In an effort to offer complementary technology for covalent biomolecule modification (bioconjugation), we have developed a method that exploits the aqueous acceleration of Diels--Alder reactions for this purpose. Three different diene phosphoramidite reagents have been synthesized that enable diene modification of synthetic oligonucleotides prepared by the phosphoramidite method. Clean and efficient Diels--Alder cycloaddition of these diene oligonucleotides with maleimide dieneophiles was carried out, and the labeled oligonucleotide bioconjugates were characterized by HPLC and electrospray mass spectrometry. Dieneophile stoichiometry, temperature, and pH are all parameters that were shown to influence the efficiency of the process.
Presented is a conceptually novel approach to oligonucleotide
synthesis referred to herein as dimethoxytrityl resin product
anchored sequential synthesis (DMT PASS). The DMT PASS
process is characterized by the reaction of a 3‘-protected
nucleoside or oligonucleotide with an excess of a nucleotide 3‘-phosphoramite or H-phosphonate which is bound to a dimethoxytrityl functionalized polystyrene resin. As a result, successfully
coupled oligonucleotide product is then attached to the solid
support, allowing for removal and potential recovery of starting
materials. The protected oligonucleotide product is then released
and subjected to an aqueous/organic extractive purification,
which serves to remove monomeric impurities. The PASS
process is anticipated to provide for the cost-effective manufacture of oligonucleotides on a scale which would allow for
clinical development and pharmaceutical product commercialization. Herein we describe some aspects of our process
development progress and discuss preliminary applications of
the process to the synthesis of short oligodeoxyribonucleotide
sequences, as well as a number of challenges confronting the
PASS development effort.
A series of boronated aluminas was prepared by the reaction of
BX3 (X = F, Cl, Br) with alumina
or by a two-step synthesis involving the reaction of
BH3·Me2S with alumina and then with
I2. The
modified aluminas,
BX
n
/Al2O3, were found to
have n = 3 (F), n = 1.5 (Cl), n
= 1 (Br), and n = 1 (I).
The acidity of the solids (indicator dyes) was large and decreased
in the order I ∼ Br > Cl > F.
The solids catalyzed the Diels−Alder reactions of methyl
acrylate with cyclopentadiene and isoprene
with methyl acrylate, both of which occurred in high yield and with
high selectivity. The
regioselectivity of the isoprene/methyl acrylate reaction in fact is
higher than seen previously. The
results of the heterogeneous reactions were compared to those catayzed
by B-bromo- and
B-chlorocatecholborane, which serve as models for the
boronated aluminas.
A Novel Reagent for the Chemical Phosphorylation of Oligonucleotides -[design, synthesis and application of the novel phosphoramidite (I) in the preparation of 5'-phosphorylated oligonucleotides]. -(LEUCK*, M.; VAGLE, K. E.; ROACH, J. S.; WOLTER, A.; Tetrahedron Lett. 45 (2004) 2, 321-324; Proligo Biochem. GmbH, D-21147 Hamburg, Germany; Eng.) -Mais 16-175
Butenal reacts with B-bromocatecholborane in CD 2 Cl 2 at -78°C to form two species in a 1:1 ratio which does not vary between -20°C and -100°C; IR and NMR experiments ( 1 H-1 H COSY and 1 H-13 C HETCOR and 1 H-1 H ROESY [CAMELSPIN]) demonstrate that the two species are π complexes in which the aldehyde exists in s-cis and s-trans conformations which interconvent slowly ( 1 H-1 H EXSY NMR).
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