*5 nucleic acids dna structure - dna replication and repair - amine prep and reactions - amino acids - peptides - protein structure and sequencing

Nucleic Acids (Intro)

Nucleic Acids store information for cellular growth and reproduction.

There are over 3 billion pairs of nucleotide monomers (base pairs) in human DNA.

If unwound to a linear structure,

DNA would have a length of about 6 feet and still fit inside the nucleus of the cell !!

1

are extremely large molecules consisting of long chains of monomers called nucleotides

Examples deoxyribonucleic acid (DNA)

ribonucleic acid (RNA)

Components of a Nucleotide monomer

2

consist of a A) pentose sugar B) base with nitrogen (nitrogenous base) C) phosphoryl group

group

Nitrogenous base

Furanose ring

phosphate

and Phosphate Esters of Adenosine Nucleotide Example: Adenosine-5’-monophosphate (AMP)

Components of a Nucleotide monomer

1

consist of a A) pentose sugar B) base with nitrogen (nitrogenous base) C) phosphoryl group

group

A) Pentose Sugars

The pentose (five-carbon) sugar

•  in RNA is ribose •  in DNA is deoxyribose

(with no O atom on carbon 2′)

Sugar carbon atoms are numbered with primes

to distinguish them from the atoms in the bases

2

B1) Nitrogenous Bases The bases in DNA and RNA are •  Pyrimidines (C, T, and U)

•  Purines (A and G)

3

B2) Nitrogenous Bases

4

B3) Formation of a Nucleoside

A nucleoside forms when a sugar combines with a base.

5

B4) Nucleosides = Nitrogenous Base + Ribose

6

. Name nucleosides by changing the end of the base name to osine for purines and idine for pyrimidines

(or deoxyribose)

(or deoxyribose)

A nucleoside is an N-glycoside of ribose

Nitrogenous Base: Sample Problem

7

8

B5) Nucleoside Summary

OHO N

HO

N

N

N

X

NH2

OHO N

HO

N

N

NH

X

O

NH2

RNA: X= OH, adenosine (A)DNA: X= H, 2'-deoxyadenosine (dA)

RNA: X= OH, guanosine (G)DNA: X= H, 2'-deoxyguanosine (dG)

OHO

HO X

N

N

O

NH2

OHO

HO

N

NH

O

OH3C

RNA: X= OH, cytidine (C)DNA: X= H, 2'-deoxycytidine (dC)

DNA: thymidine (T)

OHO

HO

N

NH

O

O

RNA: R= H, uridine (U)

1

2

34

567

8 9

1'2'3'

4'

5'

OH

1'2'3'

4'

5' 21

34

5

6

To differentiate the atoms of the carbohydrate from the base, the position number of the carbohydrate is followed by a ´ (prime).

The stereochemistry of the glycosidic bond found in nucleic acids is β.

Ribonucleosides or 2-Deoxyribonucleosides

C1) Phosphorylation A nucleotide •  has a phosphoryl group attached to the

C5′ —OH group of a nucleoside

9

Name nucleotides by using the name of the nucleoside followed by “-5′-monophosphate”

C2) Breaking the Phosphoanhydride Bond in ATP

+ energy

11

C3) Biological Phosphorylation: Kinase enzymes that catalyze the phosphoryl transfer reaction (from ATP to an acceptor substrate).

OHOHO

OHOH

OHO N

N

NN

NH2

HO OH

OPOO

OPO

OO

POO

O

OHOHO

OHOH

OPO32-

ATP

O N

N

NN

NH2

HO OH

OPOO

OPO

OO

ADPGlucose-6-phosphateGlucose

+ energy

+ HPO4 2-O N

N

NN

NH2

HO OH

OPOO

OPO

OO

POO

O

ATP

H2O O N

N

NN

NH2

HO OH

OPOO

OPO

OO

ADP

+ ~31 KJ/mol (7.4 Kcal/mol)

ATP (synthesized in the mitochondrion) is the cell’s energy store

Kinases are one of the many types of phosphotransferases.

Kinase

12

C4) Cyclase: enzymes that catalyze the INTRAmolecular phosphoryl transfer reaction.

O N

N

NN

NH2

HO OH

OPOO

OPO

OO

POO

O

ATP

O N

N

NN

NH2

HO OH

OPOO

O

AMP

O

O OH

O

PO

ON

N

NN

NH2

-P2O7

adenylylcyclase H2O

cAMP

O N

N

NNH

O

HO OH

OPOO

OPO

OO

POO

O

GTP

O N

N

NNH

O

HO OH

OPOO

O

GMP

O

O OH

O

PO

ON

N

NNH

O

-P2O7

guanylatecyclase H2O

cGMP

NH2 NH2 NH2

Nobel Prize in Medicine or Physiology (1971)

Cyclic AMP serves as a link between several hormones and certain enzymes that regulate cellular function

done by Earl Sutherland

Learning Check Give the name of the nitrogenous base and sugar, then provide the name and abbreviation for the following molecule.

13

Solution

14

Guanosine-5’-monophosphate (GMP) Sugar: ribose Base: guanine

Give the name of the nitrogenous base and sugar, then provide the name and abbreviation for the following molecule.

1

2

Names of Nucleosides and Nucleotides

3

Nucleotide: monophosphate of the Nucleoside – Adenosine (deoxyAdenosine)

4

Nucleotide: monophosphate of the Nucleoside – Guanosine (deoxyGuanosine)

5

Nucleotide: monophosphate of the Nucleoside – Cytidine (deoxyCytidine)

6

Nucleotide: monophosphate of the Nucleoside - deoxyThymidine

7

Nucleotide: monophosphate of the Nucleoside - Uridine

8

1

Primary Structure of Nucleic Acids

is a strand/sequence of nucleotides

consists of the bases A, C, G, and T or U (in RNA) in various order

linked by a phosphodiester group (between 3′ and 5′ carbons

on two furanose rings)

2

Phosphodiesters The chemical linkage between nucleotide units

in nucleic acids is a phosphodiester,

which connects the 5�-hydroxyl group of one nucleotide to the 3�-hydroxyl group of the next nucleotide.


