ucl chem2601 peptide chemistry lectures

CHEM2601: Chemistry of Biologically Important Molecules

Peptides, Peptidomimetics and Proteins

Dr Rachel Morgan

[email protected]

Background Reading

! General

• Nelson D. and Cox M. (2005) Lehninger Principles of Biochemistry, New York: Worth

• Berg J., Tymoczko J. and Stryer L. (2002) Biochemistry , New York: W H Freeman

• Creighton T. (1992) Proteins: Structures and Molecular Properties, New York: W H

Freeman

! Synthesis

• Jones J. (2002) Amino Acid and Peptide Synthesis, Royal Society of Chemistry

• Doonan S (2002) Peptides and Proteins, Royal Society of Chemistry

• Bondanszky M. and Bodanszky A. (1994) The Practice of Peptide Synthesis, Berlin:

Springer-Verlag

Proteins, Peptides and Peptidomimetics

Proteins

! Perform a vast array of functions, e.g. structure, function and regulation

! There are still many proteins who’s functions is unknown

Peptides

! Involved in defence, signalling and regulation

Peptidomimetics

! Compounds which mimic peptides

! Used to minic bioactive peptides, but provide improved increased bioavailability, biostability,

bioefficiency, and bioselectivity

Primary structure - Sequence

! Series of amino acid, usually L, units linked by amide bonds.

! Written N→C

H-Gly-Ala-Lys-Ser-Glu-OH

GAKSE

N

R

OH

http://en.wikipedia.org/wiki/Protein_structure

NH

HN

ONH

O HN

O

H2NO

OH

O

CO2H

OH

NH2

Creighton T. (1992) Chapter 1. Nelson D. and Cox M. (2005) Chapter 3

Amino acids - hydrophobic

! Ile has an additional stereocentre (3S)

! Tyr and Trp absorb UV light (λmax ~ 280 nm)

! Trp is weakly fluorescence

H3N

RO

O


Amino acids – hydrophilic! Neutral (pH 7)

• Thr has additional chiral centre (3R)

! Charged (pH 7)

• Lys & Arg – basic Asp & Glu - acidic

H3N

RO

O

LysineLysK

ArginineArgR

NHNH2

NH2HN


Amino acids – others

! Pro

• Non polar

• Secondary amine ∴

secondary amide

• Promotes β-turn

! His

• pKa (imidazole) ~ 6

• Strongest general

acid/base at pH 7

! Gly

• Very small ∴

surprisingly polar

! Cys

• Sidechain quite hydrophobic

• pKa (SH) ~ 8

• Soft nucleophile

H3N

RO

O


ProlineProP

NH

OH

O

Amino acids

! Most amino acids S

absolute

configuration ∴ L/D

notation still used


H3N

RO

O

Buffer effect

• If pH = pKa – 2 then

∴ therefore A is 99% protonated

• If pH = pKa + 2 then

∴ therefore A is 1% protonated

• If pH = pKa then A is 50% protonated

Acids and bases

∴ Keq = [H3O+][A-][AH][H2O]_________

∴ Ka = [H3O+][A-][AH]

_________

pKa = -logKa pH = -log[H+]

∴ pH = pKa + log [A-][AH]____

log = -2[A-][AH]____

= 0.01____

= 100____

log = 2____

AH + H2O A- + H3O+

[A-][AH]

[A-][AH]

[A-][AH]

