21 research outputs found
Expedient Synthesis of Core Disaccharide Building Blocks from Natural Polysaccharides for Heparan Sulfate Oligosaccharide Assembly
The complex sulfation motifs of heparan sulfate glycosaminoglycans (HS GAGs) play critical roles in many important biological processes. However, an understanding of their specific functions has been hampered by an inability to synthesize large numbers of diverse, yet defined, HS structures. Here, we describe a new approach to access the four core disaccharides required for HS/heparin oligosaccharide assembly from natural polysaccharides. The use of disaccharides as minimal precursors rather than monosaccharides greatly accelerates the synthesis of HS GAGs, providing key disaccharide and tetrasaccharide intermediates in about half the number of steps compared to traditional strategies. Rapid access to such versatile intermediates will enable the generation of comprehensive libraries of sulfated oligosaccharides for unlocking the ‘sulfation code’ and understanding the roles of specific GAG structures in physiology and disease
Expedient Synthesis of Core Disaccharide Building Blocks from Natural Polysaccharides for Heparan Sulfate Oligosaccharide Assembly
The complex sulfation motifs of heparan sulfate glycosaminoglycans (HS GAGs) play critical roles in many important biological processes. However, an understanding of their specific functions has been hampered by an inability to synthesize large numbers of diverse, yet defined, HS structures. Here, we describe a new approach to access the four core disaccharides required for HS/heparin oligosaccharide assembly from natural polysaccharides. The use of disaccharides as minimal precursors rather than monosaccharides greatly accelerates the synthesis of HS GAGs, providing key disaccharide and tetrasaccharide intermediates in about half the number of steps compared to traditional strategies. Rapid access to such versatile intermediates will enable the generation of comprehensive libraries of sulfated oligosaccharides for unlocking the ‘sulfation code’ and understanding the roles of specific GAG structures in physiology and disease
Polymers for extended-release administration
Developing strategies to deliver the required dose of therapeutics into target tissues and cell populations within the body is a principal aim of controlled release and drug delivery. Specifically, there is an interest in developing formulations that can achieve drug concentrations within the therapeutic window, for extended periods of time, with tunable release profiles, and with minimal complication and distress for the patient. To date, drug delivery systems have been developed to serve as depots, triggers, and carriers for therapeutics including small molecules, biologics, and cell-based therapies. Notably, the efficacy of these systems is intricately tied to the manner in which they are administered. For example, systemic and oral routes of administration are common, but both can result in rapid clearance from the organism. Towards this end, what formulation and administration route strategies are available to prolong the bioavailability of therapeutics? Here, we discuss historical and modern drug delivery systems, with the intention of exploring how properties including formulation, administration route and chemical structure influence the ability to achieve extended-release drug release profiles within the body
The Carbamoylmannose Moiety of Bleomycin Mediates Selective Tumor Cell Targeting
Recently,
we reported that both bleomycin (BLM) and its disaccharide,
conjugated to the cyanine dye Cy5**, bound selectively to cancer cells.
Thus, the disaccharide moiety alone recapitulates the tumor cell targeting
properties of BLM. Here, we demonstrate that the conjugate of the
BLM carbamoylmannose moiety with Cy5** showed tumor cell selective
binding and also enhanced cellular uptake in most cancer cell lines.
The carbamoyl functionality was required for tumor cell targeting.