Representing Nucleic Acids

A nucleic acid polymer has: •  a free 5′-phosphate group at one end

•  a free 3′ —OH group at the other end

•  is abbreviated by listing the letters of the bases

from the free 5′ end

3

By convention, nucleic acid sequences are written from the 5�-end (on the left) to the 3�-end (on the right)

The DNA strand/segment shown here is: 5′—A—C—G—T—3′


• Adenine (A) 30.3% Thymine (T) 30.3% • Guanine (G) 19.5% Cytosine (C) 19.9% • Total purines: 49.8% Total pyrimidines: 50.1%

For example (in Human DNA):

Purines Pyrimidines

Erwin Chargaff (at Columbia University in 1950) carefully analyses DNA from a wide variety of organisms.

Content of A,T, C & G varied widely according to the organism. However: A=T and C=G (Chargaff� Rule)

Secondary Structure of Nucleic Acids

Previously, researchers believed that there were repeating units of GACT nucleotides in DNA

(Phoebus Levene’s Tetranucleotide Hypothesis)

Chargaff met and explained his finding to Watson & Crick in 1952

2) X-ray crystallographic data (of Maurice Wilkins and Rosalind Franklin)

3) Building Molecular Models (the intermolecular distances between bases “fit”)

James&D.&Watson&and&Francis&H.&C.&Crick&proposed&a&structure&for&DNA&in&1953.&

1) Chargaff’s Observations Gives proper Chargaff ratios (A=T and G=C) Because each pair contains one purine and one pyrimidine, the A---T and G---C distances between strands are approximately equal. Complementarity between strands suggests a mechanism for copying genetic information.

Watson'and'Crick's'structure'was'based'on:


3

Hydrogen Bonds in DNA

Watson and Crick proposed that A and T were present in equal amounts in DNA because of

complementary hydrogen bonding.

Likewise, the amounts of G and C in DNA were equal because of complementary hydrogen bonding.

The G-C base pair involves THREE H-bonds

The A-T base pair involves TWO H-bonds


28- 4

The DNA Duplex Watson and Crick proposed

a double-stranded structure for DNA in which a purine or pyrimidine base in one chain

is hydrogen bonded to its complement in the other.


Forces stabilizing DNA double helix

1) Hydrogen bonding (2-3 kcal/mol per base pair)

2) Aromatic Stacking = hydrophobic interactions

(4-15 kcal/mol per base pair) 3) Electrostatic forces

(Phosphates are negatively charged).


1

one helical turn 34 Å

Major groove 12 Å

Minor groove

6 Å

Backbone: deoxyribose and phosphodiester linkage Bases: hydrogen bonding between the bases

~20 Å

5’ end of A-DNA

The strands of the DNA double helix create two

continuous grooves (major and minor)

The sugar–phosphate backbone

runs along the outside of the helix.

The amine bases hydrogen bond to one another

on the inside of the helix.

DNA: Major and Minor Grooves

Wide and deep

Narrow and deep


N

NHN

N

O

NH2

NN

H2N

O

To deoxy

ribose To deoxyribose

Major groove

Minor groove

N

NHN

N

O

NH2

NN

H2N

O

To deoxy


Major groove

Minor groove

N

NHN

N

O

NH2

NN

H2N

O

To deoxy


Major groove

Minor groove

B-DNA

N

NHN

N

O

NH2

NN

H2N

O

To deoxy


Major groove

Minor groove

Helix axis

A-DNA

• •

DNA: Major and Minor Grooves Top View

Groove binding proteins

Leucine zipper proteins bind to the DNA major groove

Helicase binding to DNA major and minor grooves


HN NH3

NH2

NH3

H2N

DAPI

5�--- A

TT

--- 3�

Other examples: Distamycin and Hoechst 33258

DAPI (4,6-diamidino-2-phenylindole) Is a synthetic antibiotic that interferes with DNA-processing enzymes (regulatory proteins)


Netropsin

Netropsin strongly binds to the minor groove of DNA

that is enriched with AT sequences.

This slightly changes the rotational conformation of DNA

thereby, interfering with DNA packing

Groove binding drugs

1

DNA: Tertiary Structure

B) DNA is �packaged�� by coiling around a core of proteins

known as histones.

C) The DNA-histone assembly is called a nucleosome.

A) Each cell contains about six feet of DNA (If it was unwound into a linear structure).

2


Loops of chromosomal DNA attached to a nuclear scaffold.

Next level of organization (after 30 nm fiber)

DNA: Tertiary Structure We are skipping this in our Orgo Course

(but keep this for reference)

4


1) Amines: Classification

Amines are classified as: NH3 (ammonia), 10 (primary), 20 (secondary), 30 (tertiary) or 40 (quaternary) based on the number of alkyl groups bonded to the nitrogen atom.

NH2

NH

NH3CH2C N

CH2CH3

CH2CH3H

HNH

H3C N

primary (1°) amines secondary (2°) amines tertiary (3°) amine quarternary (4°) ammonium ion

Examples

A) An alkylamine has a Nitrogen atom that is: sp3 hybridized, pyramidal, with bond angles of approximately 109.50.

2) Amines: Structure and Bonding

B) An arylamine has a Nitrogen atom that is bonded to an aromatic ring

C) Nitrogen heterocycles each have a Nitrogen atom within a cyclic system. (The N atom is considered to be at position �1� when naming)

If an amine nitrogen has four different groups around it, it is technically a stereogenic (chiral) center.

3A) Amines: Inversion

The barrier to nitrogen inversion is about 25 KJ/mol

(very rapid at room temperature).

3B) Amines: Inversion

In contrast, the chirality of a quaternary ammonium salt with four different groups has

no lone (nonbonded electron) pair on the nitrogen atom.

Interconversion cannot occur, and the N atom is just like

a carbon atom with four different groups around it.

nitrosoamine

�  basicity of amines (the unshared pair of electrons on the nitrogen atom)

�  form hydrogen bonds (the polarity of the carbon-nitrogen bond)

�  form nitrosamines (amines + nitrites in food + strong acid -or- heat) ~ nitrosamines tend to be carcinogens

4A) Amines: Physical Properties

4B) Amines: Physical Properties

1

1) Identify the longest carbon chain that includes the carbon attached to the Nitrogen atom 2) Use the lowest position number possible to designate where the Nitrogen atom is attached on the longest carbon chain

1A) Naming Amines - IUPAC Names

1-ethanamine is OK (but redundant)

3) Replace the �e��of the alkane name with �amine.�

4) Use the prefix �N-� to name the other (smaller) alkyl group(s) attached to the Nitrogen atom.

