CHEM2601: Chemistry of BioIogicaIIy

Important MoIecuIes
Peptides, Peptidomimetics and Proteins
Dr Rachel Morgan
r.morgan@ucl.ac.uk
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
! nvolved 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 NC
H-Gly-Ala-Lys-Ser-Glu-OH
GAKSE
N
R
O H
http://en.wikipedia.org/wiki/Protein_structure
N
H
H
N
O
N
H
O
H
N
O
H
2
N
O
OH
O
CO
2
H
OH
NH
2
Creighton T. (1992) Chapter 1. Nelson D. and Cox M. (2005) Chapter 3
Amino acids - hydrophobic
! le has an additional stereocentre (3S)
! Tyr and Trp absorb UV light (/
max
~ 280 nm)
! Trp is weakly fluorescence
H
3
N
R
O
O
Creighton T. (1992) Chapter 1. Nelson D. and Cox M. (2005) Chapter 3
Amino acids - hydrophiIic
! Neutral (pH 7)
Thr has additional chiral centre (3R)
! Charged (pH 7)
Lys & Arg basic Asp & Glu - acidic
H
3
N
R
O
O
Lysine
Lys
K
Arginine
Arg
R
NH
NH
2
NH
2
HN
Creighton T. (1992) Chapter 1. Nelson D. and Cox M. (2005) Chapter 3
Amino acids - others
! Pro
Non polar
Secondary amine !
secondary amide
Promotes -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
H
3
N
R
O
O
Creighton T. (1992) Chapter 1. Nelson D. and Cox M. (2005) Chapter 3
Proline
Pro
P
N
H
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
H
3
N
R
O
O
Buffer effect
f pH = pK
a
2 then
! therefore A is 99%
protonated
f pH = pK
a
+ 2 then
! therefore A is 1%
protonated
f pH = pK
a
then A is 50% protonated
Acids and bases
! !
"#
=
[H
3
O
+
][A
-
]
[AH][H
2
O]
_________
! !
$
=
[H
3
O
+
][A
-
]
[AH]
_________
%!
$
= -log!
$
%& = -log[H
+
]
! %& = %!
$
+ log
[A
-
]
[AH]
____
log = -2
[A
-
]
[AH]
____
= 0.01
____
= 100
____
log = 2
____
AH + H
2
O A
-
+ H
3
O
+
[A
-
]
[AH]
[A
-
]
[AH]
[A
-
]
[AH]
pH titration curves
! Alanine
! soelectric point (p) - pH at which a molecule or carries no net charge
! Cysteine
OH
O
H
3
N
O
O
H
3
N
O
O
H
2
N
Nelson D. and Cox M. (2005) Chapter 2
O
O
H
3
N
HS
O
O
H
3
N
S
O
O
H
2
N
S
OH
O
H
3
N
HS
Secondary structure: amide bond
! Amides are planar
C-N bond in peptides = 1.32
Normal C-N bond = 1.49
Normal C=N bond = 1.27
! Restricted rotation about C-N bond
Tend to adopt the trans isomer
! Form strong hydrogen bonding networks
R
1
N
H
O
R
2
R
1
N
H
O
R
2
R
1
N
H
O
R
2
R
1
NH
O
R
2
> 99 1
R
1
N
O
R
2
H
R
1
N
O
R
2
H
O
N
O
H
N
O
OH
O
bp 80 C bp 141 C
bp 204 C bp 164 C
H-bond donor
H-bond acceptor
Ramachandran pIots
! Dihedral angles ",# and $ describe
backbone shape
! $ fixed due to amide bond
! " and # governed by sterics of carbon
! Ala representative of 18 amino acids
Limited %,& pairs
! Glycine larger range " and # - No carbon
! Proline " = -60
% %% %
& && &
!
!
N
H
H
N
O
O
N
H
H
N
O
O
H
N
O
HN O
N
H
H
N
O
O
Creighton T. (1992) Chapter 5. Nelson D. and Cox M. (2005) Chapter 4
N
H
H
N
O
O
#
$
"
'
(
120
-60
60
-120
120 -60 60 -120
Secondary structure: o-heIix
! Rise (advance per residue) = 0.15 nm
! 3.6 amino acids per complete turn
! Pitch (advance per turn) = 0.54 nm
! Hydrogen bond between ' and '+4
!"#
!
!"$
!"%
!"&
Creighton T. (1992) Chapter 5. Nelson D. and Cox M. (2005) Chapter 4
Secondary structure: -sheet
! Parallel
! Alternating hydrophobic and hydrophillic amino acids results in one polar and one
hydrophobic 'face'
! Anti-parallel
H
N
O
N
O
H
H
N
O
H
O
R
R
R
N
O
H
N
O
N
O R
R
H
N
O
N
O
H
N
O
N
H
O
R
R
R
H H
H
H
N
O
R H
H
N
O
N
O
H
H
N
O
H
O
R
R
R
N
O
N
O
H
N
O
N
O
R
R
R
H H
N
O
N
O
H
N
O
N
H
