chemical synthesis of peptides and proteins: solid...
TRANSCRIPT
LG
O
H2NO
HN
R
R
PGO
NH
O
HN
R
R
PG
OH
OHN
RPG
(PG = Fmoc, Boc, Cbz, etc.)Protected Amino Acid
Activation Solid SupportLinker
Solid SupportLinker
Basic Conditions
+
Chemical Synthesis of Peptides and Proteins: Solid Support
+Ph
PhPh Ph
cross-linked polystyrene1-2 %
DBP or AIBN
+
FG
FG = CH2X (X = Cl, OH, NH2)
FG PEG-FG = (CH2CH2O)n-CH2-X
SolidSupport
FG
Synthesize solid support, add linker,
add first amino acid
These resins swell to 5 times their volume in non-polar solvents (THF, dioxan, DCM), but not in polar solvents (MeOH, EtOH). The swelling properties of the resin in polar solvents can be improved by adding polyethylene glycol (PEG-FG) chains, or by using polyacrylamide resins.
FG = -CH2Cl, -CH2OH
Polystyrene-based resins:
+Ph
PhPh Ph
cross-linked polystyrene1-2 %
DBP or AIBN
particle size depends on agitation speed
functionalize
Chemical Synthesis of Peptides and Proteins: Linkers
Linkers:Linkers are frequently used to control: 1) the functional group at the C-terminus (e.g. acid or amide), and 2) the conditions required for release of the peptide chain, which in turn determines whether side-chain protecting groups are removed or not.
SolidSupport
FG
FG = -CH2NH2, -CH2Cl, -CH2OH
SolidSupport
Linker
Cleavage conditions:
OH
OH
The following resin linkers are used to produce C-terminal carboxylic acids:
Cl Merrifield resin
O
OH
Wang resinO
OH OMe
OMe
Cl
Cl
2-Chlorotrityl-chloride resin
Loaded resin:O
ONH-PG
R O
O
O NH-PG
R
OMeMeOO
O
ONH-PG
R
O
O
ONH-PG
R
Cl
0.5 % TFA (for Fmoc)5 % TFA (Fmoc)20 – 95 % TFA (for Fmoc)HF (for Boc)
O
HN
O NH-Peptide
R
OMe
MeO
Chemical Synthesis of Peptides and Proteins: Peptide CleavageC-terminal amides can be released from :
HN
O NH-Peptide
R
CH3
95 % TFA for Fmoc(much more gentle)
Anhydrous HF for BOC(very harsh)
CH3
+
HN
HO NH-Peptide
R
H2N
O NH-Peptide
R
HN
HO NH-Peptide
RO OMe
OMe
+
H2N
O NH-Peptide
R
CH3
+
CH3
+
O OMe
OMe+
O OMe
OMe+
Same for:O
O NH-Peptide
R
Chemical Peptide Synthesis: Products of Peptide Cleavage
O
HN
O NH-Peptide
R
OMe
MeO 95 % TFA O OMe
OMe
+ +
+
CO2
Peptide
O
HN
O NH-Peptide
R
OMe
MeO95 % TFA
Si HH2O
HSSH
Peptide
Peptide x TFA
x = # basic groups
Diethyl ether
(to precipitate peptide)
NH2
RCOOH, PyBOP, DIPEA, DMF
Chemical Synthesis of Peptides and Proteins: First Residue
Normal coupling conditions:
Determination of Resin “Loading”:Calculated as moles per gram, method of determination depends upon identity of “PG” on the first amino acid.