2

N,N-Dimethylethanamine

1B) Naming Amines - IUPAC Names - Examples

3

1)  List the names of the alkyl (carbon) groups bonded to the Nitrogen atom. Use the �yl��ending

2)  Place them in alphabetical order (combine the same alkyl groups with di- or tri- prefix)

3) Add the ending �amine.�

Ethyldimethylamine

Dimethylamine

Ethylamine

2) Naming Amines - Common Names

4

The amine of benzene is called aniline •  may have alkyl groups attached to Nitrogen that

use the prefix N- and the alkyl name

Aniline 3-Chloroaniline N-Methylaniline

(m-chloroaniline)

NH2

Cl

NH2 CH3NH

3) Naming Amines – Aromatic Amines

Basicity and Nucleophilicity increase from right to left in the same row (period) of the PT.

better nucleophile

poorer nucleophile

Therefore, amines are the most common neutral organic bases

Amines Basicity/Nucleophilicity: Organic Bases We are going to make the HUGE generalization that

Basicity (attacking a proton) = Nucleophilicity (attacking a carbon/electrophile), which we know is NOT the case (look at tBuOK, LDA, NaH, others)

With an amide, the electron pair is delocalized onto the carbonyl oxygen by resonance.

Amides: delocalization onto electronegative atoms (oxygen) Amines Basicity/Nucleophilicity: 1) Resonance

This decreases the electron density on N, making an amide much less basic than an alkylamine.

Pyridine and pyrrole are both aromatic, but the nonbonded electron pair on the N atom of pyrrole

is part of the aromatic system.

Amines Basicity/Nucleophilicity: 2) Aromaticity Heteroaromatics (nitrogen lone pairs that are part of aromaticity)

The pyrrole lone pair is delocalized through the aromatic ring. Thus, pyrrole is a much weaker base than pyridine.

Compare the basicity of piperidine and pyridine (two nitrogen heterocycles).

Amines Basicity/Nucleophilicity: 3) Hybridization Nitrogen Hybridization: sp3 -vs- sp2 -vs- sp

The hybridization of the orbital that contains an amine�s lone pair

also affects its basicity/nucleophilicity.

Alkyl groups are slightly electron-donating groups. Alkyl groups increase the electron density on nitrogen,

which makes a primary amine like CH3CH2NH2 more basic than NH3 (ammonia)

Amines Basicity/Nucleophilicity: 4) Inductive Effect

Steric Hinderance: Tertiary amines tend to have about the same nucleophilicity as secondary amines,

because of the steric hinderance of the extra alkyl group (unless the alkyl groups are “tied-back” in a ring system)

Amines Basicity/Nucleophilicity: 1) Summary of Effects

Consider the steric hinderance of the alkyl groups separately

Amines react as bases with a variety of organic and inorganic acids.

pKas of Protonated Amines: 1) Amines Act as Bases

lower pKa value = stronger acid

When an amine is treated with a strong acid, it is protonated, forming an ammonium salt (an ionic compound),

which is water soluble, but insoluble in organic solvents.

pKas of Protonated Amines: 2) Water Solubility of Salts

Because amines are protonated by aqueous acid, they can be separated from other organic compounds

(by extraction using a separatory funnel).

When an amide is treated with acid, protonation occurs at the carbonyl oxygen (not the nitrogen),

because the resulting cation is resonance stabilized.

Amides are not much more basic than any carbonyl compound.

pKas of Protonated Amines: 3) Amides

The product of protonation on the NH2 group cannot be resonance stabilized.

Look at the Conjugate Acid: Higher pKa value = weaker conjugate acid

pKas of Protonated Amines: 4A) Judging Nucleophilicity

The relative basicity of different compounds (such as amines) can be compared using the pKa values of their conjugate acids.

Look at the corresponding base (amine): Weaker conjugate acid = stronger base Stronger base = better nucleophile

the pKa of CH3CH2NH3+ is higher than the pKa of NH4

+, so CH3CH2NH2 is a stronger base (better nucleophile) than NH3.

pKas of Protonated Amines: 4B) Nucleophilicity by pKa

Look at the Conjugate Acid: Higher pKa value = weaker conjugate acid

Look at the corresponding base (amine): Weaker conjugate acid = stronger base Stronger base = better nucleophile

Compare CH3CH2NH2 (ethylamine) and NH3 (ammonia)

pKas of Protonated Amines: 5) Many Examples

Which amine (in each set) is the most nucleophilic?

pKas of Protonated Amines: 6) Testing

Quinoline and isoquinoline are known as benzopyridines

pKas of Protonated Amines: 7) Another Example

pKas of Protonated Amines: 8) More pKa Data

Substitutents can greatly influence the basicity of the aniline. The effect is dependent upon the nature and position of the substitutent.

Amine Basicity/Nucleophilicity – Substituted Anilines

NH2Y NH3 + H2O + H3O+Y

+

The strength of Basicity/Nucleophilicity

of this Nitrogen on aniline

depends on the electronic effects

of the substituent (Y)

Amine Basicity/Nucleophilicity – EDGs

Whether a substituent donates or withdraws electron density depends on the balance of its inductive and resonance effects.

Whether a substituent donates or withdraws electron density depends on the balance of its inductive and resonance effects.

Amine Basicity/Nucleophilicity – EWGs

4

Electron-donating substituents (-CH3, -OH, -OCH3) make the substituted aniline more basic than aniline itself

Electron-withdrawing substituents (-Cl, -NO2)

make the substituted aniline less basic than aniline itself

Y= -NH2 pKa= 6.2 -OCH3 pKa= 5.3 -CH3 pKa= 5.1 -H pKa= 4.6 -Cl pKa= 4.0 -CF3 pKa= 3.5 -CN pKa= 1.7 -NO2 pKa= 1.0

More Basic (better nucleophile)

Less Basic

(poorer nucleophile)

NH2Y NH3 + H2O + H3O+Y

+

Amine Basicity/Nucleophilicity – Substituted Anilines

•  The Hofmann elimination converts an amine into an alkene.

