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sky, I.; Ishai-Michaeli, R.; Kovalchuk, O.; Haloun, C.;
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15. Gunnarsson, G. T.; Desai, U. R. Bioorg. Med. Chem.
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16. The introduction of non-anionic N-acyl and O-acyl groups
into heparin structure previously focused on introducing
hydrophobic, alkyl, structures. For specific examples, see
Refs. 10,11.
17. During preparation of this manuscript So¨rme et al.
reported the identification and tuning of a cation–p
interaction (an arginine–arene interaction) in the study
of aryl-substituted disaccharide inhibitors of galectin-3,
see: (a) So¨rme, P.; Arnoux, P.; Kahl-Knutsson, B.; Leffler,
H.; Rini, J. M.; Nilsson, U. J. J. Am. Chem. Soc. 2005,
127, 1737–1743; For general references on cation–p
interactions, see: (b) Ma, J. C.; Dougherty, D. A. Chem.
Rev. 1997, 97, 1303–1324; (c) Crowley, P. B.; Golovin, A.
Proteins 2005, 59, 231–239.
18. A discussion, with references, regarding our rationale for
heteroaromatic moieties interacting with lysine and argi-
nine based on oligonucleotide–protein interactions is
provided with Supplementary data.
19. (a) Inoue, Y.; Nagasawa, K. Carbohydr. Res. 1976, 46, 87–
95; (b) Nagasawa, K.; Inoue, Y.; Kamata, T. Carbohydr.
Res. 1977, 58, 47–55, During preparation of this manu-
script Naggi et al. reported graded N-desulfonation/N-
acetylation of heparin derivatives, see Ref. 9c.
10. Early studies on the chemical modification of heparin
toward modulating physical properties and anticoagulant
activity included the preparation and partial characteriza-
tion of N-acyl heparin derivatives, see for example: (a)
Hirano, S.; Ohashi, W. Agric. Biol. Chem. 1976, 40, 2501–
2502; (b) Hirano, S.; Ohashi, W. Carbohydr. Res. 1977, 59,
285–288; (c) Mallard, J.; Michel, V.; Jolly, R. Ann. Pharm.
Fr. 1963, 21, 501–504; Hydrophobization of heparin was
more recently investigated by partial N-desulfonation
followed by formation of imines with glutaraldehyde and
dodecanal or amides with cholic acid and stearic acid, see:
(d) Diancourt, F.; Braud, C.; Vert, M. J. Bioact. Compat.
Polym. 1994, 9, 267–281; (e) Diancourt, F.; Braud, C.;
Vert, M. J. Bioact. Compat. Polym. 1996, 11, 203–218; The
partial, low-level, N-acylation of heparin with oleoyl and
palmitoyl groups has been investigated toward preparing
non-anticoagulant heparin for specific applications. For
inhibition of AIDS virus, see: (f) Clayette, P.; Moczar, E.;
´
Mabondzo, A.; Martin, M.; Toutain, B.; Marce, D.;
Dormont, D. AIDS Res. Hum. Retroviruses 1996, 12, 63–
69; For inhibition or protection of heparin-binding
proteases, see: (g) Legras, S.; Diczhazi, C.; Moczar, M.
Int. J. Biol. Macromol. 1992, 14, 97–99; (h) Baici, A.;
´
Diczhazi, C.; Neszmelyi, A.; Moczar, E.; Hornebeck, W.
Biochem. Pharmacol. 1993, 46, 1545–1549; (i) Moczar, E.;
Hornebeck, W. Int. J. Biol. Macromol. 1991, 13, 261–262;
The enzymatic degradation of N-propionylated heparin
has been studied, see (j) Moffat, C. F.; Long, W. F.;
McLean, M. W.; Williamson, F. B. Arch. Biochem.
Biophys. 1997, 338, 201–206.
20. Yosizawa, Z.; Kotoku, T.; Yamauchim, F.; Matsuno, M.
Biochim. Biophys. Acta 1967, 141, 358–365.
21. The N-sulfo content of all fractions was compared to that
of parent heparin using the nitrous acid-based method as
previously reported, see: Inoue, Y.; Nagasawa, K. Anal.
Biochem. 1976, 71, 46–52, Analysis of N-sulfo content for
each heparin fraction and control heparin using this
method provided percent free amine values that were
within 5% of the values obtained and reported using the
TNBS assay.
