5CCC2010 Advanced Chemistry for Life Sciences (22~23 SEM1 000001) CHEAT SHEET
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Course
5CCC2010 Advanced Chemistry (5CCC2010)
Institution
Kings College London (KCL)
Choosing advanced chemistry was one of the worst descions of my life. As much as I like chemistry, chemistry does not like me. If you feel the same- this document is for you!
This double sided A4 "cheat" sheet contains ALL and I REALLY MEAN ALL of the content from the whole of the semester. It i...
Spectroscopy o Useful for fingerprinting of complex biological environments Shifts in Absorbances – Solvatochroism o Secondary structure gives characteristic absorptions
- Determines the structure of a molecule Surface Enhanced Raman Scattering (SERS) - Solvatochromic behaviour – absorbance is dependent on the polarity of the solvent it Ramachandran Plot
- Can improve Raman signal using nanoparticles that have surface plasmon resonances is in
Importantly, allows for the rapid detection of molecules and allows us to quickly access - Proteins are a mixture of secondary structure therefore need to quantify CD spectrum
changes in structures. o E.g. Au/Ag nanoparticles - Change in absorbance arise from how a solvent stabilises the ground (or excited) state - Mean residue ellipticity (MRE) can be used to plot data on absolute scale
What is spectroscopy? o Attach analytes to the surface or have nanoparticles in close proximity of the molecule - Can be deconvoluted to give approximate contributions of secondary structure
Interaction of electromagnetic radiation with matter Nanoparticles for SERS biosensing o Changes energy gap between them
→
Absorption = matter receiving energy from radiation - Fewer stretches visible in Raman than FTIR o
Emission = matter releasing energy as radiation o Easier to pick out signals in complex environments
[ Θ ] 222=−9500 ,Φ α ,222=0.20
- The more polar the solvent the more stable the zwitterionic ground state
Excitation from ground state to excited state is quantised. - Nanoparticles can be used for SERS in biological environments
o Fluids/cells
→ →
= have discrete bands of energy
- Targeted delivery allow detection of biomarkers without needing to purify
the larger the HOMO-LUMO gap absorbance undergoes blueshift
c
- Can be used for cancer
UV/Vis Spectroscopy (towards UV) o
λ= Molecular Orbitals
- Molecules are collections of atoms connected by shared electrons – bonds
Change the molecule, Change the absorbance
[ Θ ] 208=−8962 ,Φ α ,208=0.17
v
- A molecules absorbance can often be changed by performing simple chemical
- Bonds form by combination of atomic orbitals to give molecular orbitals reactions
- Atomic orbitals in phase = Constructive overlap - Small changes in conjugation can have a big effect on the absorbance
1
o Bonding molecular orbitals o These empirical relationships can use MRE at 222nm and 208nm to estimate the
- pH indicator e.g. Phenol Red
~v= - Atomic orbitals out of phase = Destructive overlap
o Anti-bonding molecular orbitals
o
o
pH>8.2 = red
pH<6.8 = yellow
α-helical content
Thermal stability of Proteins
λ
- Anti-bonding molecular orbitals are formed simultaneously with the bonding - Proteins are minimally stable structures
- para-Nitrophenol
molecular orbitals o ~10kJmol-1
o goes from colourless to yellow under basic conditions, forming p-nitrophenolate
- HOMO = Highest Occupied Molecular Orbital - The folded form exists in equilibrium with the denatured form
o p-nitrophenolate can be incorporated into model compounds, so that after a
hc - LUMO = Lowest Unoccupied Molecular Orbital o Under physiological conditions, the equilibrium lies in favour of the folded state
E=hv= =hc ~v
bond cleavage, p-nitrophenolate is released
- Molecular orbitals only form when the atomic orbitals are close in energy (e.g. N2) o Easily shifted by temperature, chemical denaturants solvents
Allows you to monitor the reaction and measure rate
- Non-bonding orbitals (or lone pairs) occur when atomic orbitals are far apart in energy Lipase - Temperature dependent (CD) spectra gives information on the thermal stability of
λ or are of incompatible symmetry (e.g. HF)
o p and s orbitals cannot combine therefore cannot form a bond
- Molecular orbitals apply across the whole molecule – the bigger the molecule, the
- Enzyme catalyses the hydrolysis od fatty acid esters
- Important for many industrial biotechnology applications
what is being measured
o With the correct experiment, Kc can be calculated which can lead to the free
energy
Wavelength ∝ Energy o Can be found in “bio” laundry detergents
more complex the molecule (e.g. Benzene) - Equilibrium requires 2 distinct states: beginning and end