pH titration curves

! Alanine

! Isoelectric point (pI) - pH at which a molecule or carries no net charge

! Cysteine

OH

OH3N O

OH3N

O

OH2N

Nelson D. and Cox M. (2005) Chapter 2

O

OH3N

HS

O

OH3N

S

O

OH2N

S

OH

OH3N

HS

Secondary structure: amide bond! Amides are planar

• C-N bond in peptides = 1.32 Å

• Normal C-N bond = 1.49 Å

• Normal C=N bond = 1.27 Å

! Restricted rotation about C-N bond

• Tend to adopt the trans isomer

! Form strong hydrogen bonding networks

R1 NH

OR2

R1 NH

OR2

R1 NH

OR2

R1 NH

O

R2

>99 1

R1 N

OR2

H

R1 N

OR2

H

O

N

O

HN

O

OH

O

bp 80 °C bp 141 °C

bp 204 °C bp 164 °C

H-bond donor

H-bond acceptor

Ramachandran plots

! Dihedral angles Φ,Ψ and Ω describe

backbone shape

! Ω fixed due to amide bond

! Φ and Ψ governed by sterics of β carbon

! Ala representative of 18 amino acids

• Limited φ,ψ pairs

! Glycine larger range Φ and Ψ - No β carbon

! Proline Φ = -60

φφφφ

ψψψψ 0

0NH

HN

O

O

NH

HN

O

O

HN

O

HN O

NH

HN

O

O


NH

HN

O

OΨ

Ω

Φα

β

120

-60

60

-120

120-60 60-120

Secondary structure: α-helix

! Rise (advance per residue) = 0.15 nm

! 3.6 amino acids per complete turn

! Pitch (advance per turn) = 0.54 nm

! Hydrogen bond between i and i+4 i+4

i

i+1

i+2

i+3


Secondary structure: β-sheet

! Parallel

! Alternating hydrophobic and hydrophillic amino acids results in one polar and one

hydrophobic ‘face’

! Anti-parallel

HN

ON

O

H

HN

OH

O

R

R

R

N

O

H

N

ON

O R

R

H

N

ON

O

H

N

ONH

O

R

R

R

H H

H

H

N

O

RH

HN

ON

O

H

HN

OH

O

R

R

R

N

ON

O

H

N

ON

O

R

R

R

H H

N

ON

O

H

N

ONH

O

R

R

R

H H

H

H


N-term N-term

Secondary structure: Turns! Often between two stands in β-sheet

! γ-turn

• Hydrogen bond i C=O to i+2 H-N

• Φ(i+1) = -60 ∴ often Pro

! β-turn

• Hydrogen bond i C=O to i+3 H-N

• Φ(i+1) = -60 ∴ often Pro

• Glycine often at i+2

• There are different types depending on

the confirmation of the amino acids

O

HN

HN

O

HN

O

R

R

i

i+1

i+2i+3

OH N

HN

O

ii+1

i+2

γ-turn

β-turn (type 1)


Translation

! The amino acids determined by

the codons

! Folding of the protein can be

facilitated by chaperone

proteins

! Post-translational modifications

add a further point of diversity

http://ulsfmovie.org/images/Peptide_Synthesis.jpg

Post-translational modifications

! Add a further point of diversification

! They include:• acylation

• lipidation

• alkylation

• amidation

• nitrosylation

• oxidation

• phosphorylation

• sulfation

• glycosidation

• ubiquitination

Ac-SCoA-

AcylaseH2N NH

O

FarnesylPP-[ho

transferaseOH O

OPO

OO

HOATP-[ho

phosphatase

OH-[ho

transferase

O

DP

Methods of generating peptides and proteins

Advantages

! in its’ native form

! easier to isolate and scale up

! a range of protein sizes feasible

! can easily modify

! not restricted by what the

expression system will accept

Disadvantages

! isolation difficult and hard to scale

up

! cannot easily modify

! can lack necessary PTM

! most functionalisation involves

addition of amino acids

! potential difficulties with folding

! large peptides/proteins less feasible

Isolate directly from species

Use of expression systems

Use of synthetic chemistry

Method

Simple carbonyl chemistry - revision

! Electrophilic at carbon

! Nuclophilic at oxygen

! If R' = good leaving group (X) then productive reaction with a nucleophile (Nu-) can occur.