A dye conjugate prepared from a trivalent cluster of carbamoylmannose
exhibited levels of tumor cell binding and internalization significantly
greater than those of the simple carbamoylmannose–dye conjugate,
consistent with a possible multivalent receptor
Probing the Flexibility of the Catalytic Nucleophile in the Lyase Catalytic Pocket of Human DNA Polymerase β with Unnatural Lysine Analogues
DNA
polymerase β (Pol β) is a key enzyme in mammalian
base excision repair (BER), contributing stepwise 5′-deoxyribose
phosphate (dRP) lyase and “gap-filling” DNA polymerase
activities. The lyase reaction is believed to occur via a β-elimination
reaction following the formation of a Schiff base between the dRP
group at the pre-incised apurinic/apyrimidinic site and the ε-amino
group of Lys72. To probe the steric constraints on the formation and
subsequent resolution of the putative Schiff base intermediate within
the lyase catalytic pocket, Lys72 was replaced with each of several
nonproteinogenic lysine analogues. The modified Pol β enzymes
were produced by coupled <i>in vitro</i> transcription and
translation from a modified DNA template containing a TAG codon at
the position corresponding to Lys72. In the presence of a misacylated
tRNA<sub>CUA</sub> transcript, suppression of the UAG codon in the
transcribed mRNA led to elaboration of full length Pol β having
a lysine analogue at position 72. Replacement of the primary nucleophilic
amine with a secondary amine in the form of <i>N</i>-methyllysine
(<b>4</b>) affected mainly the stability of the Schiff base
intermediate and resulted in relatively moderate inhibition of lyase
activity and BER. Elongation of the side chain of the catalytic residue
by one methylene group, achieved by introduction of homolysine (<b>6</b>) at position 72, apparently shifted the amino group to a
position less favorable for Schiff base formation. Interestingly,
this effect was attenuated when the side chain was elongated by replacing
one side-chain methylene group with a bridging S atom (thialysine, <b>2</b>). In comparison, replacement of lysine 72 with an analogue
having a guanidine moiety in lieu of an ε-amino group (homoarginine, <b>5</b>) or a sterically constrained secondary amine (piperidinylalanine, <b>3</b>) led to almost complete suppression of dRP excision activity
and the ability of Pol β to support BER. These results help
to define the tolerance of Pol β to subtle local structural
and functional alterations
Modified Bleomycin Disaccharides Exhibiting Improved Tumor Cell Targeting
The
bleomycins (BLMs) are a family of antitumor antibiotics used
clinically for anticancer chemotherapy. Their antitumor selectivity
derives at least in part from their ability to target tumor cells,
a property that resides in the carbohydrate moiety of the antitumor
agent. In earlier studies, we have demonstrated that the tumor cell
selectivity resides in the mannose carbamoyl moiety of the BLM saccharide
and that both the BLM disaccharide and monosaccharide containing the
carbamoyl moiety were capable of the delivery/uptake of a conjugated
cyanine dye into cultured cancer cell lines. Presently, the nature
of the participation of the carbamoyl moiety has been explored further
to provide compounds of utility for defining the nature of the mechanism
of tumor cell recognition and uptake by BLM saccharides and in the
hope that more efficient compounds could be identified. A library
of seven disaccharide–Cy5** dye conjugates was prepared that
are structural analogues of the BLM disaccharide. These differed from
the natural BLM disaccharide in the position, orientation, and substitution
of the carbamoyl group. Studies of these compounds in four matched
sets of tumor and normal cell lines revealed a few that were both
tumor cell selective and internalized 2–4-fold more efficiently
than the natural BLM disaccharide
Structural Features Facilitating Tumor Cell Targeting and Internalization by Bleomycin and Its Disaccharide
We
have shown previously that the bleomycin (BLM) carbohydrate
moiety can recapitulate the tumor cell targeting effects of the entire
BLM molecule, that BLM itself is modular in nature consisting of a
DNA-cleaving aglycone which is delivered selectively to the interior
of tumor cells by its carbohydrate moiety, and that there are disaccharides
structurally related to the BLM disaccharide which are more efficient
than the natural disaccharide at tumor cell targeting/uptake. Because
BLM sugars can deliver molecular cargoes selectively to tumor cells,
and thus potentially form the basis for a novel antitumor strategy,
it seemed important to consider additional structural features capable
of affecting the efficiency of tumor cell recognition and delivery.
These included the effects of sugar polyvalency and net charge (at
physiological pH) on tumor cell recognition, internalization, and
trafficking. Since these parameters have been shown to affect cell
surface recognition, internalization, and distribution in other contexts,
this study has sought to define the effects of these structural features
on tumor cell recognition by bleomycin and its disaccharide. We demonstrate
that both can have a significant effect on tumor cell binding/internalization,
and present data which suggests that the metal ions normally bound
by bleomycin following clinical administration may significantly contribute
to the efficiency of tumor cell uptake, in addition to their characterized
function in DNA cleavage. A BLM disaccharide-Cy5** conjugate incorporating
the positively charged dipeptide d-Lys-d-Lys was
found to associate with both the mitochondria and the nuclear envelope
of DU145 cells, suggesting possible cellular targets for BLM disaccharide–cytotoxin
conjugates
Detection of Dihydrofolate Reductase Conformational Change by FRET Using Two Fluorescent Amino Acids
Two fluorescent amino
acids, including the novel fluorescent species
4-biphenyl-l-phenylalanine (<b>1</b>), have been incorporated
at positions 17 and 115 of dihydrofolate reductase (DHFR) to enable
a study of conformational changes associated with inhibitor binding.
Unlike most studies involving fluorescently labeled proteins, the
fluorophores were incorporated into the amino acid side chains, and
both probes [<b>1</b> and l-(7-hydroxycoumarin-4-yl)ethylglycine
(<b>2</b>)] were smaller than fluorophores typically used for
such studies. The DHFR positions were chosen as potentially useful
for Förster resonance energy transfer (FRET) measurements on
the basis of their estimated separation (17–18 Å) and
the expected change in distance along the reaction coordinate. Also
of interest was the steric accessibility of the two sites: Glu17 is
on the surface of DHFR, while Ile115 is within a folded region of
the protein. Modified DHFR I (<b>1</b> at position 17; <b>2</b> at position 115) and DHFR II (<b>2</b> at position
17; <b>1</b> at position 115) were both catalytically competent.