IR Spectroscopy Measuring Reaction kinetics
UV/Vis o To determine the equilibrium, first need to pick a point that changes with the
- IR part of the spectrum is at higher wavelength therefore lower energy than UV/Vis - If reagent is colourimetric – releases a coloured product – kinetics can be measured
- Measures the absorbance of Ultraviolet and Visible radiation temperature (or other reactive conditions)
o Vibrational Excitation using UV/Vis spectroscopy
- The molecular orbitals is what absorbs the light and gives its colour o Wavelength chosen should ideally be representative of the molecule
λ max
- IR gives the range where bonds stretch and bend
o Most likely transition is HOMO-LUMO Transition o Plot the signal intensity at that wavelength against temperature (or other
- Usually plotted as transmission against wavelength (cm-1) - Example, Lipase, = 410nm
Typical energies = 125-650kJmol -1 conditions)
- Plots are arranged in order of wavelength
Typical wavelength = 200-800nm o Calculate fraction denatured (or generally, the fraction of product/end point in
o Higher wavenumbers to the left o Measure absorbance at 420nm with time
- Allowed electronic transitions occur from occupied orbitals to unoccupied orbitals the equilibrium
ε
- Motions available for bonds, all of which will be present in an IR spectrum
(bondto antibond) o Convert to concentration using - Using (temperature dependent) spectroscopy, it is possible to determine
- Symmetric stretching, Asymmetric stretching, Twisting, Wagging, Scissoring, Rocking
Vibrational Structure thermodynamic parameters without need for calorimetry
- When bonds vibrate, they behave like a spring
[ θ ] 208 + 4,000
- Absorptions only occur within strict selection rules between ground and excited state o Direct measurement of reaction kinetics
o Can be described by Hooke’s Law
o However, when molecules absorb electromagnetic radiation, the excited state o ∆A = ∆C = rate
Therefore, stretching frequency depends on strength of bond and mass of atoms either
end of the bond
o
causes bonds to be longer, caused by electrons in the antibonding orbitals Heme Proteins
Therefore, each electron transition is associated with change in vibrational and - Proteins that contain a heme prosthetic functional group as its functional group ϕ α , 208=
rotational states -> allows multiple transitions
Electronic transitions faster than vibrational transitions
o Heme = iron containing Protoporphyrin IX
- Key blood proteins – Myoglobin and Hemoglobin
29,000
Etotal =Ee Supramolecular Chemistry and Molecular Machines
o Iron porphyrin connected directly via histidine residues
−¿ o Active site allow for reversible binding of O2 Organic Chemistry
vib
+E ¿ Oxidises Fe 2+ to Fe3+, causes a structural shift - Organic chemistry is the chemistry of organic compounds
As stretching motions are higher energy than bending motions - The number of compounds containing carbons is greater than the number of
Franck-Condon Principle
¿
π ¿π
Chromophores - Porphyrins (when bound to proteins) have a very strong absorption due to compounds of all the rest of the elements in the periodic table combined
- Parts of a molecule that contain bonds that will absorb in the UV/Vis regions - One of the reasons for this complex chemistry is that carbon, with its 4 valence
- All molecules will have electronic absorptions electrons – can form 4 covalent bonds
Bonds as Harmonic Oscillators o Strongest istransitions
Soret band at around 400nm followed by the Q bands at higher
¿ - Carbon is also able to bond to itself to form long chains, branched chains and cyclic
σ →σ
- There are selection rules for transitions wavelengths
o Most transitions are too high in energy to be observed on structures
o Selection rule #1 ∆v±1
¿
π ¿π
o Allows large biomolecules such as proteins, lipids, carbohydrates and nucleic
o Selection rule #2 (for interactions w/IR) Must be a ∆ dipole moment o The transitions are very sensitive to changes in structure
typical spectrometer (<200nm) acids
( 12 )hv π
- It covers many broad areas of research including:
o Double and triple bond ( ) and Lone pairs (n) are observable Fluorescence Spectroscopy
E= v +
o Theoretical organic chemistry – understanding structures in terms of atoms and
Photoluminescence
electrons that bond them together
Absorption (A) - Chemiluminescence – light emitted from the decay of the excited state (e.g. Luminol)
o Synthesis – how to design and make new molecules
- The change in intensity (I) of light as it passes through a sample o If this reaction is biological, its bioluminescence (e.g. Luciferin in luciferase found
o Structural determination – how to find out the structure of new compounds,