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 (-sheet
! y-turn
Hydrogen bond ' C=O to '+2 H-N
"('+1) = -60 ! often Pro
! (-turn
Hydrogen bond ' C=O to '+3 H-N
"('+1) = -60 ! often Pro
Glycine often at '+2
There are different types depending on
the confirmation of the amino acids
O
H
N
H
N
O
HN
O
R
R
'
'+1
'+2
'+3
O
H
N
H
N
O
'
'+1
'+2
y-turn
(-turn (type 1)
Creighton T. (1992) Chapter 5. Nelson D. and Cox M. (2005) Chapter 4
TransIation
! 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-transIationaI modifications
! Add a further point of diversification
! They include:
acylation
lipidation
alkylation
amidation
nitrosylation
oxidation
phosphorylation
sulfation
glycosidation
ubiquitination
Ac-SCoA
-
Acylase
H
2
N
N
H
O
FarnesylPP
-[ho
transferase
OH O
O
P
O
O
O
HO
ATP
-[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
solate directly from species
Use of expression systems
Use of synthetic chemistry
Method
SimpIe carbonyI chemistry - revision
! Electrophilic at carbon
! Nuclophilic at oxygen
! f R' = good leaving group (X) then productive reaction with a nucleophile (Nu-) can occur.
Addition-elimination via tetrahedral intermediate
! d-protons are acidic
O
R'
O
R'
R
R
O
R'
O
R'
R
R
H HO H
2
O
N
H
R
1
O R
2
Amide bond formation
X
R
1
O
Electrophilic at carbon
Amines act as
nucleophiles
Amide bond formation from carboxyIic acids
Preformed active esters
Advantages:
Simple to use
Disadvantages:
Unstable
Expensive
Prone to racemisation
DiisopropyIcarbodiimide (DIC) /HydroxybenzotriazoIe (HOBt)
OH
R
1
O
O
R
1
O
N
N
N
DC
N
N
N
OH
HOBt
N
N
C
O
R
1
O
H N
C
N
O
R
1
O
N
C
N
H
O
R
1
O
N
HN
N
N
N
O
O
R
1
O
N
HN
N
N
N
O
O N
HN
TetramethyI-O-(1H-benzotriazoI-1-yI)uronium hexafIuorophosphate
(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
PoIypeptide synthesis
! Polypeptide always constructed CN
! PG
1
(transient PG) must be orthogonal to PG
2
and PG
3
! Synthesis approaches are named by the transient PG (PG
1
)
coupling cycle
C-terminus
Amine protecting group - Fmoc
! Fluorenylmethyloxycarbonyl (Fmoc)
O N
H
O
H
HN
O N
H
O
H
2
N
+
N
H
O
O
NH H
- CO
2
Amine protecting group - Boc
! tert-butoxycarbonyl (Boc)
Fmoc synthesis of H-GIy-GIu-AIa-OH
BocHN CO
2
H
FmocHN
O
(
Bu
O
H
2
N
O
(
Bu
O
20% piperidine
/DMF
N
H
H
N
O
OH
OH
O O
H
2
N
FmocHN CO
2
H
DC/HOBt
FmocHN
H
N
O
O
(
Bu
O
(
Bu
O
H
2
N
H
N
O
O
(
Bu
O
(
Bu
O
N
H
H
N
O
O
(
Bu
O
(
Bu
O O
BocHN
20% piperidine/DMF
coupling
global
deprotection
deprotect Fmoc
couple Fmoc-Glu(
(
Bu)-OH
deprotect Fmoc
couple Boc-Gly-OH
95% TFA/H
2
O
O O
(
Bu
O
O O
O
basic conditions
!
t
Bu unaffected
in Fmoc synthesis Boc
final N-terminal PG
removes both
Boc and tBu
Acid and aIcohoI protecting groups
! PG
1
= Fmoc
tert-butyl
! PG
1
= Fmoc
tert-butyl
O
O
TFA
OH
O
TFA
O OH
Amide protecting group
! Why?
! PG
1
= Fmoc
Trityl (Trt)
N
H
OR*
O
O
NH
2
O
N
H
O
Ph
Ph
Ph
H
NH
O
H
Ph Ph
Ph
H
+
transfer
+
N
H
O
Ph
Ph
Ph
H
+
Amine/imidazoIe/indoIe protecting group
! Why?
For Lys
For His
! PG
1
= Fmoc
Boc
Boc deprotection mechanism
analogous to that for amines
N
H
OR*
O
O H
N
H
N
N
H
OR*
O
O
NH
2
Guanidine protecting group
! Why?
! PG
1
= Fmoc
Pbf
ThioI protecting groups
! PG
1
= Fmoc
! Trityl (Trt)
Standard protecting group
! S-tert-butyl (S
t
Bu)
Orthogonal to Trityl
ThioI protecting groups
! Acetamidomethyl (Acm)
Orthogonal to Trt and StBu
! Deprotection can be oxidative, e.g.
2
, or non-oxidative, Hg
2+
.
S S
HN