Addition-elimination via tetrahedral intermediate

! α-protons are acidic

O

R'

O

R'R R

O

R'

O

R'R R

HHO H2O

NHR1

O R2

Amide bond formation

XR1

O

Electrophilic at carbon Amines act as nucleophiles

Amide bond formation from carboxylic acids

Preformed active esters

Advantages:

• Simple to use

Disadvantages:

• Unstable

• Expensive

• Prone to racemisation

Diisopropylcarbodiimide (DIC) /Hydroxybenzotriazole (HOBt)

OHR1

O

OR1

ON

NN

DIC

NN

NOH

HOBt

NNC

OR1

OH N

CN

OR1

ONCN

HO

R1

O

N

HN

NN

NO

OR1

O

N

HN

NN

NO

O N

HN

Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU)

Protecting groups

...etc

Protecting groups

Protecting groups! Protecting groups must be:

• Readily introduced

• Orthogonal to reactions of choice

• Orthogonal to other protecting groups

• Readily removed

! Significantly add to number of synthetic steps

Polypeptide synthesis! Polypeptide always constructed C→N

! PG1 (transient PG) must be orthogonal to PG2 and PG3

! Synthesis approaches are named by the transient PG (PG1)coupling cycle

C-terminus

Amine protecting group – Fmoc

! Fluorenylmethyloxycarbonyl (Fmoc)

O NH

OH

HN

O NH

O

H2N

+ NH

O

O

NHH

- CO2

Amine protecting group – Boc

! tert-butoxycarbonyl (Boc)

Fmoc synthesis of H-Gly-Glu-Ala-OH

BocHN CO2H

FmocHNOtBu

OH2N

OtBu

O

20% piperidine/DMF

NH

HN

O

OH

OH

OOH2N

FmocHN CO2H

DIC/HOBt FmocHNHN

O

OtBu

OtBu

O

H2NHN

O

OtBu

OtBu

O

NH

HN

O

OtBu

OtBu

OOBocHN

20% piperidine/DMF

coupling

globaldeprotection

deprotect Fmoc couple Fmoc-Glu(tBu)-OH

deprotect Fmoc

couple Boc-Gly-OH

95% TFA/H2O

O OtBuO

OOO

basic conditions ∴ tBu unaffected

in Fmoc synthesis Boc final N-terminal PG

removes bothBoc and tBu

Acid and alcohol protecting groups

! PG1 = Fmoc

• tert-butyl

! PG1 = Fmoc

• tert-butyl

O

OTFA

OH

O

TFA

O OH

Amide protecting group

! Why?

! PG1 = Fmoc

• Trityl (Trt)

NH

OR*

O

ONH2

O

NH

O

Ph

PhPh

H

NH

OH

Ph Ph

Ph

H+ transfer

+

NH

O

Ph

PhPh

H+

Amine/imidazole/indole protecting group! Why?

• For Lys

• For His

! PG1 = Fmoc

• Boc Boc deprotection mechanism analogous to that for amines

NH

OR*

O

O HN

HN

NH

OR*

O

O

NH2

Guanidine protecting group

! Why?

! PG1 = Fmoc

• Pbf

Thiol protecting groups

! PG1 = Fmoc

! Trityl (Trt)

• Standard protecting group

! S-tert-butyl (StBu)

• Orthogonal to Trityl

Thiol protecting groups

! Acetamidomethyl (Acm)

• Orthogonal to Trt and StBu

! Deprotection can be oxidative, e.g. I2, or non-oxidative, Hg2+.

S S

HNI2

O

S

HN

O

S

PG1 = Fmoc PG1 = BocAmino acid Functional group PG Deprotection PG Deprotection

Gly

none none none none none

Pro

Ala

Val

Leu

Ile

Met

Phe

Tyr

-OH tBu 95%TFA/H2O Bn Neat HFSer

Thr

Asn-CONH2 Trt 95%TFA/H2O Xan Neat HF

Gln

Trp indole Boc 95%TFA/H2O Formyl Neat HF

His imidazole Boc 95%TFA/H2O Ts Neat HF

Lys -NH2 Boc 95%TFA/H2O 2Cl-Z Neat HF

Arg guanidine Pbf 95%TFA/H2O Ts Neat HF

Asp-CO2H tBu 95%TFA/H2O Cy Neat HF

Glu

Cys -SHTrt

StBuAcm

95%TFA/H2OPPh3/H2O

Hg2+

Mob Neat HF

Side chain protecting groups – PG1 = Boc! Acid – cyclohexyl (Cy)

! Alcohol – benzyl (Bn)

! Amide – xanthyl (Xan)

! Amine – 2Cl-benzyl (2Cl-Z)

! Indole – formyl

! Imidazole – tosyl (Ts)