•  The Hofmann elimination consists of three steps, as shown for the conversion of propylamine to propene.

Amine Reactions: Hofmann Elimination

Overall Reaction

All Hofmann elimination reactions result in the formation of a new π bond between the α and β carbon atoms, as shown for cyclohexylamine and 2-phenethylamine.

Examples



Mechanism – E2 (Beta) Elimination


Compare the E2 elimination reactions of alkyl halides (and tosylates) -vs- quaternary ammonium salts

This result is explained by the:

Mechanism – Regiochemistry

A) size of the leaving group B) steric hinderance

5

Mechanism – Anti Periplanar Geometry


Overview Diazotization

Nitrous acid, HNO2, is a weak unstable acid formed from NaNO2 and a strong acid like HCl.

1) Making Nitrous Acid Diazotization

2) Making a Nitrosonium Ion

In the presence of acid, nitrous acid decomposes to +NO, the nitrosonium ion. This electrophile then goes on to react with amines to form diazonium salts.

Diazotization

3) Making Diazonium Salts Diazotization

•  Alkyl diazonium salts are generally not useful compounds. •  They readily decompose below room temperature and form

carbocations with loss of N2, a very good leaving group. •  These carbocations usually form a complex mixture of

substitution, elimination and rearrangement products.

Diazotization 4) Diazonium Salts from primary (1o) ALKYLamines

20 Alkylamines and 20 aryl amines react with nitrous acid to form N-nitrosamines.

Diazotization 5) NO Diazonium Salts from Secondary (2o) Amines

Nitrous acid reacts with 10 arylamines

to form diazonium salts.

Diazotization 6) Diazonium Salts from Primary (1o) ARYLamines

Aryl Diazonium Salts are useful intermediates in a series of reactions:

The Sandmeyer Reaction.

Aryl diazonium salts undergo substitution reactions.

Sandmeyer Reactions

Diazonium salts provide easy access to many different benzene derivatives.

Sandmeyer Reactions

Know this four-step sequence, because it can be used to

synthesize many substituted benzenes.

3

NX

OH I F Cl Br CN H

Cu(CN)Cu2O, H2O H3PO2HBF4NaI HCl,

CuClHBr, CuBr

N

Advantages of the aryl diazonium salt intermediate: Introduces aryl substituents that are not otherwise accessible,

such as -OH, -F, -I, and -CN.

Sandmeyer reaction: promoted by Cu(I) salts

Sandmeyer Reactions

Summary of Synthetic Transformations of Aryl Diazonium Salts

Substitution Reactions of Aryl Diazonium Salts

A diazonium salt reacts with water in the presence of Copper(I)oxide to form a phenol.

This is an alternative to direct chlorination and bromination of the aromatic ring using Cl2 or Br2 and a Lewis acid catalyst.

HBr HCl

Cu2O

Sandmeyer Reactions


A diazonium salt reacts with fluoroboric acid to form an aryl fluoride. This is a useful reaction because aryl fluorides cannot be produced by direct fluorination with F2 and a Lewis acid catalyst.

A diazonium salt reacts with sodium or potassium iodide to form an aryl iodide. This is a useful reaction because aryl iodides cannot be produced by direct iodination with I2 and a Lewis acid catalyst.

Sandmeyer Reactions


A diazonium salt reacts with copper(I) cyanide to form benzonitrile. Since the cyano group can be converted into a variety of other functional groups, this reaction provides easy access to a wide variety of benzene derivatives.

A diazonium salt reacts with hypophosphorus acid to form benzene. This reaction is useful in synthesizing compounds that have substitution patterns that are not available by other means.

Sandmeyer Reactions

When a diazonium salt is treated with an aromatic compound that contains a strong EDG

the two rings join together to form an azo compound (a compound with a nitrogen=nitrogen double bond).

Coupling Reactions of Aryl Diazonium Salts

Azo compounds are highly conjugated, rendering them colored.

Many of these compounds are synthetic dyes.

(once used to color margarine)

This reaction is another example of electrophilic aromatic substitution, with the diazonium salt acting as the electrophile.

Coupling Reactions of Aryl Diazonium Salts

Many common synthetic dyes such as , para red, and Congo red,

are azo compounds.

Natural and Synthetic Dyes

An azo compound that may be good for dying wool or silk, (both polyamides), may be poor for dying cotton, a carbohydrate.


To be classified as a dye, a compound must be colored and it must bind fabric.

Compounds that bind to fabric by some type of attractive force are called direct dyes.

The attractive forces may be: electrostatic interactions, van der Waals forces, hydrogen bonding, and sometimes even covalent bonding

The type of interaction depends on the functional groups of the dye and the fiber.

Positively charged NH3+ groups bonded to the protein backbone

are electrostatically attracted to anionic groups in a dye like methyl orange.


Wool and silk contain charged functional groups, such as NH3

+ and COO¯. Thus, they bind to ionic dyes by electrostatic interactions.

Cotton, on the other hand, binds dyes by hydrogen bonding interactions with its many OH groups.


Congo red is bound to the cellulose backbone by hydrogen bonds.

1

α-Amino Acids have an amino group (NH2) bonded to the α carbon of a carboxy group (COOH),

and so they are called α-amino acids.

The 20 amino acids that occur naturally in proteins differ only in the identity of the R group bonded to the α carbon.

Amino Acids – Intro

(Fischer Projection)

2

Common naturally occurring amino acids are called L-amino acids.

Amino Acids – Occurance in Nature

Their enantiomers, D-amino acids, are rarely found in nature.

D-Amino acids are found in the cell walls of bacteria. The D-amino acids are not genetically encoded, but derived from the epimerization of L-isomers.

All L-amino acids (except cysteine) have the S configuration

(using the Cahn-Ingold-Prelog R,S designations)

Amino Acids – Chirality

All amino acids have common names. These names can be represented by

either a one-letter or a three-letter abbreviation.