2
O
S
HN
O
S
PG
1
= Fmoc PG
1
= Boc
Amino acid FunctionaI group PG Deprotection PG Deprotection
GIy
none none none none none
Pro
AIa
VaI
Leu
IIe
Met
Phe
Tyr
-OH
t
Bu 95%TFA/H
2
O Bn Neat HF Ser
Thr
Asn
-CONH
2
Trt 95%TFA/H
2
O Xan Neat HF
GIn
Trp indoIe Boc 95%TFA/H
2
O FormyI Neat HF
His imidazoIe Boc 95%TFA/H
2
O Ts Neat HF
Lys -NH
2
Boc 95%TFA/H
2
O 2CI-Z Neat HF
Arg guanidine Pbf 95%TFA/H
2
O Ts Neat HF
Asp
-CO
2
H
t
Bu 95%TFA/H
2
O Cy Neat HF
GIu
Cys -SH
Trt
S
t
Bu
Acm
95%TFA/H
2
O
PPh
3
/H
2
O
Hg
2+
Mob Neat HF
Side chain protecting groups - PG
1
= Boc
! Acid cyclohexyl (Cy)
! Alcohol benzyl (Bn)
! Amide xanthyl (Xan)
! Amine 2Cl-benzyl (2Cl-Z)
! ndole formyl
! midazole tosyl (Ts)
! Guanidine tosyl (Ts)
! Thiol methoxybenzyl (Mob)
O OH
HF
N
H
NH
2
HF
O O O
HF
N
O
H
NH
N
H
O
O
NH
2
HF
Cl
S
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
SoIution phase synthesis
n
{ couple chromatography }
n
couple chromatography product
SoIid phase synthesis
n
{ couple filter }
n
cleave filter product
SoIution phase synthesis
! Advantages:
Easy reaction monitoring
Homogeneous reaction conditions
Good for small peptides
! Disadvantages:
solate 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
30.0
60.0
70.0
80.0
90.0
100.0
0 3 10 13 20
!
"
#
$
%

