InsulinMimetic Activity of Unnatural IPGs - PowerPoint PPT Presentation

Title: InsulinMimetic Activity of Unnatural IPGs


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Insulin-Mimetic Activity of Unnatural IPGs
Lauren Bowen, Nilanjana Chakraborty, David
Turner, Viatcheslav Azev, and Marc
d'Alarcao Michael Laboratory, Department of
Chemistry, Tufts University, Medford, MA 02155
The proposed synthesis of a second generation
thiol-containing IPG, 2,6-dideoxy-2-amino-6-(3-mer
captopropyl-(a1-6)-myo-inositol-1,2-cyclic
phosphate is shown below.
I. What are IPGs? Insulin signaling at
the target tissue is complex, involving a cell
surface insulin receptor that triggers a series
of protein and lipid phosphorylations in the
cytoplasm of the target cell. Glucose transport
and storage enzymes in the cell are controlled by
these phosphorylations. In addition to this
direct receptor-induced phosphorylation cascade,
a second signaling pathway appears to involve the
release of one or more oligosaccharides from a
cell-surface glycolipid into the solution
surrounding the cell. These oligosaccharides
contain inositol and phosphate and so are known
as inositol phosphate glycans (IPGs). IPGs, once
released, are able to signal cells to store
glucose, even without insulin! Since these
compounds work later in the signaling pathway, it
is hoped that they (or appropriate analogues)
might be useful in the treatment of type II
diabetes. The structures of natural IPGs
are not fully known, in part because they are
produced in minute amounts, but also because
there are several very structurally similar
molecules involved in signaling. Nevertheless, it
is clear that the structures contain elements
very similar to the GPI membrane anchors that
tether some proteins to cell membranes, but
differ in that the signaling IPGs may contain
either chiro-inositol or myo-inositol and either
glucosamine or galactosamine. Clearly what is
needed to sort out this issue is a study
chemically well-defined IPGs.
Synthesis of a thiol-containing IPG
Percent of maximal insulin response (MIR) by 20
mM IPG or 5 nM insulin in native rat
adipocytes. Chakraborty et al. Bioorg. Med. Chem.
2005, 13, in press.
III. IPGs can be active extracellularly. It is
know that after natural IPGs are generated in the
extracellular fluid, they are actively
transported into the cells. However, fluorescence
microscopy and FACS of a fluorescent, synthetic
IPG analog demonstrated that the fluorescent IPG
analog was confined to the extracellular solution
and further assays confirmed that the conjugate
was competent to stimulate lipogenesis in native
rat adipocytes. This confirms that although
native IPGs are internalized, this
internalization is not necessary for insulin-like
activities in adipocytes.
GPI protein membrane anchors
Fluorescent IPG analogue
a TfN3, CuSO4, K2CO3, H2O/CH2Cl2/MeOH, rt, 18h
b Ac2O, DMAP, pyridine,rt c H resin,
MeOH/H2O, 70 oC, 3d d benzaldehyde dimethyl
acetal, 10-()-camphor sulfonic acid, DMF, 50 oC,
12hr e BzOBt, Et3N, CH2Cl2, rt, 48h f BnBr,
Ag2O, CH2Cl2, rt to 0 oC to rt, 48hr g BH3/THF,
TMSOTf, 0 oC, 10hr h NaH, AllBr, DMF, 0 oC to
rt, 2hr j LiOH, H2O, THF kCl3CCN, K2CO3 l
TMSOTf m CH3OPOCl2, pyridine n BnSH, AIBN,
benzene, 70 oC o Na, NH3.
Hung et al. J. Am. Chem. Soc. 2001, 123, 3153.
Lindberg et al. Tetrahedron 2002, 58, 4245.
Due to the difficult nature of natural IPG
characterization, synthetic preparations are
necessary to both define and characterize QSARs
inherent to IPGs in the insulin-signaling
pathway. This work will be used to generate a
small library of similar thiol-containing
compounds for the further characterization and
possible use of these compounds as putative
second messengers for the treatment of type II
diabetes.
Generating a small library of compounds from a
standard template
2,6-Dideoxy-2-amino-6-mercaptoglucopyranosyl-(a1-6
)-myo-inositol 1,2-cyclic phosphate conjugated
with Lucifer yellow. Turner et al. Bioorg. Med.
Chem. Lett. 2005, 15, 2023.
II. Can Synthetic IPGs Effectively Activate
Insulin-Sensitive Cells? Several groups
worldwide are actively seeking to determine what
the structural requirements are for efficient
activation of insulin-sensitive cells by IPGs.
One strategy, which has met with rather limited
success, is to isolate and test natural IPGs. A
more effective strategy has been to synthesize
IPGs of known structure and determine their
biological activity. Our lab has been engaged in
this search for several years, and we have been
carefully refining our understanding of the
structural requirements for IPG action. Some
representative synthetic IPGs and their ability
to stimulate native rat adipocytes are shown
below.
IV. IPGs containing non-carbohydrate moieties A
compounds structural motifs are strongly
correlated to its biological properties.
Therefore, developing a structure-activity
relationship is desirable to characterize the IPG
moieties necessary for insulin-like stimulation
of cells. Although D-glucosaminyl-(a1-6)-myo-inosi
tol-1,2-cyclic phosphate has been shown to be a
minimum structural motif for insulin-like
activity, various functional groups, particularly
distal anionic groups, have been shown to
increase the ability of IPGs to stimulate glucose
utilization in insulin-sensitive tissues.
However, most IPG analogues that have been tested
are either all carbohydrate based or
glycopeptide-based. The experiment cited above,
with the fluorescent IPG analogue demonstrates,
among other things, that an IPG analogue
containing a relatively large non-carbohydrate
moiety can be active in stimulating
insulin-sensitive cells. Indeed, this conjugate
was more active than the parent disaccharide,
though still not as active as the anionic
tetrasaccharide analogues. Since oligosaccharide
synthesis remains labor intensive and therefore
time-consuming and costly, it would be very
desirable to understand the scope and limitations
for structural analogues of IPGs containing more
easily prepared non-carbohydrate portions. A goal
of this project is to synthesize a small library
of compounds obtained by conjugation of a
thiol-containing disaccharide IPG and a variety
of non-carbohydrate-based electrophiles.
Representative synthetic IPGs
Acknowledgments We are grateful to Merck Research
Laboratories for a Summer Undergraduate Research
Fellowship to L.B. Funds from Tufts University
are also acknowledged. The Tufts University NMR
and mass spectrometry facilities are supported in
part by National Science Foundation Grants CHE
032078 and CHE 9723772, respectively.