Option: ChemBio Flashcards
(159 cards)
1
Q
What are the 4 types of amino acid groups?
A
Hydrophobic
Polar
Acidic
Basic
and “others”
2
Q
What are the hydrophobic amino acids?
A
3
Q
What are the polar group amino acids?
A
4
Q
What are the acidic group amino acids?
A
Aspartic acid (Asp) - R = CH2CO2H
Glutamic acid (Glu) - R = CH2CH2CO2H
5
Q
What are the basic group amino acids?
A
Lysine (Lys) - R = CH2CH2CH2CH2NH2
Arginine (Arg) - R = CH2CH2CH2NHC(=NH)NH2
6
Q
What are the groups which are not standard regular amino acids?
A
Glycine (R=H) and
7
Q
What is ribosomal peptide biosynthesis?
A
Protein synthesis from mRNA using the ribosome
Translation
8
Q
What occurs during translation which introduces errors?
A
Different amino acid residues naturally inserted
Example: Selenocysteine (cysteine with Se instead of S)
9
Q
How can non-ribosomal peptide (NRP) synthesis occurs biologically?
A
Large multi-subunit enzymes which make peptides which aren’t from a genetic code
Often v large proteins
10
Q
When are disulfide bonds observed?
A
Between thiol groups of cysteine residues in the same (intra-chain) or other chain (inter-chain)
Most common in extracellular proteins
11
Q
What is the difference between cysteine and cystine?
A
Cysteine = thiol form
Cystine = disulfide form
12
Q
What is post-translational modifications?
A
Proteins extensively covalently modified after their synthesis
Done by enzymes
13
Q
How can amino acids be modified post-translation using phosphtaes?
A
Phosphorylation/dephosphorylation - serine, threonine, and tyrosine
This is used for signaling pathways
14
Q
What are glycoproteins and how/when are they made?
A
Protein with sugars added to OH of amino acids (serine, threonine, or amides of asn and gln)
Done via gycosylation which is post-translation
15
Q
What is collagen?
A
Extracellular protein occuring in connective tissues
Initially made as individual chains with post-translation cross-linking
16
Q
What is the mechanism for cross-linking in collagen?
A
17
Q
Why are proteins synthesised in labs?
A
Confirm structures and analysis
Prepare for biological evaluation
Used as antigens for preparing antibodies
Make peptide-based medicines
18
Q
What is the primary structure of a peptide?
A
Amino acid sequence and if there are other cross-links
19
Q
What are the different approaches for sequencing proteins?
A
Chemical analysis
Nucleic acid sequencing
X-Ray crystallography
Mass Spec
20
Q
What chemical analysis is done for amino acid sequencing?
A
N&C-terminal analysis or degredation then derivatization
Edman degredation - N-terminal modification followed by hydrolysis
21
Q
How is mass spec used for amino acid sequencing?
A
Determine total mass
Then sequential fragmentation in spectrometer can be measured
22
Q
How do you do degredation and derivatization in proteins?
A
Hydrolysis - leaves all amino acids are H-AA-OH
(AA = amino acid)
Then Derivatised by chromogenic / fluorescence label and identified by HPLC or ion exchange
(compare with standards)
23
Q
What reagents selectively react with N-terminus for analysis?
A
Sanger reagent - DNFB/FDNB
Dansyl chloride - DNSC
Edman reagent
24
Q
What reagent selectively reacts with C-terminus for analysis?
A
Akabori procedure - hydrazinolysis
25
What is Sanger's reagent?
Reacts at pH 8.5 with N-terminal amine via SnAr
Hydrolysis gives amino acids, with only terminal one being bound to reagent
26
What is the mechanism of the Sanger reagent?
27
What is dansyl chloride?
Identifies N-terminus
Via reacting with N-terminus of an amino acid (alternative to Sanger's)
28
What is the mechanism of dansyl chloride derivitization?
