Biomedical Engineering Reference
In-Depth Information
heparin. Some selectivity by AT activation protocols is possible by use of the AT glycans that are on
the exterior of the protein and have some structures not observed in the polypeptide moiety. Thus,
AT can be mildly treated with NaIO
4
, which reacts with vicinal hydroxyls (at C8 and C9) on terminal
N-acetylneuraminic acid residues of the glycans to give active aldehyde groups. The AT-aldehyde
derivative can then be reacted with diamino-alkane
NaBH
3
CN to give an AT with a prominent
alkyl-amine that will preferentially (relative to other AT R-groups) react with N-hydroxysuccin-
imidyl-4-azidobenzoate to yield a photosensitive group for heparin coupling as above. The advan-
tages of linkage through AT-glycan activation are that linkage location(s) is/are more controlled
and the critical sites on the polypeptide, where enzyme reaction and heparin-binding reside, are
unperturbed. Synthesis of ATH by simultaneous reaction with ATH by a bifunctional reagent or
spacer is more complicated than initial activation of the serpin or the GAG alone. Since AT has
R-groups with similar reactivity to functional groups on heparin, differentiation of heparin func-
tional groups from those of AT is not practical. To somewhat control linkage points, it would be
advisable to use hetero-bifunctional reagents that can select for AT R-groups not represented on
heparin. Thus, at least one end of the bifunctional agent could be directed to a particular subset of
locations on the AT as an anchor for positioning linkage on the heparin. To illustrate this concept,
a reagent terminating with a vicinal dione would selectively react with the guanidinyl R-group
of an AT arginyl residue. Linkage to heparin could then be completed at the opposite end of the
reagent by a
+
N(CH
3
)
3
+
group (attractive to polyanionic heparin) and a photoactivatable
N
3
func-
tion. The bifunctional linkage approach does allow for unmodifi ed ATH to form a native complex
prior to covalent stabilization. Unfortunately, even the most sophisticated approaches seem to lack
selectivity on the placement of linkage points, and prevention of multilinkages that lead to reduced
product activity is diffi cult.
As stated, formation of permanent linkage to AT through activated groups on the heparin spe-
cies has been the main algorithm followed for ATH synthesis. The robust nature of heparin for
performance of activation chemistries and minimal functional group types make for increased sim-
plicity in method design.
-
-
18.5.2 C
HEMICAL
S
TRUCTURES
AND
In Vitro
A
CTIVITIES
Evolution of ATH design has followed a chronology that is illustrative of issues impacting product
yield and anticoagulant functionality. Several reports have described isolation of noncovalent
AT-UFH complexes.
224-227
However, without covalent bonds to hold the ATH together, even the
most robust complexes are unlikely to be secure against dissociation by interaction with the vast
array of molecules and cell surfaces
in vivo
.
Early endeavors toward permanently bonded AT-heparin utilized knowledge gained from inves-
tigations of chemical linkage of heparin to polymer surfaces and other macromolecules. Heparin
coupling to albumin
228,229
and polysaccharides
230
gave insight into useful conjugation chemistries
to build on. About 25 years ago Ceustermans et al. gave a broad survey of potential chemical routes
attempting to realize ATH.
231
In one procedure, cyanogen bromide (CNBr) activated UFH (using
the method of Cuatrecasas et al.)
232
was mixed with AT but no signifi cant ATH was detected. Given
that CNBr is most reactive with the few amino groups
231
of heparin molecules, attachment of active
groups would be minimized. Consequently, many of the small number of CNBr activation points
might also be distant from potential linkage groups on AT during noncovalent complexation. Other
reaction schemes followed. Attempts were made to bind AT and UFH with the bifunctional 1,5-
difl uoro-2,4-dinitrobenzene,
231
according to the procedure of Zahn and Meienhofer
233,234
without
success, again likely due to a paucity of heparin amino groups for nucleophilic substitution at the
benzene ring. AT was modifi ed with active groups by photochemical reaction with either 4-fl uoro-
3-nitrophenyl azide or 4-azido-phenacyl bromide and then mixed with heparin.
235-238
Negligible
yields ensued, potentially arising from either intramolecular cross-linking reactions within AT or
from lack of heparin-amino acceptor groups.