Glycine, 2-amino-acetic acid, is achiral (since R=H)

1

Amino acids with side chains comprised of nonpolar groups (only carbon & hydrogen atoms or aromatic or thioether (R-S-R) groups)

are called nonpolar amino acids.

1) Amino Acids – Types: Nonpolar

2

Amino acids with an additional COOH group in the side chain are called acidic amino acids.

2A) Amino Acids – Types: Polar Acidic

3

Amino acids with an additional basic Nitrogen atom in the side chain

are called basic amino acids.

2B) Amino Acids – Types: Polar Basic

Why am I not including AAs that have an amide

in their side chain in this group?

Draw-In and Circle the most basic

lone pair on nitrogen in each side chain.

4

Amino acids with side chain that contain polar groups alcohols (ROH), thiols (RSH), or amides R(C=O)NH2

are called polar neutral amino acids.

2C) Amino Acids – Types: Polar Neutral

1

Amino acids are amphoteric: they can react as either an acid or a base.

Zwitterions

The amine reacts as a weak base.

Proton transfer in an amino acid forms a salt called a zwitterion.

The carboxylic acid reacts as a weak acid.

zwitterion

2

Amino acids exist as a zwitterion: a dipolar ion having both a formal positive and formal negative charge

(overall charge neutral).

Zwitterions

A B

1

the pH at which the amino acid exists predominately in its zwitterionic (net-neutral) form

1) Isoelectric Point (pI): Definition

The value for pI is largely influenced by the structure/reactivity of the side chain (R).

zwitterion

2

The pKa of the –COOH group is typically ~2

2) Isoelectric Point (pI): pKa values

The pKa of the –NH3+ group is typically ~9.

pI can be calculated from the average of the pKa values of the α-COOH and α-NH3

+ groups (for neutral amino acids only).

CO2

CH3

HH3NCO2H

CH3

HH3N

low pH

CO2

CH3

HH2N

+ +

high pH

pKa1(2.3)

pKa2(9.7)zwitterion

The isoelectric point (pI) of an amino acid is the pH at which it has no net charge

3A) Isoelectric Point (pI): Calculation for amino acids that are Polar Neutral or NonPolar

The pI of an amino acid that has an ionizable side chain is the average of the pKa values of the similarly ionizing groups

3B) Isoelectric Point (pI): Calculation for amino acids that are Polar Basic or Polar Acidic

5

4) Isoelectric Point (pI): pI Chart

You do NOT need to know the exact pI value

for each AA

You DO need to know the range of pI values

for each AA Type

You DO need to know the Structure and Type

for each AA

AA Types:

Polar

-vs-

NonPolar

Polar Neutral Polar Acidic Polar Basic

1) Gel Electrophoresis: Acid-Base Equil. A mixture of amino acids can be separated by

electrophoresis on the basis of their different net ioinc charge

The net ioinc charge at a specific pH depends upon the pI value of the AA

zwitterion Remember:

Zwitterion exists when pH = pI net charge is zero (neutral)

at lower pH value at higher pH value

2) Gel Electrophoresis: Determine Net Charge

3) Gel Electrophoresis: Separation

4

4) Isoelectric Point (pI): pI Chart

You do NOT need to know the exact pI value

for each AA

You DO need to know the range of pI values

for each AA Type

You DO need to know the Structure and Type

for each AA

AA Types:

Polar

-vs-

NonPolar

Polar Neutral Polar Acidic Polar Basic

Humans can synthesize only 10 of these 20 amino acids. Ten essential amino acids

must be obtained from the diet. Ten nonessential amino acids

can be synthesized in the body from essential amino acids.

1) Essential -vs- Nonessential Amino Acids

We will use the list of Essential Amino Acids “As Is,” but …

Histidine was once thought to be essential only for infants, but it is now known that small amounts

are also needed for adults.

These * amino acids are “conditionally essential,” if there are either inadequate concentrations of

precursors or or enzymes needed to create these in vivo.

Arginine is “essential” even though it can be

synthesized by mammals, (but it gets consumed to form urea).

Tyrosine is misclassified as nonessential, because it is derived from the

essential amino acid, L-Phenylalanine.

*

*

* * *

* (See the Special Topic)


Proteins that contain all the essential amino acids are called complete proteins.

Proteins found in most animal products (and soybeans) are complete proteins.

Proteins that lack one or more of the essential amino acids are called incomplete proteins.

Some plant proteins are incomplete proteins but a meal can be made into a complete protein

by combining foods (i.e. rice and beans).


1

Treating an aldehyde with NH4Cl and NaCN first forms an α-amino nitrile, which can then be hydrolyzed (using acid, water, heat) to an amino acid.

1A) The Strecker Synthesis Overall reaction

2

1B) The Strecker Synthesis Mechanism

3

Hydrogenation of Alkene A with the chiral rhodium catalyst Rh* forms the S isomer of N-acetyl phenylalanine in 100% ee.

2A) Knowles Asymmetric Hydrogenation

William Knowles (Monsanto), Ryoji Noyyori, and Barry Sharpless won the Nobel Prize in Chemistry (2001) for metal-catalysed, enantioselective synthesis.

Hydrolysis of the acetyl group on nitrogen then yields a single enantiomer of phenylalanine.

4

This catalyst is synthesized from a rhodium salt and a phosphorus compound BINAP (2,2′ -bis(diphenylphosphino)-1,1′ binaphthyl).

It is the BINAP moiety that makes the catalyst chiral (even though it has no tetrahedral stereogenic centers).

BINAP Catalyst 2B) Knowles Asymmetric Hydrogenation

5

2C) Knowles Asymmetric Hydrogenation DIPAMP Catalyst

Chiral reaction catalyst creates diastereomeric transition states

(one more stable than the other) that lead to an excess of one enantiomeric product

Hydrogenation of a Z enamido acid with a chiral hydrogenation catalyst produces S enantiomer selectively

7

1) Hell-Volhardt-Zellinski Reaction: Bromination of the a-carbon of a carboxylic acid by treatment with Br2 and PBr3 (and then water workup)

3A) HVZ Product + SN2 Reaction with NH3

2) Use ammonia NH3 (or phthalimide) to displace Br in an SN2 reaction

8

Usually, alkylation of ammonia with simple alkyl halides does not generally afford high yields of 1° amines.