29
What is the akabori procedure?
Identifies C-terminus amino acid
Requires addition of XS hydrazine and heat
30
What is the mechanism of the Akabori procedure?
31
What is Edman degredation?
Sequential removing of an N-terminus residue (one at a time) using limited acid hydrolysis or using enzymes
Used when large peptides
32
What is the mechanism of Edman degredation?
HPLC completed on these PTH derivatives against standards
Can then repeat on each N-terminus amino acid
33
What is required for protein sequencing with disulfide bonds?
Disulfide cross-links must be cleaved beforehand
34
How can you cleave a disulfide bond?
Reduction or oxidation then treatment to prevent disulfide bond reformation
35
What size of protein is required for Edman degradation?
10-20 residues
Larger must be fragmented using proteases then can do it
36
How can you selectively cleave at methionine residues?
Addition of BrCN
37
What does trypsin catalyse?
Peptide bond cleavage at +vely charged residues
(Arg, Lys)
Highly specific
38
What does chymotrypsin catalyse?
Peptide bond cleavage at bulky hydrophobic residues
(Phe, Trp, Tyr)
39
What does elastase catalyse?
Cleavage of peptide bond when small neutral amino acids
(Ala, Gly, Ser, Val)
40
What is nucleic acid sequencing (for protein sequencing)?
Find nucleic acid sequence of DNA
Then work out which amino acids this encodes for
This is efficient and most widely used modern method
41
How is X-ray crystallography & NMR used for protein structure?
Used for determining 3D structures of proteins
However crystallography doesnt work for disordered peptides and NMR is hard
42
What is tandem/fragmentation mass spec?
Peptides fragmented within mass spec and results are measured at each stage
Useful as accurate and post-translational modifications can be determined
43
What is the process of fragmentation/tandem mass spec?
1) Protein cleaved with protease (e.g. trypsin)
2) Compare with predicted fragments for protein sequences based on genomic data (automated)
3) Requires to be <25 residues
44
What fragmentation is seen in tandem mass spec of proteins?
Depends on sequences, charge, internal energy, etc.
Fragments only detected if charged
45
What is required for an amino acid protecting group?
* Easy to introduce
* Cleaved without amide bond hydrolysis or side-chain modification
* Must not lead to loss of stereochem (epimerisation)
46
What are the three types of protecting groups?
α - amino group
α - carboxylate group
Side-chain group
47
What is the general method for α-amino protection?
Alkoxycarbonyl protection
48
What are the α-amino protecting groups?
49
How do you protect an α-amino group (reagents)?
Need to add base to minimise side reactions
50
How can you remove protecting groups of α-amino groups?
Z: remove with Pd/H2 or HBr/AcOH
Boc: HBr/AcOH or TFA
Fmoc: base
51
What is the mechanism for removal of Z groups with Pd/H2 or HBr/AcOH?
52
How do you protect and deprotect an α-amino group with boc?
53
What is the problem with the boc PG when deprotecting?
Carbonium ion can be attacked by nuc side chains
(e.g. tyrosine)
Add anisole (Ph-OMe) as an anion scavenger
54
How do you protect an α-amino group with fmoc?
Fmoc-Cl and amino acid
(acyl chloride)
55
How do you remove a fmoc protecting group?
56
What is the general approach to protect α-carboxy groups?
Form their esters
Me/Et - too stable, so not used
Benzyl, tBu, allyl esters - more common
57
How can you protect and deprotect α-carboxyl group with benzyl esters?
58
Why is DMF used instead of DMSO in amino acid synthesis?
DMSO undergoes redox chemistry with side groups
59
How are benzyl esters deprotected?
H2/Pd/C
or
HBr/AcOH
60
How can you protect and deprotect a α-carboxy group with t-butyl?
61
What side chains typically need protecting?
Acidic: aspartic and glutamic acid (COOH)
Basic: lysine (NH2)
Cysteine (SH)
Not needed for alkyl chains
62
What side chains typically need protecting?