More difficult reaction: OH(RCO)2O, DIPEA, DMF
NH
O
RPG Solid
SupportNH
Facile SN2 reaction:Cl
R-COOH, Cs2CO3, DMF
NH
O
RPG Solid
SupportO
O
OHN
R1
PGOH
OHN
R1
PGN C N
R
CN
NH
R
RR OH
OHN
R1
PG
O
OHN
R1
PG
O HN
R1
GP
O
CNH
NH
R R
Protected Amino AcidR = cylclohexyl (DCC)R= isopropyl (EDC) Protected Amino Acid
Symmetric Anhydride "(RCO)2O"
Urea
Synthesis of (RCO)2O:
GGCYKDEIKKEIERESKRIKLNDNDDEGNKKIIAPRIFISDDKDSLKCG-NH2
O
NH2 OMe
OMe O
NH OMe
OMe
Fmoc-Gly-OHHBTU/HOBt, DIEA, NMP
GlyFmoc
a) 20% piperidine, DMFb) Fmoc-potected amino acid HBTU/HOBt (4eq.) DIEA, DMFc) Ac2O, HOBt, DIEA, DMF
O
NH OMe
OMe
GGGCYKDEIKKEIERESKRIKLNDNDDEGNKKIIAPRIFISDDKDSLKCFmoc
side chain protection: Boc (K), tBu (S,D,E,Y), Pbf (R), Trt (N,Q,C)
1) TFA, iPr3SiH, H2O EDT (94:1:2.5:2.5)
2) I2 in 80% AcOH/H2O)
From Prof. Robinson’s Lab: The aim was to prepare a fragment of the apical membrane antigen-1 (AMA-1) from the malaria parasite Plasmodium falciparum, as a potential malaria vaccine candidate to elicit an immune response against the parasite:
Chemical Peptide Synthesis: Example 1
Differences for BOC chemistry
1. BOC-Xaa-OH
2. Resin must withstand TFA
3. TFA, not piperidine deprotection
4. Side chain protecting groups mustwithstand TFA
5. Cleavage form resin in anhydrous HF and p-Cresol
Or, can incorporate pseudoproline at problem spots
Can try BOC chemistry: different resin, etc.
Yield = 0.3 %(good yield ~30%)
LG
O
H2NO
HN
R1
R2
PGO
NH O
HN
R1
R2
PG
OH
OHN
RPG
(PG = Fmoc, Boc, Cbz, etc.)Protected Amino Acid
Activation Solid SupportLinker
Solid SupportLinker
Basic Conditions
+
Chemical Synthesis of Peptides and Proteins: Monitoring Coupling Reaction
The identities of PG, LG, R1 and R2, and even the solid support can have a dramatic effect on the reaction rate and yield.
Methods for monitoring the coupling reaction:
1. Chemical stains for free amines:H2N
O
RO
NH
O
HN
R
R
PG
Solid SupportLinker
Solid SupportLinker
Stain Stain
Colored / fluorecent resin or solution No color changeNinhydrin
O
O
O
Fluorescamine
O
O O
O
2. Measuring conductivity or UV-absorbance of subsequent deprotection solution:
O
NH
O
HN
R
R
PG Solid SupportLinker
aromaticO
NH
O
H2N
R
R SolidSupportLinker+ Salt PG
Deprotect
Chemical Peptide Synthesis: Example 1
0
20
40
60
80
100
120
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29
O
NH2 OMe
OMe O
NH OMe
OMe
Fmoc-Gly-OHHBTU/HOBt, DIEA, NMP
GlyFmoc
a) 20% piperidine, DMFb) Fmoc-potected amino acid HBTU/HOBt (4eq.) DIEA, DMFc) Ac2O, HOBt, DIEA, DMF
Rel
ativ
e A
bs (3
13 n
m)
Amino acid position
Chemical Synthesis of Peptides : Problem Sequences
Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-
Ph
Ph
Ph
Ph
PhPh
H3CCH3
H3C
H3CCH3
H3C
Xaa-Xaa-
Xaa-Xaa-Xaa-Xaa-
H2N-Xaa-
Xaa-
Aggregation, secondary structure, folding, collapse, etc
By incorporating pseudoproline dipeptide units at the appropriate place in a "difficult" sequence, problems associated with peptide chain aggregation can sometimes be eliminated.