11. Acylation of free hydroxyl groups in heparin without
removal of anionic moieties has been evaluated toward of
a number of potential therapeutic applications and to
modulating physical properties of heparin. For recent
ˆ
examples, see: (a) Barzu, T.; Level, M.; Petitou, M.;
Lormeau, J.-C.; Choay, J.; Schols, D.; Baba, M.; Pauwels,
R.; Witvrouw, M.; De Clercq, E. J. Med. Chem. 1993, 36,
3546–3555; (b) Petitou, M.; Coudert, C.; Level, M.;
Lormeau, J.-C.; Zuber, M.; Simenel, C.; Fournier, J.-P.;
Choay, J. Carbohydr. Res. 1992, 236, 107–119; (c) Pukac,
L. A.; Hirsch, G. M.; Lormeau, J.-C.; Petitou, M.; Choay,
J.; Karnovsky, M. J. Am. J. Pathol. 1991, 139, 1501–1509;
22. Sudo, M.; Sato, K.; Chaidedgumjorn, A.; Toyoda, H.;
Toida, T.; Imanari, T. Anal. Biochem. 2001, 297, 42–51.
23. Heparin is a heterogeneous polymer containing primarily
N-sulfo groups, but also N-acetyl groups. Percent free
amine is based on total amine obtainable upon complete
N-desulfonation. The N-acetyl groups remain unaltered.
In long, highly charged, N-sulfonated sequences of hep-
arin the following approximations correlate percent free
amine content to alteration of heparin sequences: 10%,
one modified disaccharide per ten disaccharide units, 20%:
one modified disaccharide per five disaccharide units, 33%:
one modified disaccharide per hexasaccharide, 50%: one
modified disaccharide per tetrasaccharide, 75%: three of
every four disaccharide units modified. The random and
sequential removal of N-sulfo groups from the microhet-
erogeneous heparin chain does not allow definition of
adjacency for the N-desulfonated residues. Indeed, we
anticipated from the beginning of this proof of concept
study that hits from library screening would provide the
starting point for future studies to determine the precise
length and/or sequence of novel chemically modified
heparin sequences that bind individual heparin-binding
proteins.
ˆ
`
(d) Barzu, T.; Desmouliere, A.; Herbert, J. M.; Level, M.;
Herault, J. P.; Petitou, M.; Lormeau, J.-C.; Gabbiani, G.;
Pascal, M. Eur. J. Pharmacol. 1992, 219, 225–233; (e)
´
Saivin, S.; Petitou, M.; Lormeau, J.-C.; Dupouy, D.; Sie,
P.; Caranobe, C.; Houin, G.; Boneu, B. Thromb. Haemost.
1992, 67, 346–351.
12. For a review on the synthesis of heparin and other
glycosaminoglycans, see: Yeung, B. K. S.; Chong, P. Y.
C.; Petillo, P. A. J. Carbohydr. Chem. 2002, 21, 799–865;
Recent reports describing the synthesis of natural and/or
heparin sequences include: (a) Rele, S. M.; Iyer, S. S.;
Baskaran, S.; Chaikof, E. L. J. Org. Chem. 2004, 69, 9159–
9170; (b) Orgueira, H. A.; Bartolozzi, A.; Schell, P.;
Litjens, R. E. J. N.; Plamacci, E. R.; Seeberger, P. H.
Chem. Eur. J. 2003, 9, 140–169; (c) Lubineau, A.; Lortat-
´
Jacob, H.; Gavard, O.; Sarrazin, S.; Bonnaffe, D. Chem.
Eur. J. 2004, 10, 4265–4282.
24. For a recent report describing standard N-acetylation of
heparin, see Ref. 9c. For various other conditions reported
for the N-acylation of heparin, see Ref. 10.
25. The loss of sulfate via acetylative-desulfonation has been
studied, see: (a) Hyatt, J. A. Carbohydr. Res. 1993, 239,
291–296; Loss of N-sulfo groups was also observed, but
overcome, during previous studies looking to O-acylate
heparin-derived disaccharides using amine bases in organ-
13. Zhang, J.; Rivers, G.; Zhu, Y.; Jacobson, A.; Peyers, J.;
Grundstrom, G.; Burch, P.; Hussein, S.; Marolewski, A.;
Herlihy, W.; Rusche, J. Bioorg. Med. Chem. 2001, 9, 825–
836.
14. For recent examples, see: (a) Monien, B. H.; Desai, U. R.
J. Med. Chem. 2005, 48, 1269–1273; (b) Benezra, M.;
Ishai-Michaeli, R.; Ben-Sasson, S. A.; Vladovsky, I. J.