 MHz) δ 5.82, s, 1H; 3.88, s, 2H; 3.26, s, 2H. 13C NMR:
δ 180.4, 179.5, 164.6, 150.9, 126.0, 120.4, 46.5, 45.9. FT-IR: 1713
and 1697 (C=O), 1634 (C=C), 1520 (C–N), 1048 cm–1. Anal.
Found: C, 41.3; H, 4.0; N, 5.9%. Calc. for C8H9O7N: C, 41.6; H, 3.9; N,
6.1%.
chromatography suggesting
a significant amount of
decomposition had occurred. The C2,3 double bond is
vinylic in the glycine adduct (19) for the C4 acid, and allylic
for the C6 acid. Carboxylate activation at either the C4 or C6
carboxylic acid moiety with coupling reagents (e.g.
carbodiimide, carbonate) may have sufficiently lowered the
pKa of the C5 protons leading to deleterious coupling
reactions. Activation also produced a reactive cross-
conjugated system where double bond isomerization through
1,3-allyl shifts may be more facile. These possible side
reactions would also affect the outcome of conjugations (e.g.
(3)→(4), Scheme 1). Although the reaction of glycine adduct
(19) in the presence of diisopropylcarbodiimide and
poly(ethylene glycol) diamine gave a polymer that degraded
at acidic pH values,[4] it was highly probable this polymer
was not structurally homogeneous. Direct activation of cis-
aconityl anhydride (2) either as the C6 acid chloride (e.g.
oxalyl chloride/DMF) or C6 methyl ester (e.g.
diazomethane) to provide a low molecular weight monomer
Reaction of cis-Aconityl Anhydride with 4-Aminobutyric Acid Under
Interfacial Conditions
To a 10 mL single neck round bottom flask with a magnetic stirrer was
added cis-aconitic anhydride (2) (0.26 g, 2.1 mmol, 1.2 equiv.) and
acetonitrile (5.0 mL). To this solution was added 4-aminobutyric acid
(0.18 g, 1.7 mmol, 1 equiv.). An orange paste formed as the suspension
stirred for 36 h at ambient temperature. A slightly yellow powder was
isolated by vacuum filtration and washed with acetonitrile to give the
desired product (0.033 g, 7.3%) as an off-white coloured solid after
drying in vacuum. 1H NMR: (in D2O, Na2CO3, 250 MHz) δ 5.79, s, 1H;
3.32, t, 2H; 3.26, s, 2H; 2.33, t, 2H; 1.87, quint, 2H. Analysis of the
1
paste by H NMR indicated the presence of 4-aminobutyric acid, cis-
and trans-aconityl acids and unidentified trace products.
Acknowledgment
We are very grateful for funding from the Wellcome Trust.
also
resulted
in
complex
mixtures.
Adding
diisopropylcarbodiimide to a solution of cis-aconityl
anhydride (2) gave highly coloured solutions that implied
decomposition had occurred. No polymer was isolated when
poly(ethylene glycol) diamine was added to a solution
References
[1] R. Duncan, S. Dimitrijevic, E. Evagorou, S.T.P. Pharma 1996, 6,
237.
[2] M. Clochard, S. Rankin, S. Brocchini, Macromol. Rapid
Commun. 2000, 853.
containing
cis-aconityl
anhydride
(2)
and
diisopropylcarbodiimide.
[3] R. Tomlinson, M. Klee, S. Garrett, J. Heller, R. Duncan, S.
Brocchini, Macromolecules 2002, 35, 473.
Conclusions
[4] S. Simic, M. Clochard, S. Brocchini, Abstr. Pap.—Am. Chem.
Soc. 2001, 453.
In our efforts to develop hydrolytically labile, acid sensitive
biomedical polymers using cis-aconityl anhydride (2) it has
become apparent that this anhydride undergoes a range of
competitive side reactions during amine acylation that
include decarboxylation, isomerization and hydrolysis.
These side reactions have probably also occurred when cis-
aconityl anhydride (2) was used for the conjugation of
bioactive agents to proteins and polymers. Because the
aconityl moiety possesses excellent properties for enhanced
rates of hydrolysis at slightly acidic values, further work is
continuing to ensure that the aconityl structure can be used
in conjugation applications and as a basis for the design of
biomedical polymers.
[5] W. Shen, H. Ryser, Biochem. Biophys. Res. Commun. 1981, 102,
1048.
[6] W. Choi, P. Kopeckova, T. Minko, J. Kopecek, J. Bioact. Compat.
Polym. 1999, 14, 447.
[7] M. Wirth, A. Fuchs, M. Wolf, B. Ertl, F. Gabor, Pharm. Res.
1998, 15, 1031.
[8] D. Gaal, F. Hudecz, Eur. J. Cancer 1998, 34, 155.
[9] A. Al-Shamkhani, R. Duncan, Int. J. Pharm. 1995, 122, 107.
[10] F. Hudecz, J. Clegg, J. Kajtar, M. Embleton, M. Szekerke, R.
Baldwin, Bioconjugate Chem. 1992, 3, 49.
[11] J. Sinkule, S. Rosen, J. Radosevich, Tumor Biol. 1991, 12, 198.
[12] R. Dillman, D. Johnson, D. Shawler, J. Koziol, Cancer Res. 1988,
48, 6097.
[13] H. Yang, R. Reisfeld, Proc. Natl Acad. Sci. U.S.A. 1988, 85, 1189.
[14] E. Diener, U. Diner, A. Sinha, S. Xie, R. Vergidis, Science 1986,
231, 148.
Experimental
[15] T. Etrych, M. Jelínková, B. Ríhová, K. Ulbrich, J. Controlled
Release 2001, 73, 89.
[16] H. Yoo, E. Lee, T. Park, J. Controlled Release 2002, 82, 17.
[17] E. Lavie, D. Hirschberg, G. Schreiber, K. Thor, L. Hill, I.
Hellstrom, K. Hellstrom, Cancer Immunol. Immunother. 1991,
33, 223.
[18] R. Gaudreault, B. Bellemare, J. Lacroix, Anticancer Res. 1989, 9,
1201.
[19] P. Thorpe, 1993, PCT WO9318793 A1.
[20] P. Schoen, J. Corver, D. Meijer, J. Wilschut, P. Swart, Biochem.
Pharmacol. 1997, 53, 995.
General
All solvents were purchased from Aldrich or BDH; cis-aconityl
anhydride, itaconyl anhydride and citraconyl anhydride and the
corresponding acids were purchased from Aldrich and used as received.
The amino acids, and aliphatic and aromatic amines were also
purchased from Aldrich and used as received. NMR analysis was
performed using a Bruker AM250 or AM500 FT-NMR spectrometer
and infrared (FT-IR) spectra were obtained with a Nicolet Avatar
360 FT-IR using OMNIC E.S.P. 5.0 software.