O
N
O
Fmoc-HN
R1 O
OH
R2
R1 = anyR2 = H, CH3
Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-NH2
Ph PhPhCH3H3C
PhPh
Ph
H3CCH3
H3C
H3C
H3CCH3
H3C
H3CCH3
H3C
HO
HN
OR1 O
R2
PeptidePeptide-HN95 % TFA
O
N
OR1 O
R2
PeptidePeptide-HN
Peptide Synthesis
Limited to Xaa-Ser and Xaa-Thr
Chemical Peptide Synthesis: A Brief History
1901: Fischer's synthesis of glycyl glycine is the first reported synthesis of a dipeptide and the first use of the term "peptide" to refer to a polymer of amino acids. (Ber. Deutsch. Chem. Ges. 1901, 34, 2868).1907: Fischer's synthesis of an octadecapeptide consisting of 15 glycines and 3 leucines, but without control of the amino acid sequence. (J. Chem. Soc. Chem. Comm. 1907, 91, 1749-65).
1932: Introduction of the benzyloxycarbonyl (Z) group for α-amino protection by Max Bergmann. (Ber. Deutsch. Chem. Ges. 1932, 65, 1192-201).1953: Solution phase synthesis of the octapeptide hormone oxytocin by du Vigneaud and coworkers.(J. Am. Chem. Soc. 1953, 75, 4879-80).1963: The first stepwise solid-phase synthesis of a peptide by Bruce Merrifield (J. Am. Chem. Soc. 1963, 85, 2149-54; Science, 1986, 232, 341).
Oxytocin:
Cl
H. Kunz, Angew. Chem. Int. Ed. 2002, 41, 4439.
1966: The first automated machine for peptide synthesis by Merrifield. (Anal. Chem. 1966, 38, 1905-14).1969: The first synthesis of an enzyme: ribonuclease A by Merrifield and Gutte, and RNaseS by Ralf Hirschmannand co-workers. (J. Am. Chem. Soc. 1969, 91, 501-2; J. Am. Chem. Soc. 1969, 91, 507)1978: Introduction of the N-(a)-fluorenylmethoxycarbonyl- (Fmoc-) protecting group for solid-phase peptide synthesis. (J. Chem. Soc. Chem. Comm. 1978, 537-9; Int. J. Pept. Prot. Res. 1978, 11, 246-9).1989: The chemical synthesis and crystal structure of the HIV-1 protease. (A. Wlodawer et. al., Science 1989, 245, 616).1994: Introduction of native chemical ligation for coupling unprotected peptide fragments and so allowing routine access to synthetic proteins. (Science, 1994, 266, 776-79).
2000: The first peptide-based drug manufactured on a multi-ton scale - the anti-HIV drug T20, or Fuzeon, or Enfuvirtide. (M. C. Kang et al., (2000) US Patent 6,015,881; see also B. L. May, Nature Revs. Drug Disc. 2003, 2, 587-593).
H3N
N
NH
HO
H3N
O
H3N
N
NH
HO
H3N
O
SROH
HS
O
O
O
HSNH3
OH
HS
O
O
O
HSNH3
+
Chemical Peptide Synthesis: A Brief History
Chemical Synthesis of Peptides: Modified Residues
Phosphorylation is one of the most common post-translational modifications:
Synthetic monomers for the synthesis of phosphopeptides and proteins:
Fmoc-HNOH
O
O
PO
HOO Ph
Fmoc-HNOH
O
O
PO
HOO Ph
Fmoc-HNOH
O
O
PO
HOO Ph
Fmoc-Phosphoserine(Bzl) Fmoc-Phosphothreonine(Bzl) Fmoc-Phosphotyrosine(Bzl)
Fmoc-HNOH
O
O
PO
HOOH
Fmoc-Phosphotyrosine
NH
HN
O
O
PO
OOH
Phosphoserine
NH
HN
O
HO
Serine (Thr, or Tyr)
Kinase, ATP, Mg+2
H
Phosphatase
10 – 60 % of all proteins phosphorylated
Phosphorylation site(s) on protein surface
Each kinase has loose consensus sequence, ex. PKA phosphorylates (R/K)-(R/K)-X-S
Kinases account for ~2% of the human proteome.