I0
in fireflies)
even if they are only available in small amounts
- Bonds don’t obey Hooke’s law exactly o T1 state to S0 state
o Reaction mechanism – how the molecules react with each other (individual steps)
A=log
- Force required to compress a bond is larger than the force required to stretch it - Similar set up to UV/Vis spectroscopy
o and how to predict their reactions
- Bonds, if stretched enough will eventually break (dissociated) - Measured orthogonally (90˚) to the path of the excitation beam
I
o Biological activity – find out what nature does and how the structures of
- Can describe the energy using a Morse Curve - Need to pick excitation wavelength to get best emission spectrum
- Fluorescence detection tends to be more sensitive than absorption biologically active molecules are related to what they do
o At low energies, vibrations are essentially harmonic Hydrocarbons
Fluorophores
I0
But become more anharmonic with increasing energy - Simplest organic compounds contain just H and C – known as hydrocarbons
Energy levels get closer together = the initial light intensity - Fluorophores can be excited by chemical reactions rather than light
- Typically large organic molecules with highly conjugated bond system - Broadly divided into 3 categories:
Vibrations of Polyatomic Molecules o Aliphatic – not cyclic or aromatic; only straight, or branched chains
- Can be functionalised or incorporated into other molecules
I
- Large molecules have a more complicated stretching and bending modes – Normal Saturated – Alkanes (CnH2n+2)
Modes = the light intensity at the end o Fluorescent part is called the fluorophore
- Large range of molecules = large range of excitation + emission Unsaturated – Alkenes (CnH2n), Alkynes (CnH2n-2)
o Synchronous motion of atoms that may be excited without exciting any other o Alicyclic – cyclic compound that are not aromatic; rings with no delocalised π-
modes - Attenuation of light intensity will depend on how strongly the sample absorbs, how - Very sensitive to environment – can be used as a molecular probes
much of it is present (concentration), and how far light has to travel through the - Fluorescein (Fluorescein isothiocyanate system
o Non-linear = 3N-6 Saturated – Cyclic alkanes (CnH2n)
o Linear = 3N-5 sample (pathlength) o Basic fluorophores but robust molecules with a high fluorescence intensity
The Beer-Lambert Law o Used as a dye that can be tagged onto other molecules Unsaturated – Cyclic alkenes (CnH2n-2)
o 2 or more modes may be degenerate (have the same energy) o Aromatic – compounds with benzene rings or related systems; has a ring with a
Measuring IR spectra Absorbance (at a given wavelength, unitless) can be quantified with this equation: o Used in medical imaging
delocalised π-system
A=εcl
- Measured using a spectrometer - BODIPY
Functional groups
o Vibrational absorbances are not measured at every wavelength o Very sensitive to chemical modification
- Group of atoms in organic molecules that are responsible for characteristic chemical
- Fourier Transform o Very versatile fluorophore
- Build a calibration curve of known concentrations if extinction coefficient is not known - Nile Red reactions of those molecules
o An interferogram of IR radiation is collected
ε
Orbital Hybridisation
o Converted to a spectrum of frequencies using a Fourier Transformation o Has different forms but Nile red most common
o Gradient of A vs. C gives - Carbon can form up to 4 covalent bonds
FTIR – Fourier Transform InfraRed o Very sensitive to chemical environments – particularly polarity
o However, the modern electronic structure of carbon show there are only 2
Preparing samples Absorption in biomolecules – Proteins o Used as a molecular prob for polarity
unpaired electrons that could be contributed to covalent bonds
- To minimise background, samples are prepared with as high a concentration as - Main a.a. to look at is Tyrosine (Tyr/Y) and Tryptophan (Trp/W) - Molecular Rotors – Fluorescent Viscosity Probes - Formation of bonds releases energy so the system is more stable
possible o Are distinguishable o Greater viscosity, greater fluorescence o Forming 4 bonds rather than 2 releases more energy = more stability
- KBr pellet o Tyrosine (Tyr/Y) - GFP – Green Fluorescent Protein o Only a small energy gap between 2s and 2p orbitals
λ max
o Made by compressed sample with KBr under high pressure o Isolated from jellyfish
o The energy required to promote an electron from 2s to 2p to give 4 unpaired
- Nujol mull =274nm o Natural fluorophore
electrons is compensated by extra energy released from forming 4 bonds
