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Year 3 - Chemistry > Option: ChemBio > Flashcards

Option: ChemBio Flashcards

1
Q

What are the 4 types of amino acid groups?

A

Hydrophobic
Polar
Acidic
Basic
and “others”

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2
Q

What are the hydrophobic amino acids?

A
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3
Q

What are the polar group amino acids?

A
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4
Q

What are the acidic group amino acids?

A

Aspartic acid (Asp) - R = CH2CO2H

Glutamic acid (Glu) - R = CH2CH2CO2H

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5
Q

What are the basic group amino acids?

A

Lysine (Lys) - R = CH2CH2CH2CH2NH2

Arginine (Arg) - R = CH2CH2CH2NHC(=NH)NH2

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6
Q

What are the groups which are not standard regular amino acids?

A

Glycine (R=H) and

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7
Q

What is ribosomal peptide biosynthesis?

A

Protein synthesis from mRNA using the ribosome
Translation

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8
Q

What occurs during translation which introduces errors?

A

Different amino acid residues naturally inserted

Example: Selenocysteine (cysteine with Se instead of S)

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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

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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

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11
Q

What is the difference between cysteine and cystine?

A

Cysteine = thiol form

Cystine = disulfide form

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12
Q

What is post-translational modifications?

A

Proteins extensively covalently modified after their synthesis

Done by enzymes

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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

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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

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15
Q

What is collagen?

A

Extracellular protein occuring in connective tissues

Initially made as individual chains with post-translation cross-linking

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16
Q

What is the mechanism for cross-linking in collagen?

A
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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

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18
Q

What is the primary structure of a peptide?

A

Amino acid sequence and if there are other cross-links

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19
Q

What are the different approaches for sequencing proteins?

A

Chemical analysis
Nucleic acid sequencing
X-Ray crystallography
Mass Spec

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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

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21
Q

How is mass spec used for amino acid sequencing?

A

Determine total mass
Then sequential fragmentation in spectrometer can be measured

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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)

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23
Q

What reagents selectively react with N-terminus for analysis?

A

Sanger reagent - DNFB/FDNB
Dansyl chloride - DNSC
Edman reagent

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24
Q

What reagent selectively reacts with C-terminus for analysis?

A

Akabori procedure - hydrazinolysis

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25
Q

What is Sanger’s reagent?

A

Reacts at pH 8.5 with N-terminal amine via SnAr
Hydrolysis gives amino acids, with only terminal one being bound to reagent

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26
Q

What is the mechanism of the Sanger reagent?

A
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27
Q

What is dansyl chloride?

A

Identifies N-terminus
Via reacting with N-terminus of an amino acid (alternative to Sanger’s)

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28
Q

What is the mechanism of dansyl chloride derivitization?

A
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29
Q

What is the akabori procedure?

A

Identifies C-terminus amino acid

Requires addition of XS hydrazine and heat

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30
Q

What is the mechanism of the Akabori procedure?

A
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31
Q

What is Edman degredation?

A

Sequential removing of an N-terminus residue (one at a time) using limited acid hydrolysis or using enzymes

Used when large peptides

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32
Q

What is the mechanism of Edman degredation?

A

HPLC completed on these PTH derivatives against standards

Can then repeat on each N-terminus amino acid

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33
Q

What is required for protein sequencing with disulfide bonds?

A

Disulfide cross-links must be cleaved beforehand

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34
Q

How can you cleave a disulfide bond?

A

Reduction or oxidation then treatment to prevent disulfide bond reformation

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35
Q

What size of protein is required for Edman degradation?

A

10-20 residues

Larger must be fragmented using proteases then can do it

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36
Q

How can you selectively cleave at methionine residues?

A

Addition of BrCN

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37
Q

What does trypsin catalyse?

A

Peptide bond cleavage at +vely charged residues
(Arg, Lys)
Highly specific

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38
Q

What does chymotrypsin catalyse?

A

Peptide bond cleavage at bulky hydrophobic residues
(Phe, Trp, Tyr)

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39
Q

What does elastase catalyse?

A

Cleavage of peptide bond when small neutral amino acids
(Ala, Gly, Ser, Val)

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40
Q

What is nucleic acid sequencing (for protein sequencing)?

A

Find nucleic acid sequence of DNA
Then work out which amino acids this encodes for

This is efficient and most widely used modern method

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41
Q

How is X-ray crystallography & NMR used for protein structure?