3B) HVZ Product + SN2 Reaction with NH3

In this case, the amino group in the product is: A)  less basic; B) more sterically hindered than other 1° amines; and

C) ammonia (NH3) is used in excess.

So the single alkylation occurs and the desired amino acid is obtained.

Reaction of an α-keto acid with NH3 then reduction with sodium cyanoborohydride

produces an α-amino acid

4) Reductive Amination of α-Keto Acids

2) NaCNBH3

3) mild acid workup

1) Acid (cat.) + heat

9

1

To resolve a racemic mixture of amino acids such as (R)- and (S)-alanine,

the racemate is first treated with acetic anhydride to form N-acetyl amino acids.

1A) Kinetic Resolution of Racemates

The products are still enantiomers, so they are still inseparable.

2

Chiral reagents such as enzymes can be used to selectively react ONE of the amino acids

1B) Kinetic Resolution of Racemates

Enantiomers react differently with chiral reagents.

Pig Kidney AminoAcylase

Is used

3

A chiral amine (resolving agent) can be reacted with the free carboxy group of the N-acetyl alanine

to give a separable pair of diastereomers.

2A) Resolving Salts of Racemates

4

2B) Resolving Salts of Racemates

1

The term “Peptide” is usually reserved for amino acid polymers

of less than 50 amino acid residues.

1) Peptides: Intro

An amino group from one residue forms an amide bond

with the carboxyl of a second residue

Peptides (and proteins) are amino acid polymers in which the individual amino acid units, called amino acid residues,

are linked together by amide (peptide) bonds.

The long, repetitive sequence of !N!CH!CO! atoms that make up a continuous chain is called the protein�s backbone

2

2) Peptides: N-Terminus / C-Terminus

Peptides are always written with the C-terminal amino acid

(the one with the free !CO2H group) on the right – this is the ROOT name

Peptides are always written with the N-terminal amino acid

(the one with the free !NH2 group) on the left – this is the substituent

and gets a “-yl” ending

Serylalanine is abbreviated Ser-Ala (or S-A)

3

Since each amino acid has both an amino group and a carboxy group, two different peptides can be formed.

3) Peptides: Dipeptides

N-terminal amino acid is always written at the left end of the chain and the C-terminal amino acid at the right.

4

4) Peptides: Resonance Structures

All bond angles are ~120° and the C=O and N–H bonds

are oriented 180° from each other.

Amides are more resonance stabilized than other acyl compounds,

so the resonance structure having the C=N makes a significant contribution to the hybrid.

Resonance stabilization forces all six atoms involved in the peptide bond

to lie in the same plane.

5

Rotation about the C–N bond is restricted, because it has partial double bond character.

As a result, there are two possible conformations.

5) Peptides: Conformations

R1

HN N

O R2 HN

Oamide bond

R1

HN N

O R2 HN

O

_

+

H H

6

Bradykinin (a nonapeptide) stimulates smooth muscle contraction, dilates blood vessels and causes pain.

Arg–Pro–Pro–Gly–Phe–Ser–Pro–Phe–Arg Bradykinin

6) Peptides: Biological Function

Relatively simple peptides can have important biological functions.

Bradykinin is also a component of bee venom.

1

1) Peptide Synthesis: The Problem

The synthesis of a specific dipeptide such as Ala–Gly from alanine and glycine is complicated

because both amino acids have two functional groups.

As a result, four products Ala–Ala Ala–Gly Gly–Gly Gly–Ala

are possible.

2

To selectively join amino acids, protect the functional groups you don�t want to react

and then form the amide bond.

2A) Peptide Synthesis: The Solution

3

2B) Peptide Synthesis: The Solution (cont.)

1

Converting the N-terminal amine into a carbamate, (a functional group having a carbonyl bonded to both an oxygen and a nitrogen atom)

kills the nucleophilicity of the N-terminal amine of the amino acid.

Carbamates

Since the N atom of the carbamate is bonded to a carbonyl group,

the protected amino group is no longer nucleophilic.

1) N-Terminus Protecting Groups

2

Boc Protection

1)The Boc (tert-butoxycarbonyl) protecting group, is formed by reacting the amino acid

with di-tert-butyl dicarbonate.


3

Boc Deprotection

2) The Boc Group can be removed by acids such as trifluoroacetic acid, HCl, or HBr.


4

1) The Fmoc (9-fluorenyl-methoxycarbonyl) protecting group, is formed by reacting an amino acid

with 9-fluorenyl-methyl chloroformate.

Fmoc Protection


5

Fmoc Deprotection

Fmoc protecting group is stable to most acids

The Fmoc protecting group can be removed by treatment with a weak base (NH3 or an amine).


6

RO Cl

O+ Base

NH

O

O

Otert-butoxycarbonyl

(t-BOC)removed with

mild acid

R

NH

O

O

O

C6H5

benzyloxycarbonyl(cBz)

removed with mild acid or by hydrogenolysis

R

NH

O

O

Ofluorenylmethylcarbonyl

(FMOC)removed with mild base

(piperidine)

NH3

O

O NH

O

RO

O

OH


Carbamate Summary

We aren’t doing this one

1

The carboxy group is usually protected as a methyl or benzyl ester

by reaction with an alcohol and an acid.

1) C-Terminus Protecting Groups

Protecting as Esters

2

The esters are usually removed by hydrolysis with aqueous base.


Deprotecting Methyl or Benzyl Esters

3


Deprotecting Benzyl Esters

An advantage to using the benzyl ester for protection is that it can also be removed with H2 in the presence of a Pd catalyst.

This process is called hydrogenolysis.

Benzyl esters can also be removed with HBr in acetic acid.

The hydrogenolysis conditions are especially mild because they avoid the use of either acid or base.

•  Usually converted into methyl or benzyl esters •  Removed by mild hydrolysis with aqueous NaOH •  Benzyl esters are cleaved by catalytic hydrogenolysis of the weak benzylic C–O bond

4


Protecting as Esters: Summary

1

1) Peptide Coupling: Overview

2) Peptide Coupling: Mechanism – Part 1

Peptide bond formation can be accelerated if the carboxylic acid is activated.