Acidic: aspartic and glutamic acid (COOH)
Basic: lysine (NH2)
Cysteine (SH)
Not needed for alkyl chains
63
How do you protect the lysine sidegroup?
Lys - amino group requires protection, is more nuc and basic than α-amino (which is zwitterionic)
Protect using CuSO4 then boc-anhydride and a base, then H2S
64
What is the mechanism for protection of lysine?
65
How can the cysteine side-chain be protected?
Prevents disulfide bond or reacting as it is more nuc
Trityl chloride, Ph3C-Cl/HCl
or
Cl-CH2-anisole
66
How can you protect and deprotect cysteine?
67
How can you form disulfide bonds from Trityl-protected cysteines?
Oxidation using I2
68
How do you protect the side-groups of glutamic and aspartic acid?
Benzyl- or t-butyl esters
Works as α-carboxyl group is less reactive due to +ve charge on the amino group
69
What is the process of protecting aspartic acid with fmoc and OtBu?
70
What are the requirements for peptide bond formation reactions?
High yielding
Removal of byproducts easy
Compatible with side-chains
No racemisation
71
How does racemisation occur in peptide synthesis due to deprotonation?
Deprotonation at C instead of N stabilised only when R is Ph or stabilising group
72
How does racemisation occur in peptide synthesis due to oxazolone formation?
73
How can you prevent oxazolone formation?
Alkoxycarbonyls - inductive effect of OR hinders formation
74
What direction is peptide synthesis?
Nature: N -> C terminus
Lab: C -> N terminus
75
What are the coupling reagents for peptide synthesis?
| NEED TO LEARN
* Carbodiimides - HOBT
* Unsymm anhydrides - EEDQ
* Phosphonium salts - BOP, PyBOP
* Uronium/aminium salts - HBT, HATU
* Active esters - acyl fluorides/azides/others
76
What is the adv of using unsymm anhydrides as peptide bond coupling reagents?
Cheap
Byproducts easily removed
Useful for large scale solution
Not for use in solid phase
77
How are unsymm anhydrides used as coupling reagents for peptide synthesis?
78
What is EEDQ and how is it used in peptide synthesis?
79
What active esters are used as coupling agents in peptide synthesis?
Acyl chloride - limited use as condition not compatible with acid labile PG (BOC), and not v stable
Acyl fluoride/azide - little racemization, high yield
Acyl O-C6F5
80
How do you prepare acyl azides for protein synthesis and what is a problem?
81
How can you prepare acyl fluorides for protein synthesis?
82
What carbodiimides are used in protein synthesis?
83
What is the mech of carbodiimides coupling peptide synthesis?
Side product precipitates and is a potent allergen
84
What is the problem and solution for carbodiimide synthesis?
Intramolec acyl transfer
Solution: nuc reagent which gives RCOX intermediate which does not rearrange (DMAP and HOBt)
85
How does HOBt aid with carbodiimide-mediated coupling?
86
How does aminium/uronium salts catalyse peptide synthesis?
Carboxylate reacts with salt and then amine attacks
Wash away byproduct with water
87
How can you synthesise aminium/uronium salts?
88
How do phosphonium reagents couple amino acids?
High yield and littel racemisation
89
What are some examples of phosphonium reagents?
90
What is deamidaton?
Process increases with age of the protein
Reverser can be catalysed
91
When is solution phase synthesis used?
Large-scale synthesis of rel short sequences (< 20)
Done via convergent / fragment condensation synthesis
92
Why is covergent / fragment condensation used in solution phase synthesis?
Usually gives high yield
Purification sig easier
Min loss of stereochem - not possible to use carbamates (cant use fmoc)
Avoid adjacent sterically demanding amino acids (leucine)
93
What coupling agents are used in solution phase synthesis?