A

Used for determining 3D structures of proteins

However crystallography doesnt work for disordered peptides and NMR is hard

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42
Q

What is tandem/fragmentation mass spec?

A

Peptides fragmented within mass spec and results are measured at each stage

Useful as accurate and post-translational modifications can be determined

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43
Q

What is the process of fragmentation/tandem mass spec?

A

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

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44
Q

What fragmentation is seen in tandem mass spec of proteins?

A

Depends on sequences, charge, internal energy, etc.

Fragments only detected if charged

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45
Q

What is required for an amino acid protecting group?

A
  • Easy to introduce
  • Cleaved without amide bond hydrolysis or side-chain modification
  • Must not lead to loss of stereochem (epimerisation)
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46
Q

What are the three types of protecting groups?

A

α - amino group
α - carboxylate group
Side-chain group

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47
Q

What is the general method for α-amino protection?

A

Alkoxycarbonyl protection

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48
Q

What are the α-amino protecting groups?

A
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49
Q

How do you protect an α-amino group (reagents)?

A

Need to add base to minimise side reactions

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50
Q

How can you remove protecting groups of α-amino groups?

A

Z: remove with Pd/H2 or HBr/AcOH

Boc: HBr/AcOH or TFA

Fmoc: base

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51
Q

What is the mechanism for removal of Z groups with Pd/H2 or HBr/AcOH?

A
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52
Q

How do you protect and deprotect an α-amino group with boc?

A
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53
Q

What is the problem with the boc PG when deprotecting?

A

Carbonium ion can be attacked by nuc side chains
(e.g. tyrosine)

Add anisole (Ph-OMe) as an anion scavenger

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54
Q

How do you protect an α-amino group with fmoc?

A

Fmoc-Cl and amino acid
(acyl chloride)

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55
Q

How do you remove a fmoc protecting group?

A
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56
Q

What is the general approach to protect α-carboxy groups?

A

Form their esters

Me/Et - too stable, so not used

Benzyl, tBu, allyl esters - more common

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57
Q

How can you protect and deprotect α-carboxyl group with benzyl esters?

A
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58
Q

Why is DMF used instead of DMSO in amino acid synthesis?

A

DMSO undergoes redox chemistry with side groups

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59
Q

How are benzyl esters deprotected?

A

H2/Pd/C
or
HBr/AcOH

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60
Q

How can you protect and deprotect a α-carboxy group with t-butyl?

A
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61
Q

What side chains typically need protecting?

A

Acidic: aspartic and glutamic acid (COOH)
Basic: lysine (NH2)
Cysteine (SH)

Not needed for alkyl chains

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62
Q

What side chains typically need protecting?

A

Acidic: aspartic and glutamic acid (COOH)
Basic: lysine (NH2)
Cysteine (SH)

Not needed for alkyl chains

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63
Q

How do you protect the lysine sidegroup?

A

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

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64
Q

What is the mechanism for protection of lysine?

65
Q

How can the cysteine side-chain be protected?

A

Prevents disulfide bond or reacting as it is more nuc

Trityl chloride, Ph3C-Cl/HCl
or
Cl-CH2-anisole

66
Q

How can you protect and deprotect cysteine?

67
Q

How can you form disulfide bonds from Trityl-protected cysteines?

A

Oxidation using I2

68
Q

How do you protect the side-groups of glutamic and aspartic acid?

A

Benzyl- or t-butyl esters
Works as α-carboxyl group is less reactive due to +ve charge on the amino group

69
Q

What is the process of protecting aspartic acid with fmoc and OtBu?

70
Q

What are the requirements for peptide bond formation reactions?

A

High yielding
Removal of byproducts easy
Compatible with side-chains
No racemisation

71
Q

How does racemisation occur in peptide synthesis due to deprotonation?

A

Deprotonation at C instead of N stabilised only when R is Ph or stabilising group

72
Q

How does racemisation occur in peptide synthesis due to oxazolone formation?

73
Q

How can you prevent oxazolone formation?

A

Alkoxycarbonyls - inductive effect of OR hinders formation

74
Q

What direction is peptide synthesis?

A

Nature: N -> C terminus

Lab: C -> N terminus

75
Q

What are the coupling reagents for peptide synthesis?

NEED TO LEARN

A
  • Carbodiimides - HOBT
  • Unsymm anhydrides - EEDQ
  • Phosphonium salts - BOP, PyBOP
  • Uronium/aminium salts - HBT, HATU
  • Active esters - acyl fluorides/azides/others
76
Q

What is the adv of using unsymm anhydrides as peptide bond coupling reagents?