o Very concentrated emulsion of sample and Nujol (mineral oil) o Can be genetically modified to be different colours - The electrons then rearrange themselves again in a process called hybridisation
ε 274
- More common – Attenuated Total Reflectance (so ATR-FTIR) o Can be incorporated into other living system o Organised into 4 identical hybrid orbitals, called sp3 (from 1xs and 3xp orbitals)
o Samples loaded onto a diamond plate and measured directly = 1400M cm -1 -1
o Made up from 3 amino acids (Ser, Tyr, Gly) to make one chemical structure o Arranged themselves in space so that they are as far apart as possible – angle of
IR Sampling Methods o Excitation of the deprotonated form gives greater fluorescence intensity 109.5˚ (Tetrahedral)
ε 280
- Transmission Fluorescent Microscopy - Alkenes (unsaturated carbons)
o Bulk measurement = 1280M -1cm-1 - Multiple fluorophores can be used to probe and image systems o Only joined to 3 other atoms than 4
o KBr discs o Chemical modify fluorophores to tag different parts of the cells o Only 3 orbitals hybridised, rather than all 4
o Nujol mulls o Tryptophan (Trp/W) - Hoechst – Binds to DNA in cell nucleus o Arranged as far as possible – 120˚ to each other in a plan (Trigonal planar)
o Thin films
- Specular reflection λ max = 280nm o λ ex = 350nm
Remaining p orbital is at a 90˚ to them
o Surface measurement - Hybridised sp3 and sp2 (or sp) orbitals form
σ bonds
o Thin films on reflective surfaces
- Diffuse reflection
o Surface measurement
ε 280
= 5690M -1cm-1 o λ em = 460nm - p orbitals can overlap to form π orbitals
- Aromatic compounds
- PKH67 – Binds to cell membrane o Electrons are delocalised, which stabilises the compound
ε =nW ( 5690 ) +nY ( 1280 )
o Powdered samples
λ ex
- Attenuated Total Reflection o Bond length the same (140pm instead of 147 for C-C or 135pm for C=C)
o Surface measurement o = 490nm All the C-C bonds are midway between a single and double bond
ε
o No sample prep Nonpolar and polar covalent bonds
- The surface measurements may not always be great as the surface molecules may be - If number of Y and W is known in a protein, you can calculate
λ em
- Nonpolar covalent bond = bonded atoms are the same, or have similar
different to the internal molecules o = 502nm electronegativities – electron density is evenly shared between atoms
o You can get this from the primary sequence - Polar covalent bond = bonded atoms have significantly different electronegativities –
FTIR spectra
- Typically measured as transmittance(%T) against wavenumber o Measure absorbance at 280nm - Rhodamine Phalloidin – Binds to actin filaments (cytoskeleton) electron density is shifted towards one atom
λ ex
o Inverted axis (y-axis) o Calculate concentration using Beer-Lambert equation o The difference in electron density leads to partial charges either end of the bond
o If Mr of protein is known, concentration can be calculated in mgmL-1 −¿¿
- Can convert transmittance to absorbance: o = 540nm
+¿∧δ ¿
A=2−lo g 10 ( %T )
Absorption in biomolecules – Nucleotides – represented by
δ
o - ε 260 [ Adenosine monophosphate (AMP/A)] = 15,400 M-1cm-1 o λ em = 565nm o Difference in charge is known as a dipole
Reaction classification
FTIR Imaging
Provides more detail to images – chemical composition with spatial resolution
- Cancer Fingerprinting
- ε 260 [ Guanosine monophosphate (GMP/G)] = 11,500 M-1cm-1
Fluorescence of Proteins
- Tryptophan – weakly fluorescent
o Intensity increased when its in a hydrophobic environment
- Polymerisation – joining together lots of simple molecules to form a giant molecule
- Substitution – when one species is replaced by another
- Addition – when one species is added to another
o Nucleic acid – Phosphate group
ε 260 o Used as a measure of protein folding - Condensation – when two species joined together with the loss of water
o Proteins – Amide group
λ ex
- [ Cytodine monophosphate (CMP/C)] = 7,400 M -1cm-1 - Hydrolysis – splitting a molecule into 2 molecule by adding water
o Lipids – C-H group o = 380nm - Oxidation – when an atom loses electrons
Proteins
ε 260
- Reduction – when an atom gains electrons
- Diverse set of natural polymers – distinct structures
λ em
- [ Thymodine monophosphate (TMP/T)] = 8,700 M -1cm-1 - Redox – any reactions where electrons are transferred between 2 species
- Increasingly important for molecular sciences and biotechnology o = 300-350nm Electrophiles and Nucleophiles
o Viruses – vaccines Nucleic Acid Concentration - Nucleophile (nucleus/positive lover) – a reactant that provides a pair of electrons to
o Antibodies – immunotherapy - Concentrations of DNA and RNA are easier to determine than protein form a new covalent bond
o Temperature dependent tryptophan fluorescence
