The Two Bioinorganic Projects of Research Methodology Fall 2012
Ligand synthesis
MeOH
20 h or so
in NaOH pH ~11
Solvent : aqueous ethylene glycol
What is the significance
of this project?
Tris-chelate Ru compexes
bind to DNA…
… where the strength of
binding corresponds to a
match of chirality
between the Ru complex
and DNA
Look at how snugly this ‘red’ complex fits into
the groove:
B-DNA is a right hand helix
and
that red complex is “right-handed” too!
How can that be?
[Ru(bpy)2(DPPZ)]2+
Simplify first to see chirality
Simplify again
to see handedness
[Ru(bpy)3]2+
What is the point symmetry?
To have complex oriented
properly to see this, arrange
complex so that one end of
each chelate makes a triangle.
‘lambda’ isomer, left handed
‘delta’ isomer, right handed
Λ - [Ru(bpy)3]2+
Δ - [Ru(bpy)3]2+
What we could see for [Ru(bpy)3]2+
Λ - [Ru(bpy)2(DPPZ)]2+
Δ - [Ru(bpy)2(DPPZ)]2+
Is also true for [Ru(bpy)2(DPPZ)]2+
And also for all the other [Ru(bpy)2(L)]2+ complexes you will make
Bioinorganic Research in the Burgmayer Labs
then we add them to DNA and see what happens . . . . .
Do they bind to DNA?
How?
Are they able to damage
DNA?
What aspect of Ru
complexes causes DNA
damage?
Intercalation mode
Groove binding mode
Metals Work with DNA
DNA is the target of anti-cancer metal drugs
University of Richmond, November 2010
1. cis-Platin, the most famous example,
used since 1977
binds to DNA, distorts,
prevents DNA repair,
leads to cell death
Pteridines meet Ru
University of Richmond, November 2010
Why we decide to jump into the game
So let’s see if
we can make this,
and see what happens
with DNA
Some questions about the reaction:
1) Why are Cl- ligands on Ru replaced?
2) What role do Cl- ions play after step 1?
3) Why is the reaction heated?
Theses two complexes are special;
They are made from different ligands that
undergoes a reaction
on the metal during heating.
2+
N
N
N
4
NH
N
2
1
Ru(bpy)2Cl2
N
N
N
OH
H2O / HO
N
N
N
140 oC, 2.5 hours
NH
Ru
O
N
O
N
O
N
N
N
N
N
N
NH2
Ru
N
N
NH2
N
N
110 oC, 3 hr
L-keto ligand
"Ru(L-keto)"
"Ru(L-aap)"
Theses two complexes are special;
The ligand undergoes a reaction
on the metal during heating.
2
2+
N
N
N
N
N
N
4
N
N
1
2
Ru(bpy)2Cl2
N
N
N
N
Ru
H2O / HO
OH
N
N
N
N
N
140 oC, 2.5 hours
N
N
N
N
N
Ru
N
N
110 oC, 3 hr
L-UN ligand
"Ru(L-UN)"
very unstable!!!
"Ru(L-alap)"
O
NH2
How will we know when reactions are done?
ESI-MS
Electrospray Ionization Mass Spectrometry ESI-MS
black box
Molecules in gas phase as ions + or -
magnetic field
separates
light and heavy
mass ions
mass
detector
Electrospray ionization method
makes tiny droplets
Vacuum removes solvent
Electric potential
creates + and - ions
For discussion:
Porphyrin
Pteridinyl
Phenanthroline
1)
What did you think about the two very different procedure formats last week?
Reaction scale is 2.0 mmol using reagents in an equimolar ratio.
Phen dione is dissolved in 15 mL MeOH.
Diamine reagent is dissolved in either MeOH or water made basic with dilute NaOH.
Two reagent solutions combined in round bottom flask.
Mixture is stirred overnight.
Product is isolated by filtration (Hirsch funnel, filter paper).
Product is washed with water, methanol and diethylether.
**All manipulations including isolation of the product must be performed in a hood.**
A stir bar and 0.3L propionic acid are added to a 500-mL three-neck, round-bottom flask fitted
with a reflux condenser. Put the addition funnel in the center neck, the condensor on a side
neck and a septum on the third neck. The acid is brought to a reflux and 0.1 mol of the
appropriate substituted benzaldehyde is added.
Quantities of aldehydes:
benzaldehyde [for TPP synthesis] = 10.16 mL
p-tolualdehyde [for TTP synthesis] = 11.8 mL
4-chlorobenzaldehyde [for TClPP synthesis] = 14.06 g
To the refluxing solution, 10 mL (0.1 mol) freshly distilled pyrrole is cautiously added via a
dropping funnel. A recommended rate is about 1 drop/sec.
Caution! The condensation reaction of the pyrrole with the aldehyde is extremely exothermic,
and the rate of pyrrole addition must be continuously monitored. The solution is refluxed for
30 min, cooled to ambient temperature, and chilled in an ice bath before vacuum filtering off
the purple, crystalline product. [Note: Filtrations can be slow. Try adding small volumes of
For discussion:
1) What did you think about the two very different
procedure formats last week?
2) Why were there two ligand synthesis methods?
3) How best to clean glassware?
Brief Outline of Ru reaction steps:
1)
2)
3)
4)
Purge reactants in solution with N2 gas.
Heat / reflux / cause reaction to occur
Cool then filter to remove unwanted solids
Add NH4PF6 in excess to ppt the complex,
[Ru(bpy)y(L-)]2+ (PF6)2
5) Filter to isolate next week