Fmoc-HNOH
O
O
SO
O
O
Fmoc-Sulfotyrosine
- (nBu)4N+
For sulfopeptides:
Chemical Synthesis of Peptides: Modified Residues
Some post translational modifications are highly complex and not fully amenable to standard solid-phase synthesis:
Glycopeptides and Proteins
(Ser or Thr)
Asn-Xaa-(Ser/Thr/Cys)
Approx. 50% of all proteins are glycosylated.
Essential for proper folding of certain proteins, as well as cell-cell communication.
Variable linkages and monosaccharide units allow many glycoforms of the same protein. This often complicates biological production and isolation.
Chemical Synthesis of Peptides: Modified ResiduesAlthough the O-glycosidic and interglycosidic linkages are acid labile, the glycosidic linkages of fully acylatedcarbohydrates can withstand a short treatment with TFA. Hence:
Fmoc-HNOH
O
OO
NHAcAcO
AcO
OAc
R OO
NHAcHO
HO
OHR = H, CH3
Ac-Ser-Tyr-Pro-Thr-Ser-Pro-Ser-Tyr-Ser-NH2
SPPS
TFA
NaOH
CH3
In eukaryotes, one of the most prevalent types of O-linked glycosylation is where N-acetylgalactosamine (GalNAc) or N-acetylglucosamine (GluNAc) is linked in the α-anomeric configuration to the ß-hydroxy group of Ser or Thr. Formation of the O-glycosyl amino acid requires a suitably protected monosaccharide, with a leaving group at the anomeric position:
ORO
RO
XOR
H
OR
ORO
ROOR
ORO
RORO
OR'OR
H
OR
ORO
RO
HOR
OR'
ORActivator
+ R'-OH +
Note that α- and ß-glycosides may be produced, depending upon the starting materials and conditions. But when a neighbouring group is present that can participate in stabilizing the intermediate carbocation, then often the 1,2-trans-glycosides (ß-gluco) anomer is preferred:
ORO
RO
XO
H
OR
OMe
ORO
ROO
OR
O
Me
ORO
RO
O
OR
O
Me
ORO
RO
OR'AcO
H
OR
ORO
RO
HAcO
OR'
OR
preferred
+
Main product
Minor product
X = Halogen (Cl)X = S-RX= O
NH
CCl3
(trichloroacetimidate)
For the synthesis of more complex glycopeptides, it is an advantage to use a relatively simple glycosyl-amino acid, and then to elaborate the glycopeptideenzymatically, e.g.:
Chemical Synthesis of Peptides: Modified Residues
UDP-Galactose
O
NH
O
ONO
OH
OPOH
O
OH
POO
OH
O
HO
HO
OH
HO
CMP-Sialic Acid
N
NH2
ONO
OH
O
OH
POO
OHO CO2H
HOAcHN
HOOH
OH
GDP-Fucose
O O
OH
OPOH
O
OH
POO
OH
N NH
NN
O
NH2O
HOHO
OH
OH
For the synthesis of more complex glycopeptides, it is an advantage to use a relatively simple glycosyl-amino acid, and then to elaborate the glycopeptideenzymatically, e.g.:
Chemical Synthesis of Peptides: Modified Residues
The glycopeptide can then be made into a glycoprotein using native chemical ligation:
But how is the C-terminal thioester made?
For proteins: see inteins..