! Guanidine – tosyl (Ts)

! Thiol – methoxybenzyl (Mob)

O OHHF

NH

NH2

HFOO O

HFN

OH

NH

NH

O

O

NH2

HF

ClS

HF

OMe

SH

Peptide synthesis – Boc vs Fmoc

Boc synthesis

! Advantages:

• Generally high coupling yields

• Cheap building blocks

• Good for ‘difficult’ sequences

• Fast

• Good method for peptide thioesters

! Disadvantages:

• Hazardous side chain deprotection

conditions i.e. neat HF

• Need specialist equipment

• Non-orthogonal deprotection

Fmoc synthesis

! Advantages:

• Mild deprotection conditions

• Orthogonal deprotection

• Relatively safe

• Monitor deprotection

! Disadvantages:

• Slower

• Aggregation

Solution phase synthesis

n

couple → chromatography n → couple → chromatography → product

Solid phase synthesis

n

couple → filter n → cleave → filter → product

Solution phase synthesis

! Advantages:

• Easy reaction monitoring

• Homogeneous reaction conditions

• Good for small peptides

! Disadvantages:

• Isolate at each step

• Purify at each step

• Solubility

• Excess of reagents to drive reaction to completion need to be

removed

• Limited by solubility of growing peptide chain (10 amino acids)

40.0%

50.0%

60.0%

70.0%

80.0%

90.0%

100.0%

0 5 10 15 20

Yiel

d /%

Coupling cycles

99.5%

98.0%

Solid phase peptide synthesis

Advantages:

• Fast, high yielding reactions – excess reagents

• Simple purification – filtration

Disadvantages:

• Reaction monitoring limited

• Reaches limit around 50 amino acids


! Resin

• Cross linked polystyrene

• 40-150 µm

• 0.2-1.2 mmol/g amine

• Solvent permeable

≡2-Chlorotrityl resin

Solid phase linkers ! Wang (Fmoc/tBu)

• Final peptide with C-terminal acid

! 2-Chlorotrityl (Fmoc/tBu)

• Mild deprotection thus final peptide with C-terminal acid still N-terminal and side chain

protected

O

O

OHN

R

0.5% TFA

CH2Cl2peptidePG1HN

PG3

Ph

Cl

OH

OHN

R

peptidePG1HN

PG3

Solid phase linkers ! Rink (Fmoc/tBu)

• Final peptide with C-terminal amide

! MBHA (Boc)

• Final peptide with C-terminal amide


! PG1 = Fmoc

linker and PG3 must be stable to Fmoc

deprotection conditions (20% pip/DMF)

! PG1 = Boc

linker and PG3 must be stable to Boc

deprotection conditions (neat TFA)

PG1HNO

NH

HN

O

OH

OH

OOH2N

global deprotectionand resin cleavage

deprotect PG1 couplePG1-Glu(PG3)-OH

couplePG1-Gly-OH

O

linkerO

HN

O

linkerOO

PG1HN

O OPG3

H2NO

linkerO

HN

O

linkerOO

H2N

O OPG3

HN

O

linkerOO

NH

O OPG3

OPG1HN

deprotect PG1

Example – Synthesis of H-Thr-Lys-Cys-Ala-OH

FmocHNO

WangOHN

O

WangOO

FmocHN

S1) 20% pip/DMF

2) HBTU, DIPEASTrt

CO2HFmocHNHN

O

WangOO

NH

SO

FmocHN

PhPh

Ph

HN O

1) 20% pip/DMF2) HBTU, DIPEA

CO2HFmocHN

BocHN

HN

O

WangOO

NH

SOH

N

PhPh

Ph

HN O

Ph PhPh

ONH

O

O

O

CO2HBocHN

tBuO

O

OHN

O

OHO

NH

SHOH

N

NH2

OH2N

HO95%TFA/H2O

1) 20

% pip/D

MF

2) HBTU, D

IPEA

commerciallyavailable

protected L-amino acidscommercially available

in Fmoc SPPS lastamino acid oftenBoc protected

Further potential problems/challenges:

! Synthesis of large peptides

! Side reactions under cleavage conditions

! Racemisation

! Incomplete coupling

Synthesis of large peptides – Fragment coupling

! Solid phase synthesis limited to

maximum 50 AAs

∴couple multiple fragments in solution

! Classical amide bond formation

• Requires N-terminal, C-terminal and

side chain protection of fragments

• Racemisation often problematic ∴

often R = H

• Peptides must be soluble in organic

solvent

BocHN peptide 1

PG3

peptide 2

PG3

CO2tBu

HN

HATUDMF

95% TFAscavengers

NH

O

CO2H

R+

BocHN peptide 1

PG3

NH

O R

O

O

R'

peptide 2

PG3

CO2tBu

H2NO

R'

peptide 2 CO2HHN

H2N peptide 1 NH

O R

O

O

R'

Biopolymers (Peptide Science) 1999 51, 266-278

Synthesis of large peptides – Staudinger ligation

! Functions with unprotected peptide fragments

! Reaction occurs in water

! Requires

• C-terminal thioester

• N-terminal glycine azide

Tetrahedron Lett. 2003, 44, 4515–4518

Mechanism – Staudinger ligation

Tetrahedron Lett. 2003, 44, 4515–4518

SR

O

NH

O

O

Synthesis of large peptides – Native chemical ligation

! Functions with unprotected peptide fragments

! Reaction occurs in water

! Requires

• C-terminal thioester

• N-terminal cysteine

! Ligation slow if R is bulky

• e.g. Val, Leu

! Multiple ligations using Cys protected fragments

! Cys to Ala via radical desulfurisation

! Also used in Expressed Protein Ligation

H2N peptide 1

peptide 2 CO2HHN

NH

O R

+

H2N peptide 1 NH

O R

O

O

peptide 2 CO2HH2N

O

O

SBn

PhSHH2OpH ~ 8

HS

SH

Science 1994, 266, 776–779. PNAS 1998, 95, 6705–6710

Mechanism – Native chemical ligation

Science 1994, 266, 776–779. PNAS 1998, 95, 6705–6710

SPh

O

H2NO

S

OPhSH

thioesterexchange

H2NO

SH

O

H2N

S

ONH2

O

O

SH++

transthioesterification

SR

O

NH

O

O

SH

H2NO

SH




! Racemisation


Cleavage side reactions - Tyrosine

! What is happening?

OH O OH- H+

H

electrophillic aromatic subsitution

Cleavage side reactions - Tryptophan

! Also with tryptophan and Pbf cation

! How do we get around the problem?

• Add ethanedithiol (EDT) – a good nucleophile, reacts with cations faster than Trp or Tyr

NH

SO

O

O NH

O

SO O

+

HN

O

RinkNHO

NH

SOH

N

PhPh

Ph

O

ONH

OOH

NO

O

HN

O

NH2

ONH

SHOH

N

OH

ONH

HOO

H2N95% TFA/H2O

2%HS

SH




! Racemisation


Racemisation

! Conversion of an enantiomerically pure mixture into a mixture where more than one enantiomer

is present

! Direct enolization - Base catalysed

! will occur if OR is strongly electron withdrawing and there is a strong unhindered base

O

ORHN

O

O

ORHN

O

O

ORHN

O+

acid or basic conditions

Racemisation

! Oxazolone mechanism

! Racemization via stabilised anions fast compared with peptide bond formation

! Less of a problem when the N is functionalised with -CO2R




! Racemisation


Capping

! Target H-A8-A7-A6-A5-A4-A3-A2-A1-OH

! If A6 A5 coupling poor then observe deletion sequence ∴ difficult to purify.

Capping

! If coupling is incomplete by then cap with acetic anhydride (Ac2O)




! Racemisation


Synthesis non natural amino acids

! Synthesise amino acids for:

• Animal feed additives (e.g. lysine as a growth additive for pigs, methionine for poultry)

• Laboratory and industrial scale production of synthetic peptides

• Building blocks for drugs (e.g. β-lactam antibiotics)

• Food additives (e.g. Aspartame, MSG)

! Allow the production of D-amino acids and unusual amino acids.

