SBM

What does oligo refer to?

General prefix for more than 2 monomers

Peptide bond formation

Condensation reaction

Peptide sequences written from N (-NH2) to C (-COOH) terminus

amino acids can be L or D depending on the stereochemistry at the α-carbon

Most stable conformation of hexose sugars?

Ring in chair conformation. Equatorial favoured.

In solution, free monosaccharides exist as an equilibrium of two dif anomers

Determined by hydroxyl group at the C1 (anomeric) position

Occurs through mutarotation where the ring converts to an acyclic aldehyde intermediate and back

Mutarotation requires a _______ at the anomeric position. The anomeric stereochemistry (α or β) is fixed once a ____________ is formed, i.e. there is a substituent other than __ at the anomeric position:

free OH, glycosidic bond, OH

For efficient & selective coupling between AAs for peptide synthesis, we need to:

1) increase reactivity by activating the -COOH by turning the carboxylic -OH into a better leaving group

2) encourage regio-/chemoselectivity by protecting groups

3) avoid loss of stereochemistry / racemisation

What groups need protecting (on amino acids)?

N terminus on one

C terminus on the other

Functional groups on the side chains (R groups) - especially nucleophiles (OH/SH), acids (carboxylates) & bases (NH groups)

Activation of the C-terminal carboxylic acid as an acid chloride

Causes racemisation of the activated amino acid so don’t use acyl chlorides

How does racemisation happen?

Oxazolone formation leads to keto-enol tautomerisation, which causes the chiral center at the α-carbon (adjacent to the carbonyl group) to racemise

Can minimise racemisation by using a coupling reagent to activate the C terminus

• generates a less reactive leaving group (but still better than OH)

• allows one-pot reaction without isolation of the activated species

Peptide synthesis is preferred in the ______ terminal direction:

C to N

Coupling reagents: DCC

Coupling reagents: HATU + base (DiPEA)

• Base lp to H to O on AA’s -OH (carboxylate)

• O- to C=N

• Cleave AA off by forming a pyridine

• O- from benzobriazole goes to C on C=O on AA forming activated intermediate which has a good leaving group

• AA + AA makes peptide

Protecting group criteria

1. Stability towards the conditions used in subsequent steps (all assessed are)

2. Orthogonality: being able to remove protecting groups separately, i.e. without affecting other groups that are present.

3. Selectivity for installation of protecting groups at specific positions.

Protection of the amino acid N-terminus (carbamates): General

Installation of the protecting groups: acylation with an acyl chloride or dicarbonate under basic conditions

Deprotection conditions: variable, dependent on the structure

Protection of the amino acid N-terminus (carbamates): Boc

Protection of the amino acid N-terminus (carbamates): Fmoc

Protection of the amino acid C-terminus (esters): General

Installation of the protecting groups: generally by acid-catalysed esterification w/ an alcohol

Deprotection conditions: variable, dependent on structure

All esters are sensitive to hydrolysis with aqueous base

Protection of the amino acid C-terminus (esters): methyl ester

Protection of the amino acid C-terminus (esters): tert-butyl ester

Protection of amino acid side chains: R = carboxylic acid

Same protective groups as for C- terminus: methyl ester, tert-butyl ester

Protection of amino acid side chains: R = amines

Same protective groups as for C- terminus: Boc & Fmoc

Protection of amino acid side chains: R = hydroxyl groups - general

Installation of the protecting groups: by nucleophilic substitution of an alkyl/silyl halide under basic conditions

Deprotection conditions: depend on structure

Alkyl ethers are more stable than analogous esters & not sensitive to hydrolysis. Ethers can be removed using specific conditions.

Protection of amino acid side chains: R = hydroxyl groups - tert-butyl ether

Protection of amino acid side chains: R = hydroxyl groups - benzyl ether

Why is PMB more sensitive to acidic hydrolysis than the benzyl ether?

+M effect of OMe

Protection of amino acid side chains: R = hydroxyl groups - silyl ethers

Other silyl ethers carry different substituents that change their relative stability:

Solid phase peptide synthesis (SPPS):

Construction of peptides on insoluble functionalised polymer beads (‘resin’)

• Resin immobilises peptide

• Reagents are in solution & can be washed away after reaction (no work-up required)

• Product can be cleaved off after synthesis is complete

What is a resin?

• A crosslinked polystyrene

• C-terminus of the first amino acid is used as the site of attachment to the solid support

• Need a functionalised resin that can be covalently linked to the carboxylate of an amino acid

Merrifield Resin

Wang resin and Sasrin resin:

OH functionalised

Rink amide resin:

NH2 functionalised

Peptides are commonly released from the resin under _____ conditions.

acidic

Cleavage from Merrifield resin requires __

HF

Wang & Sasrin resins can be cleaved using ___ instead of HF

TFA

Sarsin requires less TFA conc as it is more acid labile due to having more +M effects

Will the N-terminal protecting group be removed concurrently with peptide release from the resin?