'
(
)*+,$"-. /0/$#1
99.3
98.0
SoIid phase peptide synthesis
Advantages:
Fast, high yielding reactions excess reagents
Simple purification filtration
Disadvantages:
Reaction monitoring limited
Reaches limit around 50 amino acids
SoIid phase peptide synthesis
! Resin
Cross linked polystyrene
40-150 m
0.2-1.2 mmol/g amine
Solvent permeable
=
2-Chlorotrityl resin
SoIid phase Iinkers
! Wang (Fmoc/
!
Bu)
Final peptide with C-terminal acid
! 2-Chlorotrityl (Fmoc/
!
Bu)
Mild deprotection thus final peptide with C-terminal acid still N-terminal and side chain
protected
O
O
O
H
N
R
0.5% TFA
CH
2
Cl
2
peptide
PG
1
HN
PG
3
Ph
Cl
OH
O
H
N
R
peptide
PG
1
HN
PG
3
SoIid phase Iinkers
! Rink (Fmoc/
!
Bu)
Final peptide with C-terminal amide
! MBHA (Boc)
Final peptide with C-terminal amide
SoIid phase peptide synthesis
! PG
1
= Fmoc
linker and PG3 must be stable to Fmoc
deprotection conditions (20% pip/DMF)
! PG
1
= Boc
linker and PG3 must be stable to Boc
deprotection conditions (neat TFA)
PG
1
HN
O
N
H
H
N
O
OH
OH
O O
H
2
N
global deprotection
and resin cleavage
deprotect PG
1
couple
PG
1
-Glu(PG
3
)-OH
couple
PG
1
-Gly-OH
O
linker O
H
N
O
linker O
O
PG
1
HN
O OPG
3
H
2
N
O
linker O
H
N
O
linker O
O
H
2
N
O OPG
3
H
N
O
linker O
O
N
H
O OPG
3
O
PG
1
HN
deprotect PG
1
ExampIe - Synthesis of H-Thr-Lys-Cys-AIa-OH
FmocHN
O
Wang O
H
N
O
Wang O
O
FmocHN
S
1) 20% pip/DMF
2) HBTU, DPEA
STrt
CO
2
H FmocHN
H
N
O
Wang O
O
N
H
S
O
FmocHN
Ph
Ph
Ph
HN O
1
)

2
0
%
p
ip
/
D
M
F
2
)

H
B
T
U
,

D

P
E
A
CO
2
H FmocHN
BocHN
H
N
O
Wang O
O
N
H
S
O
H
N
Ph
Ph
Ph
HN O
Ph Ph
Ph
O
N
H
O
O
O
CO
2
H BocHN
!
BuO
O
O
H
N
O
OH
O
N
H
SH
O
H
N
NH
2
O
H
2
N
HO
95%TFA/H
2
O
1
)

2
0
%

p
i
p
/
D
M
F
2
)