A

Cheap
Byproducts easily removed
Useful for large scale solution

Not for use in solid phase

77
Q

How are unsymm anhydrides used as coupling reagents for peptide synthesis?

78
Q

What is EEDQ and how is it used in peptide synthesis?

79
Q

What active esters are used as coupling agents in peptide synthesis?

A

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
Q

How do you prepare acyl azides for protein synthesis and what is a problem?

81
Q

How can you prepare acyl fluorides for protein synthesis?

82
Q

What carbodiimides are used in protein synthesis?

83
Q

What is the mech of carbodiimides coupling peptide synthesis?

A

Side product precipitates and is a potent allergen

84
Q

What is the problem and solution for carbodiimide synthesis?

A

Intramolec acyl transfer

Solution: nuc reagent which gives RCOX intermediate which does not rearrange (DMAP and HOBt)

85
Q

How does HOBt aid with carbodiimide-mediated coupling?

86
Q

How does aminium/uronium salts catalyse peptide synthesis?

A

Carboxylate reacts with salt and then amine attacks
Wash away byproduct with water

87
Q

How can you synthesise aminium/uronium salts?

88
Q

How do phosphonium reagents couple amino acids?

A

High yield and littel racemisation

89
Q

What are some examples of phosphonium reagents?

90
Q

What is deamidaton?

A

Process increases with age of the protein

Reverser can be catalysed

91
Q

When is solution phase synthesis used?

A

Large-scale synthesis of rel short sequences (< 20)
Done via convergent / fragment condensation synthesis

92
Q

Why is covergent / fragment condensation used in solution phase synthesis?

A

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
Q

What coupling agents are used in solution phase synthesis?

A

DCC/HOBt or RCON3

Where possible need to couple at Gly-X or Pro-X residues to prevent epimerization

94
Q

What is protein splicing?

A

Splicing mech is thioester to amide rearrangements

95
Q

What is the mechanism of protein ligation?

A

Occurs on N-terminus of Cys

96
Q

How can protein ligation be used in synthesis?

A

C-terminal thioester and N-terminal cysteine

Occurs with unprotected peptides/proteins

Same 5-exo trig

97
Q

What is the direction of solid-phase peptide synthesis (SPPS)?

A

C -> N terminus

1 amino acid added at a time

98
Q

What is the setup of SPPS?

A

C-terminal PG in SPPS rendered insoluble by linking to inert solid polymer support

Reaction occurs between solid and solution

99
Q

What are the adv of SPPS?

A

Purification efficient (as solid support)
High yield
Can automate
C to N maintains stereochem

100
Q

What are the steps of SPPS?

A

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
Q

What is the Merrifield method?

A

Boc/benzyl chemistry used in SPPS
αN-Boc protection (acid labile), benzyl PG on side chains (removed by Pd/H2)

102
Q

What are reactions in the Merrifield mech?

A

React solid support (polystyrene) to allow for protection of αN-Boc

103
Q

How does HF effect PG?

A

Deprotects all side-chains
Cleaves peptide-resin anchorage

104
Q

What is Sheppard’s method?

A

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
Q

Why is Sheppard’s method used instead of the Merrifield method?

A

Can monitor release of Fmoc

Uses more polar amide-based resin (rather than polystyrene), so avoids aggregation with hydrophobic resin

106
Q

What is the process of Sheppard’s method?

107
Q

What is a linker in SPPS?

A

Reversible linkage between synthetic peptide chain and solid support

Cleaved along with acid (TFA) in final step

108
Q

What are the types of linker in SPPS?

A

Wang - produces acid at C terminus

Rink amide - produces amide at C terminus

109
Q

What is the structure of Wang resin linker?

A

Requires that benzyl esters not used as side PG, so then don’t need HF

110
Q

What is the structure of a rink amide linker?

111
Q

What side-chain groups can you use when Fmoc used for amino?

A

Boc, trityl, and t-butly

All removed by TFA unlike Fmoc (base)

112
Q

What is the mech of Fmoc deprotection?

A

Piperidine in DMF

113
Q

What are the main problems of SPPS?

A

Cost/volume of a reaction
Incomplete orthogonality between PGs
Side reactions
Not 100% yield

114
Q

How can you tune SPPS to get higher yields?

A

XS protected AA to avoid interactions between long peptides

115
Q

What is the Kaiser test?