• if PG = Fmoc No

• if PG = Boc Yes

In SPPS, the N-terminus of the amino acids is protected with Fmoc, not Boc. Why?

Cleavage of Boc would cause release of the peptide

In SPPS, we use DIC as a coupling reagent instead of DCC. Why?

DCC would produce an insoluble urea byproduct, which precipitates with the solid support.

• DIC follows the same activating mechanism as DCC

• The urea byproduct that forms is soluble:

In SPPS, protecting groups for side chains are chosen so that deprotection and release of the peptide (from the resin) simultaneously

So they have to be labile under acidic conditions

Challenges in glycosidic bond formation: Reactivity

OH- is a poor leaving group

Need to increase donor reactivity by activation of the anomeric hydroxyl group

Challenges in glycosidic bond formation: Regioselectivity

Need to react at the correct hydroxyl groups so need to install appropriate protecting groups

Challenges in glycosidic bond formation: Stereoselectivity

Need to control formation of the α or β anomer

β-anomer favoured if PG₁ =

ester (e.g. acetyl group)

α-anomer favoured if PG₁

= ether (e.g. benzyl ether)

Protection of (non-anomeric) hydroxyl group

Similar to peptide synth

Selective protection of the primary hydroxyl group

Triphenyl methyl ether (Trityl)

• Very sterically demanding → selectively added onto the least hindered OH

Non-selective protecting groups: Acetyl esters

Non-selective protecting groups: Benzyl ethers

Selective protection & deprotection of the anomeric hydroxyl group:

Anomeric hydroxyl group is a _________ with unique reactivity so can use for selective protection/deprotection

hemiacetal

Fisher glycosylation:

Deprotection of anomeric acetyl ester

Selective protection of 1,3-diols: Benzylidene acetal

Prefers 1,3 over 1,2-diols → adds onto the hydroxyl groups at C4 & C6

Can selectively deprotect C4 or C6 & leaves the other hydroxyl group protected with a benzyl ether

• Selective cleavage of the benzylidene acetal from only one of the two hydroxyl groups is possible by using specific reducing agents:

C4 - OH, C6 - OBn requires _________ + AlCl₃

C4 - OBn, C6 - OH requires _______ + AlCl₃

NaCNBH₃, LiAlH₄

Activation of the anomeric hydroxyl group

• Install a good leaving group (‘X’) at the anomeric position of the glycosylation donor to enable reaction with the acceptor molecule.

• Activation gives a oxocarbenium ion (reactive carbocation intermediate)

The oxocarbenium ion can be attacked by an acceptor from __________ of the sugar ring, generating ____ the α & β anomers via an ___-type mechanism

either face, both, S_N1

Leaving groups for glycosylation? Glycosyl bromide

Donor can be activated with silver salts to enable reaction with an acceptor molecule (ROH)

(Keep in mind that equivalent reactions with non-participating donors like Bn will involve direct attack of the acceptor at the oxocarbenium ion)

Leaving groups for glycosylation? Thioethers

Thioethers can be made into good leaving groups by:

1. Iodination with NIS + acid

2. Methylation with MeOTf or TMSOTf

3. Oxidation followed by Tf₂O

Each can eliminate the activated leaving group to form an oxocarbenium ion which then reacts w/ the acceptor

Leaving groups for glycosylation? trichloroacetimidate

Installed using trichloroacetonitrile (CCl3CN) + NaH

Activated by a Lewis acid such as TMSOTf

Factors driving stereochemical outcome of glycosylation to consider

1. Is there neighbouring group participation? (favours 1,2-trans products)

2. Is there intramolecular aglycon delivery? (favours 1,2-cis products)

3. If not, assume the anomeric effect drives formation of the α-anomer

Stereochemical outcome of glycosylation: anomeric

• α is most stable so major product

Explanation for anomeric effect

1. Dipole minimisation: In β, dipoles are partially aligned causing repulsion

2. Hyperconjugation: Orbital overlap between axial lp on the endocyclic oxygen & the σ* of the axial (α) C-O bond leads to a stabilising interaction

Stereochemical outcome of glycosylation: neighbouring group participation

Activation of donors w/ an ester protecting group at C2 causes formation of a bicyclic oxonium ion intermediate, sterically hindering attack by the acceptor (ROH) from 1 side, leading to exclusive formation of the 1,2-trans isomer

Stereochemical outcome of glycosylation: intramolecular aglycon delivery

• Used to make 1,2-cis glycosidic bonds

• A temporary linker between donor & acceptor molecules is positioned so that it restricts attack by the acceptor to the correct face of the donor ring