H
B
T
U
,

D

P
E
A
commercially
available
protected L-amino acids
commercially available
in Fmoc SPPS last
amino acid often
Boc protected
Further potentiaI probIems/chaIIenges:
! Synthesis of large peptides
! Side reactions under cleavage conditions
! Racemisation
! ncomplete coupling
Synthesis of Iarge peptides - Fragment coupIing
! 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
PG
3
peptide 2
PG
3
CO
2
!
Bu
H
N
HATU
DMF
95% TFA
scavengers
N
H
O
CO
2
H
R
+
BocHN
peptide 1
PG
3
N
H
O R
O
O
R'
peptide 2
PG
3
CO
2
!
Bu
H
2
N
O
R'
peptide 2
CO
2
H
H
N
H
2
N
peptide 1 N
H
O R
O
O
R'
!"#$#%&'()* ,-($."/( 01"(21(3 2333 31, 266-278
Synthesis of Iarge peptides - Staudinger Iigation
! Functions with unprotected peptide fragments
! Reaction occurs in water
! Requires
C-terminal thioester
N-terminal glycine azide
4(.)56(/)#2 7(..8 4556, 44, 4313-4318
Mechanism - Staudinger Iigation
4(.)56(/)#2 7(..8 4556, 44, 4313-4318
SR
O
N
H
O
O
Synthesis of Iarge peptides - Native chemicaI Iigation
! 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
H
2
N
peptide 1
peptide 2 CO
2
H
H
N
N
H
O R
+
H
2
N peptide 1 N
H
O R
O
O
peptide 2
CO
2
H
H
2
N
O
O
SBn
PhSH
H
2
O
pH ~ 8
HS
SH
01"(21( 2337, 266, 776-779. nAS !""#9 93, 6703-6710
Mechanism - Native chemicaI Iigation
01"(21( 2337, 266, 776-779. nAS !""#9 93, 6703-6710
SPh
O
H
2
N
O
S
O
PhSH
thioester
exchange
H
2
N
O
SH
O
H
2
N
S
O
N
H
2
O
O
S
H
+
+
trans
thioesterification
SR
O
N
H
O
O
SH
H
2
N
O
SH
Further potentiaI probIems/chaIIenges:
! Synthesis of large peptides
! Side reactions under cleavage conditions
! Racemisation
! ncomplete coupling
CIeavage side reactions - Tyrosine
! What is happening?
OH O OH
- H
+
H
electrophillic aromatic subsitution
CIeavage 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
S
O
O
O
NH
O
S
O
O
+
H
N
O
Rink N
H
O
N
H
S
O
H
N
Ph
Ph
Ph
O
O
N
H
O
O
H
N O
O
H
N
O
NH
2
O
N
H
SH
O
H
N
OH
O
N
H
HO
O
H
2
N
95% TFA/H
2
O
2%
HS
SH
Further potentiaI probIems/chaIIenges:
! Synthesis of large peptides
! Side reactions under cleavage conditions
! Racemisation
! ncomplete 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
OR
H
N
O
O
OR
H
N
O
O
OR
H
N
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 -CO
2
R
Further potentiaI probIems/chaIIenges:
! Synthesis of large peptides
! Side reactions under cleavage conditions
! Racemisation
! ncomplete coupling
Capping
! Target H-A
8
-A
7
-A
6
-A
5
-A
4
-A
3
-A
2
-A
1
-OH
! f A
6
A
5
coupling poor then observe deletion sequence !difficult to purify.
Capping
! f coupling is incomplete by then cap with acetic anhydride (Ac
2
O)
Further potentiaI probIems/chaIIenges:
! Synthesis of large peptides
! Side reactions under cleavage conditions
! Racemisation
! ncomplete coupling
Synthesis non naturaI 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. -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 - GabrieI
Br
EtO
O
OEt
O
H
2
N
OH
O
H
R
i)
ii) NaOEt,
N
O
O
K
Br R
iii) NaOH, H
2
O
Br
EtO
O
OEt
O
N
O
O
K
(i)
N
EtO
O
OEt
O
O O
H
N
EtO
O
OEt
O
O O
(ii)
Br R
N
EtO
O
OEt
O
O O
R
(iii) NaOH, H
2
O
Kinetic resoIution - ChemicaI
! !"# diastereomeric salts
! Only generate max 50% chemical yield
Diastereomeric salts
Enzymatic Kinetic resoIution - o-Chymotrypsin
! Selective hydrolysis of hydrophobic N-Ac-L-amino acid esters e.g. phenylalanine
! Hydrolysis of L- over D- and d- over -
! d-Chymotrypsin is relatively promiscuous
N
H
O
O
O
Ac-L-Asp(Et)-OEt
+
N
H
O
O
O
Ac-D-Asp(Et)-OEt
N
H
O
OH
O
Ac-L-Asp(Et)-OH
N
H
O
O
O
Ac-D-Asp(Et)-OEt
!"chymotrypsin
+
O
O
O O O
O O O
N
H
O
O
O
Ph
Ac-L-Phe-OEt
+
N
H
O
O
O
Ph
Ac-D-Phe-OEt
N
H
O
OH
O
Ph
Ac-L-Phe-OH
N
H
O
O
O
Ph
Ac-D-Phe-OEt
!"chymotrypsin
+
o-Chymotrypsin
! 25 kDa protein
! Serine protease
! Activity of the enzyme is due to a catalytic triad
PDB D: 1GCT
o-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 D: 1GCT
o-Chymotrypsin mechanism
N
O
O
H
O
H
N
O
N
H
Ser-195
N
N
O
O