Chemical Synthesis of Site-Specifically Modified ProteinsWe can use native chemical ligation (review previous slide and homework) to fuse peptide fragments into proteins, this requires an N-terminal Cys and a C-terminal thioester which can be prepared according to the choice of linker:
NH
O
S NH
O
O
N
Nucleophile:(ex. R2-SH)
ONH-Peptide
R
SR2
From previous examples, one might think the following is a good route:
O
HS
OMeMeO R NH
O NH-PG
R
O
S
O Peptide
R
OMeMeO
HS
O Peptide
R
95 % TFA Br-CH2-RS
O Peptide
RR
However, thioesters are not very stable in the presence of primary amines:
O
S
O NH-PG
R
OMeMeO
R NH2
Alkylation:(ex. ICH2CN)
NH
O
S N
O
OO
NH-Peptide
R
N
RCO2HPyBOP/DIPEA Peptide synthesis
NH
O
SHN
O
OO
NH-Peptide
R
This has motivated the development of a “saftycatch” resin:
NH
O
S NH2
O
O
Chemical Synthesis of Site-Specifically Modified Proteins
By modifying this approach, the same resin can be used to make cyclic peptides:
DeprotectNH
O
S N
O
OO
Peptide-NH2
R
N
"Backbone" Cyclic Peptide
Alkylation:(ex. ICH2CN)
NH
O
S N
O
OO
NH-Peptide
R
N
RCO2HPyBOP/DIPEA Peptide synthesis
NH
O
SHN
O
OO
NH-Peptide
R
This has motivated the development of a “saftycatch” resin:
NH
O
S NH2
O
O
What To Do With Modified Peptides and Proteins?
Biophysical Analysis (in vitro):Structure (NMR, X-ray crystallography, CD)Stability Dynamics
Biological Analysis (in vitro or in cell lysates):Enzymatic activityIdentification of binding partnersInhibition of binding interactionsSignal transduction
How do each of these properties change upon site-specific modification (glycosylation, phosphorylation, etc.)?
Biological Analysis (in living cells):Enzymatic activity?Identification of binding partnersInhibition of binding interactionsSignal transductionDrug-like properties
Requires cellular uptake of modified peptides and proteins.
Alternatively, these studies can be enabled by site-specific modification of proteins produced by the cell.Biological Analysis (in animals):
Drug-like propertiesImmunological response
O OHO
CO2H
NH
O
HN
OI
SHR(pH = 7-8)
O OHO
CO2H
NH
O
O
ON
O
O
NH2R
(pH = 8-10)
succinimidyl esteriodoacetamide
Labeling of Proteins with Fluorescent Probes In VitroHere are some very common methods involving residue-selective electrophiles:
O OO
CO2
NH
O
HN
OSR
_
_
O OO
CO2
NH
OO
HNR
_
_
Angew Chem Int Ed Engl. 2009, 48, 6974-98.
Methods for Labeling Proteins in VivoMethod 1
Genetic engineering to introduce new sequence into a “fusion protein” which can be detected directly:
Genetic fusion (like GFP) “Any” protein
Method 2
Introduction of modified metabolite that is incorporated into protein:
Method 3
Combination of Methods 1 and 2 where fusion proteins are subsequently modified:
Method 4
Use of modified enzyme substrates for covalent enzyme modification followed by ligation with reporter molecule.
“Any” protein*
*“Any” protein “Any” protein*
*
4-Hydroxybenzylidene-imidazolidone
Q = 0.10 – 0.76ε = 32,000 – 138,000 cm-1M-1
τ = ~3 ns
Disadvantages
Requires transfection of DNA
Large size (238 amino acids)
Slow fluorophore maturation
Multimerization Nature Biotechnology 1999, 17, 969.
In vivo imaging
Variable Fluorescence Properties
Methods for Labeling Proteins in Living Cells: Method 1
Methods for Labeling Proteins in Living Cells: Method 2
In addition to Schultz’s approach (review “Expanded Genetic Code”), a number of other examples have been reported:
Introduction of modified metabolite that is incorporated into protein:
“Any” protein**
H2NOH
O
Azidohomoalanine
N3
Media depleted of Met
“All” proteins
N3 N3N3
Azido groups (R-N3) and terminal alkynes are examples of “bioorthogonal functional” groups.
