HATU Mechanism

Ck. 84 – Bioorganic Chemistry

Lecture Notes on Peptide Synthesis Oligosaccharides: Roughly 10 natural building blocks; multiple stable stereogenic centres; branching; α/β problem in synthesis. Oligopeptides: 20 natural building blocks (amino acids); a single stereogenic centre per unit (except Thr/Ile) that can be prone to scrambling during synthesis; no branching. Proteinogenic amino acids are all α-amino acids, which refers to there being only one carbon (the α-carbon) between the amine and the carbonyl function. They are all L-amino acids, which in Cahn-Ingold-Prelog nomenclature corresponds to S with the

exception of cystein (Cys, C). Cysteine is an L-amino acid where the α-carbon has the R-configuration.

Amino acids/peptides are written according to convention written with its N-terminus to the left and the C-terminus to the right. Three letter codes or even shorter one letter codes are normally used for short representation.

Example: OH O

H N

H2N

N H

O O

OH O

OH

HValGluTyrOH = VEY

The peptide bonds are luckily stable to peptide synthesis conditions and scrambling (epimerisation/racemisation) is not a problem.

If scrambling occurs it is usually takes place in the state of an activated ester:

R'

R' R''NH2

OLG

RHN

NHR''

RHN O

O

scrambled amino acid fragment

active ester

Amides are known to be able to swing around and to form intermediate oxazolones that easily scramble.

R

R O R

N H

OLG O

HN

O

N

base

R N

O O

O protonated oxazolone

O OH

R'NH2

O R

N H

NHR' O

Active ester leaving groups that are too labile can furthermore be expected to react through a ketene that will again cause scrambling to occur.

OLG

RHN O

active ester

R''NH2 RHN O

OR O RO

N H

HN

OLG O

O O

carbamate

Building of a peptide from the N-terminus: R'

O R

N H

H2N

OLG O

O CO2R''

R

possible scrambling

N H

H N O

CO2R'' R'

growing peptide

Building of a peptide from the C-terminus: R' NHR CarbamateHN

H2N

R'

CO2LG

O

H N

CarbamateHN

growing peptide

O NHR

O

Formation of the active ester: Carbodiimides: The classical reagent is DCC (dicyclohexyl carbodiimide), a solid: Urea by-product (DCU) is highly insoluble in most solvents, which might sometimes be of benefit and sometimes not.

DIC (diisopropyl carbodiimide), a liquid: Urea by-product (DIU) somewhat more soluble than DCU.

EDC (ethyl-dimethylaminopropyl carbodiimide): Urea by-product easily removed as its ammonium ion by implementing an acid wash in the work-up procedure.

N C N

N C N

N C N N

DIC

DCC

EDC

The carbodiimide activated ester is very reactive. In the case of slowly reacting nucleophiles, rearrangement is a common side reaction.

O R

OH

O

R N C N

N

R

O

O

rearrangement R

R

O

NH R

R

N N H R acyl urea

R

This can be avoided by using (usually in stoichiometric amounts) the nucleophilic additive 1-hydroxybenzotriazole (HOBt). This reagent reacts quickly with the active ester and forms a less reactive species that is still reactive enough to react with the intended nucleophile without formation of the stable rearranged acyl urea.

O

N

R

R

O

O

N

+ NH R

O +

N

R

N OH HOBt

N N N O

R

major

O

N N N

+

O R

N H

N H

R

minor

The acyl-HOBt adduct have been found in two isomeric forms one being the N-acyl and the other the O-acyl compounds. It is believed that in polar aprotic media as DMF the N-acyl compound is dominating (J. Chem. Soc. Perkin Trans. 2, 2001, 113-120).