Reagent: DCC (dicyclohexylcarbodiimide)

3) Peptide Coupling: Mechanism – Part 2

Amino acids can be added to the growing C-terminal end by repeating these two steps

4) Peptide Coupling: Growing the C-Terminal End

When the desired number of amino acids has been added to the chain, the protecting group can be removed

5) Peptide Coupling: Removing the Protecting Group

6

C6H11 N C N C6H11

R O

O

C6H11 N C NH

C6H11

R O

O

+

R O

O

CNC6H11

C6H11

NH

R'-NH2••

R O

O

CHN

C6H11

C6H11

NH

NHR' +

R NH

OR'

NH

NH

OC6H11 C6H11+

(DCC)

Amide DCU

"activated acid"

R O

O

CN

C6H11

C6H11

NH

NR'

++HH

H

NH

Ala

H2N

Val

+ NH

HN

OcBz OH

O

OBn

O

cBz OBn

O

selectivelyremove N-protecting

group

H2NHN

OOBn

O

NH

cBz OH

O

Ph

Phe (F)

NH

HN

OOBn

OOHN

Ph

cBz

DCC

peptidecoupling

CF3CO2H

DCCH2, Pd/C

NH

HN

OOH

OOH2N

PhPhe - Ala - Val

(F - A - V)

6) Peptide Coupling: Summary

The disulfide bridges in proteins contribute to the overall shape of a protein

1) Disulfide Bonds: Protein Shape

2

2) Disulfide Bonds: Cross-Link Two Ways

Disulfide bonds are covalent bonds that stabilize the protein structure.

3) Disulfide Bonds: Formation

Primary structure (Amino acid sequence)�

Secondary structure �α-helix, β-sheet�

Tertiary structure �Three-dimensional structure formed by assembly of secondary structures��

Quaternary structure �Structure formed by more than one

polypeptide chains�

Protein Structure

Protein Structure: Shapes

Protein Structure: Stabilizing Interactions

Protein Structure: Subunits in Quaternary Structures

Hemoglobin Tetramer An assembly

of four globular protein (heme) subunits

Separation is achieved with:

- extreme pH

- 8 M urea

- 6 M guanidine HCl (guanidinium chloride)

- high salt concentration (usually ammonium sulfate)

Denaturation: Separating peptide chains and subunits

Interactions between protein subunits depend on weak forces

2) Reduce with Sulfhydryl reducing agents

To prevent any disulfide bond(s) being reformed, use the alkylating agent, iodoacetate

Cleavage of Disulfide bridges

Mercaptoethanol Dithiothreitol or Dithioerythritol (Cleland’s reagent)

1) Oxidize with Performic acid

2

Cleavage of Disulfide bridges: Oxidation -or-Reduction

NO INFORMATION is gained about the sequence, or order of attachment of the amino acids.

Complete hydrolysis tells us the identity and relative amount of each amino acid

present in the peptide

Subject the peptide/protein to hydrolysis conditions

that will break all amide (peptide) bonds

Complete Hydrolysis: Goals

Complete Hydrolysis: Protocol

Liquid&Phase&Hydrolysis!a.  Place!the!protein!sample!in!a!hydrolysis!tube,!and!dry.!!b.!Add!200!μL!of!Hydrolysis!Solution!per!500!μg!of!protein.!!!c.!Freeze!the!sample!tube!in!a!dry!iceBacetone!bath,!and!!

flame!seal!under!vacuum.!!d.!Samples!are!typically!hydrolyzed!at&110ºC&for&24&hours&!

in!a!vacuum!or!inert!atmosphere!to!prevent!oxidation.!!!Longer!hydrolysis!times!(e.g.,!48!and!72!hours)!are!investigated!if!there!is!a!concern!that!the!protein!is!not!completely!hydrolyzed.!

!

Hydrolysis Solution: 6 N hydrochloric acid (containing 0.1% - 1.0% of phenol to prevent halogenation of tyrosine)

Complete Hydrolysis: conversion of a peptide into a mixture of its component amino acids

Complete Hydrolysis: Example

1

Chromatography: Ion Exchange

Chromatography: Ion Exchange

Aspartic Acid elutes first, followed by Alanine; Lysine elutes last,

but only after pH of the buffer is increased (and Lysine gets deprotonated).

Under the pH=6 buffer conditions

2

There is a problem in detecting amino acids; they are colorless, since they have no (or very little) conjugation.

(even the the four aromatic amino acids barely absorb in the UV)

To overcome this difficulty, amino acids are converted (after separation by ion exchange chromatography)

to a derivative using ninhydrin.

H3N C CO

H O

R(any)

O

O

OH

OH2 +

O

ON

O

O

Ninhydrin (2 mol) reacts with ANY amino acid (1 mol) to give the

SAME purple colored product.

Chromatography: Detect AAs

3

Chromatography: Ninhydrin Mechanism

4

Chromatogram of Amino Acid Analysis

5

Chemical Analysis: Identify the N-terminal residue

Chemical tests to identify the N-terminal AA:

Edman's reagent

Dansyl chloride method

Dabsyl chloride method

Sanger’s Reagent

2

Pehr Edman devised a chemical method for cleaving the amino-terminal of a peptide without distrupting the peptide bonds between the other a.a. residues.

Chemical Analysis: Identify the N-terminal residue

You could potentially remove one residue at a time (sequentially) from the amino end of the peptide chain

in an automated fashion (using a sequenator) to determine the entire primary structure of the peptide.

The nucleophilic NH2 group of the N-terminal amino acid reacts with the electrophilic carbon of phenyl isothiocyanate.