! Methods include: Strecker, Gabriel

Synthetic approaches - Strecker

Synthetic approaches - Gabriel

BrEtO

O

OEt

O H2NOH

O

HR

i)

ii) NaOEt,

N

O

O

K

Br R

iii) NaOH, H2O

BrEtO

O

OEt

O

N

O

O

K

(i)N

EtO

O

OEt

O

O O

H

NEtO

O

OEt

O

O O(ii)

BrR

NEtO

O

OEt

O

O O

R

(iii) NaOH, H2O

Kinetic resolution - Chemical

! via diastereomeric salts

! Only generate max 50% chemical yield

Diastereomeric salts

Enzymatic Kinetic resolution - α-Chymotrypsin

! Selective hydrolysis of hydrophobic N-Ac-L-amino acid esters e.g. phenylalanine

! Hydrolysis of L- over D- and α- over β-

! α-Chymotrypsin is relatively promiscuous

NH

OO

OAc-L-Asp(Et)-OEt

+NH

OO

OAc-D-Asp(Et)-OEt

NH

OOH

OAc-L-Asp(Et)-OH

NH

OO

OAc-D-Asp(Et)-OEt

α−chymotrypsin+

O

O

O O O

OOO

NH

OO

O

Ph

Ac-L-Phe-OEt

+NH

OO

O

Ph

Ac-D-Phe-OEt

NH

OOH

O

Ph

Ac-L-Phe-OH

NH

OO

O

Ph

Ac-D-Phe-OEt

α−chymotrypsin+

α-Chymotrypsin

! 25 kDa protein

! Serine protease

! Activity of the enzyme is due to a catalytic triad

PDB ID: 1GCT

α-Chymotrypsin

• hydrogen bonds between enzyme and substrate

• Hydrophobic pocket recognises hydrophobic side chain

• Ser His Asp catalytic triad

• Ser is responsible for bond cleavage

PDB ID: 1GCT

α-Chymotrypsin mechanism

N

O O

HO

HN

ONH

Ser-195

NNO

O

His-57Asp102

NHO

R

OH H

NO

HO

HN

ONH

Ser-195

NHNO

O

His-57Asp102

NHO

R

O

OH

α-Chymotrypsin mechanism

NO

HO

HN

ONH

Ser-195

NHNO

O

His-57Asp102

NHO

R

O

HOH

α-Chymotrypsin selectivity

N

O O

HO

HN

ONH

OH

Ser-195

NNHO

O

His-57Asp102

NHO

R

NO

H

HN

ONH

OH

Ser-195

NNHO

O

His-57Asp102

NHO

R

H

OO

Ac-L-Phe-OEt

H

Ac-D-Phe-OEt

Chemically modified peptides and proteins

Why?

! Label with a dye

• can be tracked within cell

• interactions with other proteins

• fluorescence quenching or FRET

! Attach an affinity tag

• enable imobilisation

! Attach a post translational modification

• Phosphate, sugars, lipids, etc

! Make hybrid proteins

• connect two functional units or two

structural units, investigate properties

! Attach peptides to particles

• e.g. liposomes or quantum dots

selective?

X

Chemically modified peptides and proteins

How?

! During synthesis

• Suitable monomers and/or protecting

groups

selective?

X

! Selective reaction on synthetic or WT

protein

Modifying native functional groups - Amines

! Not selective i.e. can react with N-terminus or

Lys side chain

! Generally introduced during peptide synthesis

! Isothiocyanate

! N-hydroxysuccinate (NHS) ester

! Sulfonyl chloride

RO

NHNH2

R

O

ON

O

O

thiourea sulfonamide

! Thiols are attractive groups for functionalisation due to relatively low abundance of free cysteine

! They can also be selectively reacted with compared with amine residues:

Calculating at:

pH 7.4,

pKa (SH) 8.4

→ 10% S-

pKa (+NH3) 10.8

→ <1% NH2

Modifying native functional groups - Thiol

! Iodoacetamide

• React faster with thiols than amines or

imidazoles as pH ~ 7

• Not entirely specific for cysteine

Modifying native functional groups - Thiol ! Maleimide

• At pH 7, thiols react with maleimides 1000x

faster than amines

• Reaction almost completely selective

SN2

Modifying native functional groups - Thiol

! Pyridyl disulfides

• Reversible

SHN S

S R

SS R

HO

HO

SHSH

DTTSH

S

N SS R

N S

pH~7

Modifying native functional groups - Reagent examples

Fluorophores

! Fluorescein

• Most common class of fluorophore

• Often introduced as maleimide or ITC SCl

O O

NMe2

! Dansyl

Native functional groups - Reagent examples

Biotin

! Sulfo-NHS biotin

• Water soluble NHS ester for N-biotinylation

SS N

HN

O NH

O S

HN NH

O

Biotin-HPDP

O

ON

O

ONa

HN NH

S

O

O3S

sulfo-NHS biotin

! Biotin-HPDP

• For protein purification

• Employs avidin-biotin interaction

• Reversible

Ligations

! Staudinger ligation

! Native chemical ligation

+R SR

ONH

peptide OHN3

OR

HN

ONH

peptide OH

O

HS

PPh

Ph

H2O

R SR'

ONH

peptide OHH2N

OR

HN

ONH

peptide OH

O

+

HS

H2O

SH

Modification via non-natural functional groups

Photoreactive groups

! Form highly reactive intermediate

! Often non-selective i.e. react with any residue in close proximity

! Thus, often used to interrogate protein-ligand or protein-protein interactions

! Aryl azide

• React with proximal nucleophiles, e.g. amines, via nitrene


! Diazirine

• Generates highly reactive carbene

! Benzophenone

• Diradical less reactive than diazirine derived carbene

hνO

R

O

R R

HOH


Hydrazones

! Stable at pH 7

! Hydrolysed at pH < 6

! Often used to synthesise peptide/DNA constructs


! Catalysed by Copper (I) generated in situ

! Peptide can contain azide or alkyne

N peptide OH N peptide OHNNR NN

RCuSO4TCEPH2O

H peptide OHN NR

CuSO4TCEPH2O

NH peptide OH

NNN

R

! Rapid and high yielding reaction

! Copper free versions are available

Azide-alkyne cycloaddition – also called ‘click chemistry’

Overall Summary

! Amino acids

! Primary peptide structure

! Secondary structure

! Post translational modifications

! Methods of generating peptides and proteins

! Carbonyl chemistry revision

! Mechanism of amide bond formation

! Methods of forming peptide bonds - coupling

! N-terminal protecting groups

! Side chain protecting groups

! Solution phase synthesis

! Solid phase synthesis

! Choice of linkers



! Racemisation


! Non natural amino acid synthesis

! Resolution of L and D isomers

! Chemically modified peptides

! Native functional groups

! Non-natural functional groups

Example

! Questions

• Unnatural amino acids?

• Non-natural groups?

• Linker?

• N-terminal protecting groups?

• Side chain protecting groups?

• Coupling reagents?

• Resin cleavage step?

H2NHN

NH

HN

NHO

O

O

OS

OHNH2

O

OH

Ph

linker

OFmocHN

OtBu

?

Example

i) 20% piperidine/DMFii) HBTU, Fmoc-Tyr(tBu)-OH iii) 20% piperidine/DMF

iv) HBTU, Fmoc-Ala-OHv) 20% piperidine/DMFvi) HBTU, Boc-Cys(StBu)-OH

OHN

OtBu

NH

HN

BocHNO

O

OtBu

O

SStBu

ClTrityl

OFmocHN

OtBu

ClTritylOO

OHN

OtBu

NH

HN

BocHNO

O

OtBu

O

SH

ClTritylOOH

N

OtBu

NH

HN

BocHNO

O

OtBu

O

S

ClTritylO

NH2

O

Example

H2NNH

HN

NH

HN

O

O

O

OS

OHNH2

O

OH

Ph

OHN

OtBu

NH

HN

BocHNO

O

OtBu

O

S

OH

NH2

O

OHN

OtBu

NH

HN

BocHNO

O

OtBu

O

S

NH

NH2

O

PhHBTUDIPEA

0.5% TFA/CH2Cl2

H2N Ph

95% TFA, 2% HSSH

ucl chem2601 peptide chemistry lectures

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