DCC/HOBt or RCON3
Where possible need to couple at Gly-X or Pro-X residues to prevent epimerization
94
What is protein splicing?
Splicing mech is thioester to amide rearrangements
95
What is the mechanism of protein ligation?
Occurs on N-terminus of Cys
96
How can protein ligation be used in synthesis?
C-terminal thioester and N-terminal cysteine
Occurs with unprotected peptides/proteins
Same 5-exo trig
97
What is the direction of solid-phase peptide synthesis (SPPS)?
C -> N terminus
1 amino acid added at a time
98
What is the setup of SPPS?
C-terminal PG in SPPS rendered insoluble by linking to inert solid polymer support
Reaction occurs between solid and solution
99
What are the adv of SPPS?
Purification efficient (as solid support)
High yield
Can automate
C to N maintains stereochem
100
What are the steps of SPPS?
1) Coupling of first protected PG-AA to solid support
2) Deprotect αN-AA1
3) AA coupling
4) Repeat 2&3
Extensive washing in each step
101
What is the Merrifield method?
Boc/benzyl chemistry used in SPPS
αN-Boc protection (acid labile), benzyl PG on side chains (removed by Pd/H2)
102
What are reactions in the Merrifield mech?
React solid support (polystyrene) to allow for protection of αN-Boc
103
How does HF effect PG?
Deprotects all side-chains
Cleaves peptide-resin anchorage
104
What is Sheppard's method?
Most widely used
αN Fmoc protected
Side chain protected by Boc
Weak base iteratively deprotects Fmoc, whereas TFA used to remove from resin and all PGs
Produces C-terminal amide
105
Why is Sheppard's method used instead of the Merrifield method?
Can monitor release of Fmoc
Uses more polar amide-based resin (rather than polystyrene), so avoids aggregation with hydrophobic resin
106
What is the process of Sheppard's method?
107
What is a linker in SPPS?
Reversible linkage between synthetic peptide chain and solid support
Cleaved along with acid (TFA) in final step
108
What are the types of linker in SPPS?
Wang - produces acid at C terminus
Rink amide - produces amide at C terminus
109
What is the structure of Wang resin linker?
Requires that benzyl esters not used as side PG, so then don't need HF
110
What is the structure of a rink amide linker?
111
What side-chain groups can you use when Fmoc used for amino?
Boc, trityl, and t-butly
All removed by TFA unlike Fmoc (base)
112
What is the mech of Fmoc deprotection?
Piperidine in DMF
113
What are the main problems of SPPS?
Cost/volume of a reaction
Incomplete orthogonality between PGs
Side reactions
Not 100% yield
114
How can you tune SPPS to get higher yields?
XS protected AA to avoid interactions between long peptides
115
What is the Kaiser test?
Measure completeness fo coupling of N-terminal residue
Uses ninhydrin which reacts with primary amine and turns purple
116
What is the main difference between Boc (Merrifield) and Fmoc (Sheppard) chemistry?
Iterative αN amino deprotection and degree of acid stability of side-chain and peptide-resin linkages
Boc - HF used, dangeous, and could degrade peptides, C-term carboxythioesters
Fmoc - no HF, more dimensions of orthogonality, milder TFA deprotection allows acid-labile mods to survive
117
What is reverse-phase HPLC?
Separation of molecules due to hydrophobicity
Stationary phase - hydrophobic ligands immobilized on silica
Mobile phase - polar/aqueous with the peptides (ACN and MeCN)
118
How does elution rate change in reverse-phase HPLC?
Goes via gradient elution
So amount of ACN:MeCN or using ionic modifier like TFA
119
How effective is reverse-phase HPLC?
Excellent resolution under many conditions
Easy to optimise and change conditions
High recoveries
Good reproducibility
120
What is the asprtimide problem in Fmoc-SPPS?
Aspartimide forms in side reaction
This can then react
121
Learn the purines
122
Learn the pyrimidines
123
What is the sugar unit in nucleosides/nucleotides?