1A) Edman Degradation

3

Chemical Analysis: Identify the N-terminal residue 1B) Edman Degradation

Chemical Analysis: Identify the N-terminal residue 2) DansylChloride

Chemical Analysis: Identify the N-terminal residue 3) DabsylChloride

6

The peptide is first treated with 1-fluoro-2,4-dinitrobenzene (Sanger�s reagent),

which selectively reacts with the N-terminal amino group. NO2

O2N

FH3N

O

R1 HN CO2+

Δ

NH O

R1 HN CO2nucleophilic

aromaticsubstitution

NO2

O2N

NH O

R1NH2

NO2

O2N

+plus other unlabeled

amino acids

enzymaticdigestion

-or-H3O+, Δ

Chemical Analysis: Identify the N-terminal residue 4A) Sanger’s Reagent

The peptide is then hydrolyzed to their amino acids and

the N-terminal amino acid identified as its N-(2,4-dinitrophenyl) derivative (DNP).

S G MVFA

Sanger's Reagent(2,4-dinitrofluorobenzene)

C C

OH

CH2OH

H

NH3N C C

H O

CH3 CH2CH2SCH3

H O

CC ON

H

C C

OH

H

N

H

C C

OH

CH(CH3)2

N

HH

N

CH2

H O

CC

C C

OH

CH2OH

H

N

H

N C C

H O

CH3

O2N

NO2

CH2CH2SCH3

H O

CC OHN

H

C C

OH

H

N

H

C C

OH

CH(CH3)2

N

HH

N

CH2

H O

CC

Chemical Analysis: Identify the N-terminal residue 4B) Sanger’s Reagent

S

H3O, heat(total hydrolysis)

C C

OH

CH2OH

H

N

H

N C C

H O

CH3

O2N

NO2

CH2CH2SCH3

H O

CC OHN

H

C C

OH

H

N

H

C C

OH

CH(CH3)2

N

HH

N

CH2

H O

CC

C CO

OH

CH2OH

N N

CH2

H O

COC C CO

OH

CH(CH3)2

N C CO

OH

H

NCH2CH2SCH3

H O

COCNH3H3H3 H3 H3

+

++ + +

"tagged" A

F V G M

) S, F, V, G, and M("tagged" A plus an equimolar mixture of

H

N C C

H O

CH3

O2N

NO2

OH

Chemical Analysis: Identify the N-terminal residue 4C) Sanger’s Reagent

Peptidases: Exo and Endo Enzymatic cleavage of specific Amino Acid Residues

1) EXOpeptidases catalyze the cleavage of a terminal amino acid

A) Aminopeptidases cleave the amino acid from the N-terminal end

B) Carboxypeptidases cleave the amino acid from the C-terminal end

2) ENDOpeptidases catalyze the fragmentation of a peptide by cleaving an internal (nonterminal) amino acids.

Enzymes from the brush border of the small intestine

Digestive enzymes from the pancreas

Exhibit selectivity towards specific side chains

A F V MG

C C

OH

CH2OH

H

NH3N C C

H O

CH3 CH2CH2SCH3

H O

CC ON

H

C C

OH

H

N

H

C C

OH

CH(CH3)2

N

HH

N

CH2

H O

CC

S

A (first aa released) F V GS

Leucineaminopeptidase, 10 min

Leucineaminopeptidase, 10 more min

F VS (2nd aa released) G

+

M

C C

OH

CH2OH

N

CH2CH2SCH3

H O

CC ON

H

C C

OH

H

N

H

C C

OH

CH(CH3)2

N

HH

N

CH2

H O

CCH3N C CO

H O

CH3

H3

C CO

OH

CH2OH

N

M

CH2CH2SCH3

H O

CC ON

H

C C

OH

H

N

H

C C

OH

CH(CH3)2

N

H

N

CH2

H O

CCH3 H3+

1A) Peptidases: EXOpeptidase Leucineaminopeptidase cleaves the N-terminal AA

2) Peptidases: EXOpeptidase Carboxypeptidases cleaves the C-terminal AA

Carboxypeptidase B (hog pancreas)

only works on Arg and Lys

Carboxypeptidase A cleaves any residue

except Pro, Arg, and Lys

4

Chymotrypsin: cleaves at the C-terminal side of aromatic residues Phe, Tyr, and Trp

NH O

HN

O

NH

R3

O

HN

R1O

H3NCO2

NH3

NH O

HN

O

H3N

R3

O

HN

R1O

H3NCO2

NH3

O +trypsin

H2O

NH O

HN

O

NH

R3

O

HN

R1O

H3NCO2 N

H O

HN

O

H3N

R3

O

HN

R1O

H3NCO2O +chymotrypsin

H2O

Trypsin: cleaves at the C-terminal side of basic residues, Arg -or- Lys (but not His)

3A) Peptidases: ENDOpeptidase

Unless followed by Proline


5

Chymotrypsin: cleaves at the C-terminal side of aromatic residues Phe, Tyr, and Trp

Trypsin: cleaves at the C-terminal side of basic residues, Arg -or- Lys (but not His)

3B) Peptidases: ENDOpeptidase



From Lehninger Principles of Biochemistry

4) Peptidases: ENDOpeptidase Summary

1.  CNBr is useful because proteins usually have only a few Met residues"2.  CNBr is specific for (it acts only on) methionine residues"

Chemical Analysis: Cyanogen Bromide

Cyanogen bromide causes the hydrolysis of the amide bond

on the C-side of a methionine residue

R C

O

CH C

O

N R1N

HH

CH2CH2S

CH3N C Br

R C

O

CH C

O

N R1N

HH

CH2CH2S

CH3N C Br

R C

O

CH C

O

N R1N

HH

CH2CH2S

CH3N C Br

R C

O

CH C

O

N R1N

HH

CH2CH2S

CH3

C N

Br


Mechanism – Part 1

S

CH3

C N

R C

O

CH C N R1N

HH

H2C OCH2

(Schiff Base)

R C

O

CH C

O

N R1N

HH

CH2CH2S

CH3

C N

R C

O

CH C

O

N R1N

HH

CH2CH2S

CH3

C N

R C

O

CH C

O

N R1N

HH

CH2CH2S

CH3

C N

R C

O

CH C

O

N R1N

HH

CH2CH2S

CH3

C N



R C

O

CH C N R1N

HH

H2C OCH2

R C

O

CH CN

H

H2C OCH2

OH2O

R1 N

H

H



Education

*5 nucleic acids dna structure - dna replication and repair - amine prep and reactions - amino acids - peptides - protein structure and sequencing