ucl chem2601 peptide chemistry lectures
TRANSCRIPT
CHEM2601: Chemistry of Biologically Important Molecules
Peptides, Peptidomimetics and Proteins
Dr Rachel Morgan
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
Creighton T. (1992) Chapter 1. Nelson D. and Cox M. (2005) Chapter 3
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
Creighton T. (1992) Chapter 1. Nelson D. and Cox M. (2005) Chapter 3
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
Creighton T. (1992) Chapter 1. Nelson D. and Cox M. (2005) Chapter 3
ProlineProP
NH
OH
O
Amino acids
! Most amino acids S
absolute
configuration ∴ L/D
notation still used
Creighton T. (1992) Chapter 1. Nelson D. and Cox M. (2005) Chapter 3
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
Creighton T. (1992) Chapter 5. Nelson D. and Cox M. (2005) Chapter 4
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
Creighton T. (1992) Chapter 5. Nelson D. and Cox M. (2005) Chapter 4
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
Creighton T. (1992) Chapter 5. Nelson D. and Cox M. (2005) Chapter 4
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)
Creighton T. (1992) Chapter 5. Nelson D. and Cox M. (2005) Chapter 4
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
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
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
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
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
Solid phase peptide synthesis
! 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
Solid phase peptide synthesis
! 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
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
Further potential problems/challenges:
! Synthesis of large peptides
! Side reactions under cleavage conditions
! Racemisation
! Incomplete coupling
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
Further potential problems/challenges:
! Synthesis of large peptides
! Side reactions under cleavage conditions
! Racemisation
! Incomplete coupling
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
Further potential problems/challenges:
! Synthesis of large peptides
! Side reactions under cleavage conditions
! Racemisation
! Incomplete coupling
Capping
! Target H-A8-A7-A6-A5-A4-A3-A2-A1-OH
! If A6 A5 coupling poor then observe deletion sequence ∴ difficult to purify.
Further potential problems/challenges:
! Synthesis of large peptides
! Side reactions under cleavage conditions
! Racemisation
! Incomplete coupling
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 - 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 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
Modification via non-natural functional groups
! Diazirine
• Generates highly reactive carbene
! Benzophenone
• Diradical less reactive than diazirine derived carbene
hνO
R
O
R R
HOH
Modification via non-natural functional groups
Hydrazones
! Stable at pH 7
! Hydrolysed at pH < 6
! Often used to synthesise peptide/DNA constructs
Modification via non-natural functional groups
! 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
! Synthesis of large peptides
! Side reactions under cleavage conditions
! Racemisation
! Incomplete coupling
! 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