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Organic Chemistry 2 > Chromophores, Amino Acids > Flashcards

Chromophores, Amino Acids Flashcards

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1
Q

When pH>pI, most of the molecules in a solution have a net ____ charge

A

most molecules have a net negative charge

More = Negative

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2
Q

most of the molecules in a solution will have a net positive charge when
pH___ pI

A

pH

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3
Q

Why do we use electrophoresis?

How does electrophoresis work?

A

Helps us to identify and separate amino acids.
A sample is placed into the middle of the tray, and then a current is applied. This is being run at neutral pH. Amino acids (eg, Aspartate) that have a net negative charge (low pI) will go to the anode. Amino acids (eg, Arginine) with a net positive charge (aka, are protonated, high pI) travel to cathode. And the neutral ones will stay in middle.

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4
Q

If a pigment has a lower Rf, where will it be situated on the tablet?
How do you calculate Rf?

A

Low Rf = lower on plate.

Rf = (distance spot travelled) / (distance solvent front travelled)

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5
Q

If all the points are clumped at the bottom of the plate, what’s wrong?

A

The solvent was not polar enough.

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6
Q

If all the points are at the top of the plate, what’s wrong?

A

The solvent was too polar. (just think toooo polar, too high)

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7
Q

Fully protonated state when:

A

pH is low

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8
Q

pKa1

A

Protonated NH3+, de-protonated CO2-

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9
Q

What are the proportions of pH and pKas when Protonated NH3+, de-protonated CO2-?

A

pKa1(carboxyl)

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10
Q

pKa1

A

Fully de-protonated state

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11
Q

Fully de-protonated state when:

A

pKa1

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12
Q

What’s the pKa for carboxyl group?

Why does it have this value?

A

2

Is so low because of the inductive effect of electron density towards the ammonium group.

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13
Q

What’s the pKa for ammonium group?

Why does it have this value?

A

9 or 10

Very electron-withdrawing

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14
Q

What is Ka?

A

the acid dissociation constant (how much it takes for the acid to dissociate into its conjugate base)

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15
Q

What happens if pKa=pH?

A

[Conjugate Base] = [acid]

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16
Q

Calculating pI for neutral side chains.

Calculating pI for acidic/basic side chains.

A

neutral: avg the strongest NH3 and the strongest CO2H?

acidic/basic: avg the pKas that are the most similar in number

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17
Q

When proteins are denatured, what is lost?

A

Tertiary and sometimes secondary. Primary not lost unless hydrolyzed.

18
Q

What is Amino Acid Analysis used for?

What are the 3 steps to this?

A

To determine the composition and relative amounts of amino acids present in a peptide.
DOES NOT TELL US CONNECTIVITY/no primary structure.

  1. Acid Hydrolysis of peptide to amino acid
  2. Separation of AAs by Ion-Exchange HPLC
    • based on ionic interactions and AA net charge
  3. Quantification
    • determine how many of each AA is here because after it leaves column it reacts with triketone form of ninhydrin (via Nucleophilic Addition/Imine Formation then Decarboxylation to give an amine again) to produce Ruhemann’s Purple (a chromophore), and its absorption is measured. (intensity proportional to amount)
19
Q

Primary Structure
Secondary Structure
Tertiary Structure
Quaternary Structure

A

Primary: sequence/order of AAs – only ruined by hydrolysis
Secondary: folding of proteins into conformations that are stabilized by non-covalent forces (eg, H bonds btwn non-adjacent AAs)
- alpha helix: NH bonded to C=O 4 AAs away
- beta/pleated sheets: NH bonded to C=O on neighbouring B strand
Tertiary: folding into 3D shape/native conformation.
-stabilized by non-covalent interactions (electrostatic, hydrophobic, H bonds) & covalent (disulfide bridges)
Quaternary: aggregate of proteins forming a molecule (ex, hemoglobin). If the protein exists as a single molecule, won’t have a quaternary structure.

20
Q

How are disulfide bridges formed?

Where do we see them?

A

Oxidation of thiols in Cysteine creates cyclic disulfide bridges in Tertiary Structure. Can be reduced by using excess of another thiol.

21
Q

Which amino acids will leave the column first and last?

What is this column made of?

A

Amino acids that are the same charge of the column will be repulsed and jetted out.
Amino acids that have opposite charge will do a lot of interacting inside the column, and come out slower.

Column filled with insoluble polymer that has permanent charges.

22
Q

What is Edman Degradation?

A

It’s N-Terminal Sequencing.
It’s a way to figure out the sequence of AA in a peptide by removing one AA at a time from the N terminus and chemically converting it into a PTH derivative that will have an R group specific to that AA.

23
Q

What AA’s side chain will react with Sanger’s Reagent?

A

Lysine

24
Q

How to protect and deprotect COOH

A

Using solution-phase synthesis

Protect: introduce benzyl ester through Fischer Esterification

Deprotect: treatment of strong acid without breaking/hydrolyzing amide bond that will be made during synthesis. So yea if not too strong of an acid, then good because esters are more acid-labile (will undergo change readily) than amides.

25
Q

Specificity of cleavage of Chymotrypsin and Trypsin

What are they used for?

A

Chymotrypsin: after Phe, Tyr, Trp
Trypsin: after Arg, Lys

Endopeptidase enzymes (proteases) used to cleave/hydrolyze peptide bonds (for breaking peptides into smaller fragments for sequencing purposes)

26
Q

Specificity of cleavage of Chymotrypsin and Trypsin

What are they used for?

