Chemical Synthesis of Peptides and Proteins: Solid...

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:


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.:


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.:


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


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

+


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


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


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


++

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).


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

+


Labeled streptavidin

Keq = ~1014

NH2

ATPBirA

O

OS

NHHN

O N N

NN

NH2

O

HO

OP

OO

OH

-




NH

O

S

NHHN

O

NH2

Angew ChemInt Ed Engl. 2009, 48, 6974-98.

E. Coli biotin ligase (BirA):

Other examples:

Chemical Synthesis of Peptides and Proteins: Solid...

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