His-57 Asp102
N
H
O
R
O
H
H
N
O
H
O
H
N
O
N
H
Ser-195
N
HN
O
O
His-57 Asp102
N
H
O
R
O
O
H
o-Chymotrypsin mechanism
N
O
H
O
H
N
O
N
H
Ser-195
N
HN
O
O
His-57 Asp102
N
H
O
R
O
HO
H
o-Chymotrypsin seIectivity
N
O
O
H
O
H
N
O
N
H
O
H
Ser-195
N
N
H
O
O
His-57 Asp102
N
H
O
R
N
O
H
H
N
O
N
H
O
H
Ser-195
N
N
H
O
O
His-57 Asp102
N
H
O
R
H
O
O
Ac-L-Phe-OEt
H
Ac-D-Phe-OEt
ChemicaIIy 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
ChemicaIIy modified peptides and proteins
How?
! During synthesis
Suitable monomers and/or protecting
groups
selective?
X
! SeIective reaction on synthetic or WT
protein
Modifying native functionaI groups - Amines
! Not selective i.e. can react with N-terminus or
Lys side chain
! Generally introduced during peptide synthesis
! sothiocyanate
! N-hydroxysuccinate (NHS) ester
! Sulfonyl chloride
R O
NH NH
2
R
O
O
N
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 (+NH
3
) 10.8
<1% NH
2
Modifying native functionaI groups - ThioI
! odoacetamide
React faster with thiols than amines or
imidazoles as pH ~ 7
Not entirely specific for cysteine
Modifying native functionaI groups - ThioI
! Maleimide
At pH 7, thiols react with maleimides 1000x
faster than amines
Reaction almost completely selective
S
N
2
Modifying native functionaI groups - ThioI
! Pyridyl disulfides
Reversible
SH
N S
S R
S
S R
HO
HO
SH
SH
DTT
SH
S
N S
S R
N S
pH~7
Modifying native functionaI groups - Reagent exampIes
FIuorophores
! Fluorescein
Most common class of fluorophore
Often introduced as maleimide or TC
S
Cl
O O
NMe
2
! Dansyl
Native functionaI groups - Reagent exampIes
Biotin
! Sulfo-NHS biotin
Water soluble NHS ester for N-biotinylation
S
S N
H
N
O N
H
O
S
HN
NH
O
Biotin-HPDP
O
O
N
O
O
Na
HN NH
S
O
O
3
S
sulfo-NHS biotin
! Biotin-HPDP
For protein purification
Employs avidin-biotin interaction
Reversible
Ligations
! Staudinger ligation
! Native chemical ligation
+
R SR
O
N
H
peptide
OH
N
3
O
R
H
N
O
N
H
peptide
OH
O
HS
P
Ph
Ph
H
2
O
R SR'
O
N
H
peptide OH
H
2
N
O
R
H
N
O
N
H
peptide
OH
O
+
HS
H
2
O
SH
Modification via non-naturaI functionaI 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, !"# nitrene
Modification via non-naturaI functionaI groups
! Diazirine
Generates highly reactive carbene
! Benzophenone
Diradical less reactive than diazirine derived carbene
h#
O
R
O
R R
HO
H
Modification via non-naturaI functionaI groups
Hydrazones
! Stable at pH 7
! Hydrolysed at pH < 6
! Often used to synthesise peptide/DNA constructs
Modification via non-naturaI functionaI groups
! Catalysed by Copper () generated in situ
! Peptide can contain azide or alkyne
N peptide OH N
peptide
OH N N
R N
N
R
CuSO
4
TCEP
H
2
O
H peptide OH
N N R
CuSO
4
TCEP
H
2
O
N
H peptide OH
N
N N
R
! Rapid and high yielding reaction
! Copper free versions are available
Azide-aIkyne cycIoaddition - aIso caIIed 'cIick chemistry'
OveraII 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
! ncomplete coupling
! Non natural amino acid synthesis
! Resolution of L and D isomers
! Chemically modified peptides
! Native functional groups
! Non-natural functional groups
ExampIe
! Questions
Unnatural amino acids?
Non-natural groups?
Linker?
N-terminal protecting groups?
Side chain protecting groups?
Coupling reagents?
Resin cleavage step?
H
2
N
H
N
N
H
H
N
N
H
O
O
O
O
S
OH
NH
2
O
OH
Ph
linker
O
FmocHN
O
t
Bu
?
ExampIe
i) 20% piperidine/DMF
ii) HBTU, Fmoc-Tyr(
$
Bu)-OH
iii) 20% piperidine/DMF
iv) HBTU, Fmoc-Ala-OH
v) 20% piperidine/DMF
vi) HBTU, Boc-Cys(S
$
Bu)-OH
O
H
N
O
$
Bu
N
H
H
N
BocHN
O
O
O
$
Bu
O
S
S
$
Bu
ClTrityl
O
FmocHN
O
$
Bu
ClTrityl O
O
O
H
N
O
$
Bu
N
H
H
N
BocHN
O
O
O
$
Bu
O
SH
ClTrityl O
O
H
N
O
$
Bu
N
H
H
N
BocHN
O
O
O
$
Bu
O
S
ClTrityl O
NH
2
O
ExampIe
H
2
N
N
H
H
N
N
H
H
N
O
O
O
O
S
OH
NH
2
O
OH
Ph
O
H
N
O
$
Bu
N
H
H
N
BocHN
O
O
O
$
Bu
O
S
OH
NH
2
O
O
H
N
O
$
Bu
N
H
H
N
BocHN
O
O
O
$
Bu
O
S
N
H
NH
2
O
Ph
HBTU
DPEA
0.5% TFA/CH
2
Cl
2
H
2
N Ph
95% TFA, 2% HS
SH