A

Chymotrypsin: after Phe, Tyr, Trp (prefers hydrophobic=nonpolar, and aromatic residues)
Trypsin: after Arg, Lys (basic residues)
*after, when written in the N–>C direction

Endopeptidase enzymes (proteases) used to cleave/hydrolyze peptide bonds (for breaking peptides into smaller fragments for sequencing purposes)

27
Q

What’s TFA?

What is it used for?

A

CF3COOH — an acid
To remove BOC (to deprotect NH2)
Its first step is to protonate the carbonyl (using one of its Hs). Then it’ll do an E1 reaction (thus needs stable carbocation to form) to remove the BOC.
It’ll ultimately create isobutylene and CO2 which are let out at gas, and an amino acid/peptide chain

28
Q

What represents the catalytic triad?

What does each do?

A

Asp, His, Ser.
Present in chymotrypsin mechanism.

Asp holds the imidazole ring of His in proper orientation. This is done using a low-barrier H bond (short and strong)

His deprotonates Ser (acts as a base). because Ser-OH is a poor Nu —>O-

Ser acts as the base for the hydrolysis.

29
Q

In the chymotrypsin mechanism of action, what does the enzyme backbone do?
What is the backbone made out of?
What is the purpose of chymotrypsin’s hydrophobic pocket?

A

Enzyme backbone Ser H-bonds with the oxyanion tetrahedral intermediate formed during the hydrolysis rxn to stabilize it. This area known as the oxyanion hole.
Good H donor
Polyamide backbone

Hydrophobic pocket in active site recognizes hydrophobic AAs (Tyr, Phe, Trp only)

30
Q

Steps of Chymotrypsin Mechanism to break amide bond and create acid: (to give new N and new C terminal for respective fragments)

A
  1. Ser-OH is a poor Nu so His deprotonates it==>Ser-O-. Concomitantly, Ser-O- Nuc attack to the substrate to avoid alkoxide anion formation.
    Ser (enz backbone) covalently H-bonds to tet intermediate to stabilize it.
  2. NH- of C-term fragment of substrate leaves as NH2 by deprotonating His and taking its H.
  3. Known as the acyl enzyme bc the substrate is bonded to the enzyme (Ser) as an ester.
    The amine group with C-term peptide chain leaves, being replaced by water
  4. Water is a poor Nu so His deprotonates it. Concomitantly (to avoid generation of hydroxide anion), the water (aka, OH) now Nuc attacks the ester from Step 3.
  5. The same oxyanion intermediate produced is again stabilized by H-bonding to amide backbone. Collapse of this tetrahedral regenerates the C=O. Ser-O- is a poor LG, so His protonates it so that it can leave. (same mechanism as #2)
  6. The enzyme is returned to its original state, the N-term from OG substrate is cleaved and can diffuse away ==>

acid formed and amide bond broken :)

31
Q

In the chymotrypsin catalyzed hydrolysis of a peptide bond: Contrast and Compare
What’s the first tetrahedral intermediate formed from?
What’s the second one formed from?

A

When the OH is O-, the intermediate is being stabilized by the Ser enzyme backbone

32
Q

What is the L configuration?
AA that are L will usually also be what other classification/configuration? (what’s the exception to this?)
And what handed?

A

H3N—+—-H (with R at bottom and COO- at top)
Most L configurations have an S configuration.
Cysteine is the exception: cysteine is L but R.
Most Ls will be right-handed.
But note, the LD and SR are not a direct correlation

33
Q

Amphoteric/Amphiprotic

A

A dual behaviour
Amino acids have both an acidic (RCOOH) and a basic (RNH2) functional group, allowing them to accept a proton from a strong acid, or donate a proton to a strong base.

Proteins are also amphoteric because they can contain acidic (Asp, Glu) and basic (His, Lys, Arg) side chains on their neutral backbone.

34
Q

Draw an ester.

What functional group is associated with it?

A

R-C(=O)-OR’

35
Q

Give the convention of Fischer Projections

A

Vertical lines pointing in/down

Horizontal lines pointing out/up

36
Q

What is the order in which amino acids would leave a negatively charged column?

A

basic (neg) , polar, nonpolar, acidic (pos)

37
Q

Which amino acid will not react with ninhydrin to form Ruhemann’s Purple?

A

Proline because it is a 2 amine, but you need a primary one.

38
Q

What is Sanger’s Reagent?
What is its structure like?
What is it used for?
What reacts with it?

A

DNFB.
It is a benzene ring with 2NO2s and an F. The NO2s are EWGs that allow the compound to undergo nucleophilic substitutions. (The F is its LG)
It is used for N-Terminal Identification (no sequencing). IT hydrolyzes the N-terminal AA which, combined with DNP (the thing formed once it has attached to ammonium. benzene ring with 2NO2s) will be analyzed with HPLC.
It reacts with the (primary) ammonium group at the N-terminal.

39
Q

What type of molecule is the PITC derivative?

What is PITC used in?

A

A thiourea.
PITC for Edman Degradation (N-Terminal Sequencing).
Used to separate the AA from the peptide so that it can be identified. You’ll see its ring and then the amino acid’s side chain hanging off of it – each AA gives a different PTH derivative!! then to be analyzed using HPLC

40
Q

What does a BOC group look like?

What does it turn into during the process of deprotection?

A

that cross structure with a carboxyl group

BOC leaves as isobutylene.

Organic Chemistry 2 (2 decks)

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