Both compounds are able to react with amines to form amides. O R'

N H

O R

R

O N N N O

R'NH2

R

O

N N N

O R'

N H

R

Modern peptide coupling reagents: There exists a large number of peptide coupling reagents. Two of the most popular are the analogous compounds HATU and HBTU. They are carbodiimides and HOBt in one in the sense that a urea and HOBt (or in the case of HATU a nitrogen analogue of HOBt) are their by-products. Their structures were initially believed to be, as their names seem to suggest, uronium reagents. Later crystallographic analysis, however, have shown this not to be true (J. Chem. Soc. Perkin Trans. 2, 2001, 113-120).

Mechanism:

N

O N

O

R

N X

N N O

+

R

O

N O

N

+

N N N

N N N OH

X X = CH, HBTU X = N, HATU

N H

O

X

R'NH2

O R'

O R

R

Solid phase peptide synthesis: Peptide synthesis can successfully be carried out in solution (solution phase synthesis) but is typically more laborious than using solid phase peptide synthesis (SPPS). The benefits of SPPS are plentiful. Among them are i) ease of purification between steps (a simple filtration and wash will do); ii) possibility of full automatisation; and iii) no problems associated with using excess reagents and conducting multiple couplings to force particular reluctant reactions to completion.

Merrifield Strategy (Bruce Merrifield: Nobel Prize 1984) Uses polystyrene solid support. Cl

Cl

polymerisation reaction +

Ph

Ph

Ph

Ph

= P

Cl styrene

polymer with a few functionalised residues

p-chloromethyl styrene

(many)

(few)

Attachment to solid support via caesium salt followed by a series of Boc-removal, neutralisation, and coupling reactions results in an oligopeptide attached to the Merrifield resin. R OH

BocHN P Cl

R

O

P'

= Cl

1/2 Cs2CO3

P' O

BocHN O

R OCs

BocHN

TFA/DCM

O

R

P' O

H3N O

Mechanism: R oligopeptide

P

R

H-F

oligopeptide

O

N H

O

P O

N H

O

H

F R H2N

oligopeptide

N H

R

OH

oligopeptide

O

+

N H

OH O

+

P

P

F

Side chain protection - A few examples:

O N H

(H/CH3)

HO H N H

O

HO

(H/CH3)

N H

O

(H/CH3)

O

Ser/Thr benzyl ether protection

H O O

O 1,2

N H

O

Asp/Glu benzyl ester protection

H

O

O 1,2

N H

O

HO

1,2 N H

O

Fmoc Strategy on Rink Solid Support Currently this approach is probably the most widespread for peptide synthesis.

P

P

FmocHN

Piperidine/DMF

MeO

H2N =

MeO

OMe

H2N

P'

O

OMe O

OH HBTU

FmocHN O O

O

O

H N

H2N

1) FmocNHCHRCO2H HBTU 2) Piperidine/DMF

O P

n O

H2N

O oligopeptide

N H

H N O

MeO OMe

Piperidine/DMF P'

O

H N

FmocHN O

P'

Mechanism for cleavage (release) from resin: The Rink linker works like a trityl group. Treatment with TFA gives a stabilised carbocation. P

P

H N

R

H N

R O

O

MeO H O

O

H

CF3

MeO

OMe

OMe

P

P O O

O R

NH2

+

F3C

F3C

O

O

O

R

NH2

+ MeO

MeO

OMe

OMe

Side-chain protection for Fmoc-strategy: Protecting groups chosen so they cleave during resin cleavage. A few examples: Cysteine protected as its tritylthioether. Serine/Threonine protected as their tbutylethers. Glutamic acid/aspartic acid: protected as their tbutylesters. All cleaved in the resin cleavage step. Mechanism for tbutylether deprotection

O

O

(H/CH3) H O

N H

O

Ser/Thr

CF3

H

O N H

(H/CH3)

O

HO

(H/CH3)

N H

+ O

H +

H

Mechanism for tbutylester deprotection:

O O

1,2 N H

O

O

O

Asp/Glu

H O

CF3

O

H

O 1,2

N H

O

H

HO

1,2 N H

O

+

+

H

Total Synthesis of Proteins: Fragments consisting of more than 40-50 amino acid residues are difficult to synthesise using SPPS. For larger peptides/proteins a procedure referred to as Native Chemical Ligation (NCL) is used. The key to NCL is formation of two peptide fragments using e.g. SPPS. One fragment must have an N-terminal cysteine residue, while the other fragment must possess a C-terminal thioester functionality.