A

Measure completeness fo coupling of N-terminal residue

Uses ninhydrin which reacts with primary amine and turns purple

116
Q

What is the main difference between Boc (Merrifield) and Fmoc (Sheppard) chemistry?

A

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
Q

What is reverse-phase HPLC?

A

Separation of molecules due to hydrophobicity

Stationary phase - hydrophobic ligands immobilized on silica

Mobile phase - polar/aqueous with the peptides (ACN and MeCN)

118
Q

How does elution rate change in reverse-phase HPLC?

A

Goes via gradient elution
So amount of ACN:MeCN or using ionic modifier like TFA

119
Q

How effective is reverse-phase HPLC?

A

Excellent resolution under many conditions
Easy to optimise and change conditions
High recoveries
Good reproducibility

120
Q

What is the asprtimide problem in Fmoc-SPPS?

A

Aspartimide forms in side reaction
This can then react

121
Q

Learn the purines

122
Q

Learn the pyrimidines

123
Q

What is the sugar unit in nucleosides/nucleotides?

124
Q

What is a nucleotide and nucleoside?

A

Nucleoside - nucleic acid and sugar

Nucleotide - nucleic acid, sugar, and phosphate group

125
Q

What changes in DNA and RNA compounds?

A

Uracil replaces thiamine

Ribose repleaces deoxyribose

126
Q

What is the 5’ or 3’ end of a DNA strand?

A

End of DNA strand with OH in either 5 position or 3

127
Q

How does DNA dissolve in water?

A

Phosphodiester - salt of phosphoric acid ester

Polyanion so dissolves in water

128
Q

How is DNA usually read?

A

5’ to 3’ end

129
Q

What is the H-bonding in AT base pair?

130
Q

What is the H-bonding in GC base pair?

131
Q

What is the structure of DNA in general?

A

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
Q

What is the major and minor groove of DNA?

A

Major - deeper and wider channel in helix where proteins interact

Minor - smaller channel in helix where small molecules interact

133
Q

What mechanism of DNA replication?

A

Uses DNA polymerases and nucleotide triphosphates, with Pi is leaving group

From 3’ to 5’

134
Q

How can incorrect base pairing occur?

A

Rare in replication but repaired by enzymes

135
Q

What occurs to H-bonding when purine:purine?

A

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
Q

What occurs to DNA structure if pyrimidine:pyrimidine base pairing occurs?

A

Helix constricts and worse base-stacking

137
Q

What is the tautomer hypnothesis with bases?

A

1 in 104 bases exist as their minor tautomer

Allows for incorrect base pairing

138
Q

What is the minor tautomer base pairing?

139
Q

What does adding new nucleotides allow for?

A

Non-nautral side chains in proteins

But remember these bases dont H-bond

140
Q

What is the mech for adding an amino acid at the ribosome?

141
Q

What is the mech for adding an amino acid at the ribosome?

142
Q

Why might you stabilise a base duplex?

A

Used in diagnostic and therapeutic settings

143
Q

How can you increase the stability of A.T duplex?

A

Dont use S - cannot H bond

144
Q

How can you increase the stability of G.C duplex?

145
Q

Why is the phosphodiester backbone in that form?

A

Entropically favoured
Correct shape to bind to RNA

146
Q

What is the use of analogues of DNA/RNA backbones?

A

Binds to complementary DNA or RNA with high affinity

147
Q

What is the structure of the DNA triple helix?

A

Not watson-crick
Transiently formed but not v stable

148
Q

What are the adv/disadv of triple helix DNA?

A

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
Q

How do antisense oligos prevent protein function?

150
Q

What is the key advantage of targeting RNA in pharma?

A

Pharmacophore (DNA sequence) and dianophore (molecular features that determine tissue distribution and metabolism) can be optimized separtely

151
Q

What are the main problems of antisense therapeutic oligos?

A

Limited cellular uptake
Toxicity
Off-target effects
Unstable to nuclease degredation

152
Q

What are Lipinski’s rules for an active drug?

A

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
Q

How can you stabilise oligos?

154
Q

How can you stabilise oligos?

155
Q

Where is chirality introduced in stablised antisense oligos?

156
Q

What is a gapmer?

A

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
Q

When are phosphorothioate linkages used?

A

Resistant to degradation by nucleases

158
Q

What is the therapeutic effect of methylating C/U?

A

Reduces toxciticy

159
Q

What is the therapeutic effect of methylating C/U?

A

Reduces toxciticy

Year 3 - Chemistry (12 decks)

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