o Enzymes – biocatalysts o All nucleobases absorb o Has a -ve charge, partial -ve charge or a π-bond
Used to probe changes in tertiary structure
- Structure very important – dictates function o Have slightly more bases than average proteins have amino acids - Electrophile (electron/negative lover) – a reactant that accepts a pair of electrons to
Tertiary structure denatures before secondary structure
o Difficult to assess quickly - Generic Extinction coefficients: forma a new covalent bond
Can be monitored by CD spectroscopy
Secondary structures
ε 260 - Protein aggregation (forming fibrils) is the cause of neurodegenerative diseases o Has a +ve charge, partial +ve charge or an empty valence orbital
- Proteins are polymers of L-amino acids connected by peptide bonds o [dsDNA] = 0.020mlug -1cm-1 o Aggregation creates new hydrophobic domains Mechanisms
- Typically no structure - Arrows show the movement of electrons as bonds between atoms are broken and
o Fluorophores can be trapped in aggregated fibrils
o Described as being “random coils” or “unordered”
ε 260 Increase in fluorescence formed
- Amino acids can hydrogen bond to each other o [ssDNA] = 0.027 mlug-1cm-1 o Single headed arrow = movement of one electron
CD Spectroscopy
Hydrogen bonding patterns o Double headed arrow = movement of a pair of electron
Circular Polarised Light
- α-helix
ε 260
- Light can be polarised so that the plane of the polarised light (perpendicular to the - This is a formalism – electrons (more precisely, electron density) do not move around
o H bonds between amino acids 4 residues apart on same peptide strand o [ssRNA] = 0.025 mlug-1cm-1 so neatly and discretely in reality
direction of wave) rotates at a steady rate
- Β-sheet o Can be left or right handed - Rules:
o H bond between adjacent peptide strands Shifts in Absorbances – Conjugation 1. Arrow must start from electrons and point to an atom or bond
- Chiral molecules will absorb left and right handed light differently
The peptide bond - Molecules with multiple chromophores have greater extinction coefficient o Absorption will depend on orientation of the chiral bind 2. Electrons and bonds form bonds, not charges
- All proteins have an abundance of peptide bonds
π
o Repeated dihedreal angles will give stringer absorption o However, common convention in organic chemistry is to use a shorthand
o Very visible stretching in IR - When chromophores are conjugated (usually -bonds) notation of a -ve charge to represent a lone pair in mechanisms – for charged
o The difference in absorption = Circular Dichroism
- Amide I and II are the strongest of the vibrational modes atoms either show all lone pairs or none at all
o Characteristic peaks for each give secondary structure
Amide I Amide II
o
o
Energy between HOMO and LUMO decreases
Absorbance undergoes a redshift Δ A= A L −A R Supramolecular Chemistry
- “Chemistry beyond the molecule”
λ max ε
- The most important feature of supramolecular systems is that the components are
α-helix
β-sheets
1648-1657cm-1
1623-1641cm-1
1540-1550cm-1
1525-1535cm-1
o As number of double bonds increase, values of and increase
Θ=32.98 Δ A held together by reversible, intermolecular forces, not by covalent bond
- Important concept in supramolecular chemistry is complementarity e.g. base pairing
- Retinal, bound to protein rhodopsin, main receptor for vision – absorbs light CD of Proteins in DNA double helix
Unordered/Random 1642-1648cm-1 1535-1540cm-1 o Only aldehyde form is active however, greatest conjugation (more redshifted so - Proteins are polymers of L-a.a. connected by peptide binds o Combines 2 antiparallel strands held together by complementary H-bonds
→
o Typically, no structure, “random coil”/”unordered” between pairs of bases
Raman Spectroscopy UV Visible) o Can H-bond to each other o H-bond donors and acceptors are arranged that adenine forms 2xH-bonds with
→
- Also measures vibrational states thymine, whilst guanine forms 3xH-bonds with cytosine
o Many birds and insects (particularly bees) can see in UV - R group dictate allowed dihedreal angles very specific secondary structure - A receptor (the host) is complementary to the substrate (the guest)
o Uses a form of Rayleigh scattering
Change in polarity of the binding pocket of rhodopsin so is blueshifted – o Its size, shape, and position of binding sites are ideal for substrate recognition
- Provides complementary knowledge to IR
o Show vibrations that do not break a molecules symmetry
- There a fewer transitions
Glutamic acid
→ tyrosine/phenylalanine
- Peptide bonds is chromophore – chiral and planar
o Repeated dihedral angles give string absorptions
o “lock and key” principle
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