124
What is a nucleotide and nucleoside?
Nucleoside - nucleic acid and sugar
Nucleotide - nucleic acid, sugar, and phosphate group
125
What changes in DNA and RNA compounds?
Uracil replaces thiamine
Ribose repleaces deoxyribose
126
What is the 5' or 3' end of a DNA strand?
End of DNA strand with OH in either 5 position or 3
127
How does DNA dissolve in water?
Phosphodiester - salt of phosphoric acid ester
Polyanion so dissolves in water
128
How is DNA usually read?
5' to 3' end
129
What is the H-bonding in AT base pair?
130
What is the H-bonding in GC base pair?
131
What is the structure of DNA in general?
Regular double helix - right handed
Base pairs on inside & phosphate backbone
No gaps between bases - nuc acids stabilised by H-bonding and electrostatic base stacking
132
What is the major and minor groove of DNA?
Major - deeper and wider channel in helix where proteins interact
Minor - smaller channel in helix where small molecules interact
133
What mechanism of DNA replication?
Uses DNA polymerases and nucleotide triphosphates, with Pi is leaving group
From 3' to 5'
134
How can incorrect base pairing occur?
Rare in replication but repaired by enzymes
135
What occurs to H-bonding when purine:purine?
Both are larger base pairs so must be longer and bulkier
A.A gives 1 H-bond
A.G/G.G give 2 H-bonds
136
What occurs to DNA structure if pyrimidine:pyrimidine base pairing occurs?
Helix constricts and worse base-stacking
137
What is the tautomer hypnothesis with bases?
1 in 104 bases exist as their minor tautomer
Allows for incorrect base pairing
138
What is the minor tautomer base pairing?
139
What does adding new nucleotides allow for?
Non-nautral side chains in proteins
But remember these bases dont H-bond
140
What is the mech for adding an amino acid at the ribosome?
141
What is the mech for adding an amino acid at the ribosome?
142
Why might you stabilise a base duplex?
Used in diagnostic and therapeutic settings
143
How can you increase the stability of A.T duplex?
Dont use S - cannot H bond
144
How can you increase the stability of G.C duplex?
145
Why is the phosphodiester backbone in that form?
Entropically favoured
Correct shape to bind to RNA
146
What is the use of analogues of DNA/RNA backbones?
Binds to complementary DNA or RNA with high affinity
147
What is the structure of the DNA triple helix?
Not watson-crick
Transiently formed but not v stable
148
What are the adv/disadv of triple helix DNA?
Adv: binds to one strand fo duplex (blocks function), recognises duplex sequence without denaturation
Disad: pH dependent (on C+), low affinity, limited sequence recognition
149
How do antisense oligos prevent protein function?
150
What is the key advantage of targeting RNA in pharma?
Pharmacophore (DNA sequence) and dianophore (molecular features that determine tissue distribution and metabolism) can be optimized separtely
151
What are the main problems of antisense therapeutic oligos?
Limited cellular uptake
Toxicity
Off-target effects
Unstable to nuclease degredation
152
What are Lipinski's rules for an active drug?
Cannot vioolate more than one of:
* `<=5` H-bond donors (N-H and O-H bonds)
* `<=10` H-bond acceptors (N/H atoms)
* Mr `<500` daltons
* partition coefficient logP`<5`
Oligos break all rules
153
How can you stabilise oligos?
154
How can you stabilise oligos?
155
Where is chirality introduced in stablised antisense oligos?
156
What is a gapmer?
Short DNA antisense oligos with RNA segments on either side fo a DNA one
Allows for bonding to target mRNA then attacts RNase H for degredation
157
When are phosphorothioate linkages used?
Resistant to degradation by nucleases
158
What is the therapeutic effect of methylating C/U?
Reduces toxciticy
159
What is the therapeutic effect of methylating C/U?
Reduces toxciticy