However, DHFR II containing the potentially rotatable biphenylphenylalanine
moiety at sterically encumbered position 115 was significantly more
active than DHFR I. Irradiation of the modified DHFRs at 280 nm effected
excitation of <b>1</b>, energy transfer to <b>2</b>, and
emission by <b>2</b> at 450 nm. However, the energy transfer
was substantially more efficient in DHFR II. The effect of inhibitor
binding was also measured. Trimethoprim mediated concentration-dependent
diminution of the emission observed at 450 nm for DHFR II but not
for DHFR I. These findings demonstrate that amino acids containing
small fluorophores can be introduced into DHFR with minimal disruption
of function and in a fashion that enables sensitive monitoring of
changes in DHFR conformation
Fluorescent Biphenyl Derivatives of Phenylalanine Suitable for Protein Modification
In a recent study, we demonstrated
that structurally compact fluorophores
incorporated into the side chains of amino acids could be introduced
into dihydrofolate reductase from Escherichia coli (<i>ec</i>DHFR) with minimal disruption of protein structure
or function, even when the site of incorporation was within a folded
region of the protein. The modified proteins could be employed for
FRET measurements, providing sensitive monitors of changes in protein
conformation. The very favorable results achieved in that study encouraged
us to prepare additional fluorescent amino acids of potential utility
for studying protein dynamics. Presently, we describe the synthesis
and photophysical characterization of four positional isomers of biphenyl-phenylalanine,
all of which were found to exhibit potentially useful fluorescent
properties. All four phenylalanine derivatives were used to activate
suppressor tRNA transcripts and incorporated into multiple positions
of <i>ec</i>DHFR. All phenylalanine derivatives were incorporated
with good efficiency into position 16 of <i>ec</i>DHFR and
afforded modified proteins that consumed NADPH at rates up to about
twice the rate measured for wild type. This phenomenon has been noted
on a number of occasions previously and shown to be due to an increase
in the off-rate of tetrahydrofolate from the enzyme, altering a step
that is normally rate limiting. When introduced into sterically accessible
position 49, the four phenylalanine derivatives afforded DHFRs having
catalytic function comparable to wild type. The four phenylalanine
derivatives were also introduced into position 115 of <i>ec</i>DHFR, which is known to be a folded region of the protein less tolerant
of structural alteration. As anticipated, significant differences
were noted in the catalytic efficiencies of the derived proteins.
The ability of two of the sizable biphenyl-phenylalanine derivatives
to be accommodated at position 115 with minimal perturbation of DHFR
function is attributed to rotational flexibility about the biphenyl
bonds
Fluorescent Biphenyl Derivatives of Phenylalanine Suitable for Protein Modification
In a recent study, we demonstrated
that structurally compact fluorophores
incorporated into the side chains of amino acids could be introduced
into dihydrofolate reductase from Escherichia coli (<i>ec</i>DHFR) with minimal disruption of protein structure
or function, even when the site of incorporation was within a folded
region of the protein. The modified proteins could be employed for
FRET measurements, providing sensitive monitors of changes in protein
conformation. The very favorable results achieved in that study encouraged
us to prepare additional fluorescent amino acids of potential utility
for studying protein dynamics. Presently, we describe the synthesis
and photophysical characterization of four positional isomers of biphenyl-phenylalanine,
all of which were found to exhibit potentially useful fluorescent
properties. All four phenylalanine derivatives were used to activate
suppressor tRNA transcripts and incorporated into multiple positions
of <i>ec</i>DHFR. All phenylalanine derivatives were incorporated
with good efficiency into position 16 of <i>ec</i>DHFR and
afforded modified proteins that consumed NADPH at rates up to about
twice the rate measured for wild type. This phenomenon has been noted
on a number of occasions previously and shown to be due to an increase
in the off-rate of tetrahydrofolate from the enzyme, altering a step
that is normally rate limiting. When introduced into sterically accessible
position 49, the four phenylalanine derivatives afforded DHFRs having
catalytic function comparable to wild type. The four phenylalanine
derivatives were also introduced into position 115 of <i>ec</i>DHFR, which is known to be a folded region of the protein less tolerant
of structural alteration. As anticipated, significant differences
were noted in the catalytic efficiencies of the derived proteins.
The ability of two of the sizable biphenyl-phenylalanine derivatives
to be accommodated at position 115 with minimal perturbation of DHFR
function is attributed to rotational flexibility about the biphenyl
bonds