“All” proteins
R – C CH / Cu(I)
N
N
N
R
N
N
N
R
N
N
N
R
Bacteria
+
Methods for Labeling Proteins in Living Cells: Method 2
Introduction of modified metabolite that is incorporated site-specifically into a protein using the “expanded genetic code” approach, followed by a “bioorthogonal chemical reaction”:
“Any” protein** expanded genetic code
Biomolecule N N N
Biomolecule C CH
Biomolecule N N N
Biomolecule
R-N3 Biomolecule NNN
R
Cu(I) Biomolecule NR
RNN
R
Biomolecule NNN
R
O
ON N
NNR
-N2Biomolecule
O
ON
NH
RR
R
Biomolecule IPd(0)
R BOH
OH
Biomolecule IPd(0)
RBiomolecule
Biomolecule R
R
Cu(I)
Biomolecule N N N
P
R O
OPh
PhP
RO
NH
PhPh
O
Biomolecule
bioorthogonal reaction“Any” protein
*R
Metal-Catalyzed Reactions Metal-Free Reactions
Other Examples of Bio-orthogonal Functional Groups
ketone
hydrazide
expanded geneticcode
ketonealkoxyamine
expanded geneticcode
Methods for Labeling Proteins in Living Cells: Method 2
An example using modified saccharides is from Bertozzi’s group:
Media lacking ManNAc
Human cells
O
NHAcO
AcO
OAc
OAc
O
N3
The azide-modified ManNAc was converted into azide-modified sialic acid units expressed on cell surface proteins
O
CO2-
O
HO
HNO
N3
HO
OHHO
O
NH
PPh 2 O
O
BIOTIN
O
NH
PPh 2 O
BIOTINO
CO2-
O
HO
HNO
NH
HO
OHHO
O
CELLSURFACE
Capture with S.A.agarose beads
Methods for Labeling Proteins in Living Cells: Method 3
Combination of Methods 1 and 2 where fusion proteins are subsequently modified:
*“Any” protein*
-CCPGCC-
B. A. Griffin, S. R. Adams, R. Y. Tsien, Science. 1998, 28, 269-272.
+ +
O OOAs
SSAs
SS
CO2
-
-
FlAsH
HSSH
HSSHCys4 protein
The second example utilizes thiol-arsenic exchange chemistry:
O
S Ph
“Any” proteinO
HN
SH
“Any” protein
The first example utilizes native chemical ligation:
Protease siteExpressProtease
CysMet
Methods for Labeling Proteins in Living Cells: Method 3
++
FLNCCPGCCMEPN
O OOAs
SSAs
SS
-
ReAsH
HSSH
HSSHOptimized Cys4 motif
The second example utilizes thiol-arsenic exchange chemistry:
HeLa cells expressing tetracysteine-fused connexinwere treated with FlAsH (green), incubated in medium for 4 hours, then treated with ReAsH (red) and imaged. This two-color pulse-chase labeling experiment demonstrated that newly synthesized connexin is incorporated at the outer edges of existing gap junctions (indicated by white arrows).
Methods for Labeling Proteins in Living Cells: Method 4
The last example utilizes biotin, a 15 residue biotinylation sequence and E. Coli biotin ligase (BirA):
“Any” proteinATPBirAGLNDIFEAQKIEWH
OH
OS
NHHN
O
Biotin
+
“Any” protein GLNDIFEAQKIEWH
NH
O
S
NHHN
O
BirA
+
“Any” protein GLNDIFEAQKIEWH
Labeled streptavidin
Keq = ~1014
NH2
ATPBirA
O
OS
NHHN
O N N
NN
NH2
O
HO
OP
OO
OH
-
Methods for Labeling Proteins in Living Cells: Method 4
“Any” protein GLNDIFEAQKIEWH
“Any” protein GLNDIFEAQKIEWH
NH
O
S
NHHN
O
NH2
Angew ChemInt Ed Engl. 2009, 48, 6974-98.
E. Coli biotin ligase (BirA):
Other examples: