Biomedical Engineering Reference
In-Depth Information
2004 [
140
], in a landmark paper that also reported the first structure-activity studies
on AFGPs, and thus provided data that are essential to understanding the mechanism
of action and to guiding the design of AFGP mimics. It was reported in an earlier
communication polymeric AFGP of molecular weight (
M
W
)
D
6-7 k could be
efficiently synthesized by simple polymerization of the repeating glycopeptides
unit of AFGP with diphenylphosphoryl azide (DPPA) as a promoter [
141
]. In the
presence of the unprotected hydroxyl groups in the sugars, DPPA selectively acti-
vates the peptide carboxylic acid and by using this method, glycopeptides polymers
of different ranges of molecular weight were produced. Through optimization of
the chemistry, polymerization occurs most efficiently when using the glycoside of
Ala-Thr-Ala; steric effects are minimized in the polymerization reaction compared
with Ala-Ala-Thr with a glycoside C terminus [
143
]. Additional sugars were also
appended enzymatically to the disaccharides [
143
].
The details of the overall synthesis is described in reference [
140
]. Low-
molecular-weight fractions containing between two and eight repeating units
(syAFGP2-syAFGP8) were obtained using a lower temperature and shorter reaction
times. Consistent fractions of precise chain lengths containing up to seven repeating
units were achieved by using recycling preparative size-exclusion chromatography.
The effectiveness of 1-isobutoxycarbonyl-2-isobutoxy-1,2-dihydroquinoline
(IIDQ) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4 methylmorpholinium chloride
(DMTMM) versus DPPA was also assessed as promoters for the formation of
the glycopolymers [
33
]. DMT-MM-mediated condensation reactions proceeded
smoothly, even though the average molecular weights of the polymers obtained
with IIDQ were lower than those obtained with DPPA, and therefore provides a
more general reagent for the synthesis of AFGP analogues consisting base-labile
functional groups.
The recent NMR solution structure of syAFGP3 showed a significant popu-
lation of a folded conformation with hydrophilic and hydrophobic faces [
140
].
This solution structure, and clear evidence for the important role of hydrophobic
interactions in the interactions of several classes of AFPs with ice [
44
,
131
,
144
,
145
] has refocused attention on understanding the possible role of hydrophobicity
in the mechanism of action of AFGPs. Of particular note is the high prevalence of
Ala and Thr residues in both type I AFPs [
145
] and AFGPs. It should be noted
that AFGP8 at high concentrations (20-40 mM) exists predominately as dimer
aggregates with higher thermal hysteresis activity as shown through dynamic light
scattering and CD studies [
44
]. Overall solution conformation does not appear to
be affected by these aggregates, and diffusion NMR studies of AFGP8 revealed no
evidence for aggregation in solution at biologically relevant conditions [
97
]. In light
of these structural studies, recent solution studies by a variety of techniques have
looked for any proof for a preferred conformation of AFGPs at close to 0
ı
C, or a
rise in population of folded conformers that might be important in the interaction
with the ice/water interfaces. The dynamics, structure, and conformational free
energy of AFGP8, the smallest 14-residue AFGP containing two Pro residues, were
studied by molecular dynamics [
146
]. The results confirmed that N-acetyl group
plays an important role, with hydrogen bonding between the N-acetyl group and