Boc-approach: As NCL use thioesters and since these are unstable to base (piperidine used in Fmoc SPPS) but not acid, a Boc-approach have been proven successful in the preparation of these needed fragments. The thiocarboxylic acid released from the solid support can then be alkylated (in aqueous buffer) with BnBr to form a thioester. P

R OH

BocHN =

HS

HS

R

O P'

HBTU

S

BocHN

P'

O

1) TFA/DCM 2) neutralisation 3) BocNHCHR'CO2H n

oligopeptide

SH

anhydrous HF

O

BnBr, aqueous buffer

S O

thioester

S O

released thioacid in solution

oligopeptide

oligopeptide

P'

For the second peptide fragment (up to 40-50 amino acid residues in length) the requirement is that the N-terminal residue is cystein. This fragment can be prepared using a Boc- or an Fmoc-approach. The first step in NCL is trans-thioesterification i.e. a dynamic exchange of thioesters. S-N-cyclisation gives the thermodynamically stable native peptide. Benyl thioesters are not fantastically reactive thioesters in this context so thiophenol or like thiols are usually used as catalysts. HS oligopeptide

SR O

H N

+ H N 2

S

oligopeptide

H N

O H2N

oligopeptide

O

oligopeptide

O

HS

native linkage

H N

NH

oligopeptide O

oligopeptide

O

For the ligation step it is important to use a reagent like guanidinium chloride in high concentration (6 M). Guanidinium chloride is a chaotropic agent that denatures (unfolds) the large peptide whereby the reacting groups are made accessible.

Peptides for NCL using an Fmoc-appraoch: Since thioesters are unstable during normal Fmoc-deprotectin (20% piperidine in DMF) it is essential in this approach to introduce the thioester functionality in the last step. R O

O O S H2N

N H

P

O O S = P' H2N

OH

FmocHN

O O S P' NH

R O FmocHN

HBTU

O

sulphonamide 1) Piperidine/DMF 2) FmocNHCHRCO2H HBTU n

1) Piperidine/DMF 2) BocNHCHRCO2H/HBTU

O FmocHN

R

O

BocHN

HN

oligopeptide

O O S P' NH

R

O N H

O

oligopeptide

BocHN =

O O S P' NH

R N H

O

O O S P' NH

oligopeptide O

safety catch "on"

The last peptide is coupled on as a Boc-amino acid. Treatment with iodoacetonitrile in this way will only alkylate the sulphonamide function and not a free nucleophilic amine. After alkylation with iodoacetonitrile the acyl group of the sulphonamide is prone to nucleophilic attack by a thiol, which will release the desired thioester bearing acid labile side-chain- and N-terminus protection. These can now be removed by TFA treatment under which the thioester remains stable.

BocHN

O O S P' NH

oligopeptide

I

CN base

BocHN

oligopeptide

O safety catch "off"

most acidic proton

O O S P' N O NC

SH

H2N

SBn

oligopeptide

TFA

BocHN

SBn

oligopeptide

O

O

fully deprotected, ready for NCL

The sulphonamide is said to be a safety-catch linker because the safety-catch is “on” because it is unreactive to nucleophiles when in its NH-state and “off” and reactive to nucleophiles when in its NCH2CN-state. Native chemical ligation has proven to be an immensely powerful tool for the synthesis of large polypeptides and even proteins. It is possible to ligate glycopeptide fragments to other glycopeptide fragments via the safe-catch strategy even though it ultimately involves acid treatment. This is due to the limited and not repeated use of a relatively mild acid (TFA) under which the glycosidic linkage it relatively stable.

Henrik Helligsø Jensen, Ck. 84, Bioorganic Chemistry, February 2009.