Solid phase peptide synthesis Part II Application of Fmoc/ t Bu strategy Gábor Mező Research Group of Peptide Chemistry Hungarian Academy of Sciences Eötvös

Solid phase peptide synthesis

Part II

Application of Fmoc/tBu strategy

Gábor Mező

Research Group of Peptide ChemistryHungarian Academy of Sciences

Eötvös L. University Budapest, Hungary

Outline

Resins;

Protecting groups;

Synthetic protocol;

Monitoring;

Cleavage technics;

Side reactions;

Fmoc/tBu:

NH

C

CH2 O C NH CH

CH2

C

O

O

C

O

H

C

CH3

CH3CH3

NH CH C

O

O

CH2

CH2 O CH2

O

O

C

CH3

CH3CH3

C

R

Fmoc

tert-butyl

..

piperidine

TFA

Wang-resin

Fmoc-Asp(OtBu)-Tyr(tBu)-Wang resin

Type of resins for Fmoc-chemistry

There are many different resins and most of them are used for special cases and in individual laboratories. Here only the most widely applied resins will be presented. Resins are based on PS-DVB (1%) copolymer.4-Alkoxybenzyl alcohol (Wang) resin:

CH2O PCH2HO

Attachment of the first amino acid:

Fmoc-Aaa(X)-OH:DIC:DMAP (2:2:0.2 equiv to the resin OH content)in DMF, 1h at RT.

The final cleavage results in peptides with COOH group at the C-terminus

The resin is not available for the synthesis of peptides with a sequence on the C-terminal that is sensitive for diketopiperazine formation !

SASRIN (Super Acid Sensitive ResIN)

(2-methoxy-4-alkoxybenzyl-alcohol resin)

CH2O PCH2HO

OCH3

Peptide is cleavable with0.5-1.0% TFA in DCMresulted in protected peptide fragments.

CH2OCH2HO C OOH

4-Hydroxymethylphenoxyacetic acid (HMPA) linker:

Attach to aminomethyl PS-DVB resin

(CH2)3OCH2HO C OOH

OCH3

4-(4-Hydroxymethyl-3-methoxyphenoxy)butyric acid (HMPB) linker:

Removal of the peptide with TFA

Removal of the peptide with diluted TFA

Attach to aminomethyl PS-DVB resin

2-Chlorotrityl chloride (ClTrt) resin:

ClCl

P

Attachment of the first amino acid:

The final cleavage results in peptides with COOH group at the C-terminus

Cleavage with 90-95% TFA + scavangers results in free peptides

Cleavage with AcOH:MeOH(TFE):DCM (1:1:8 or 2:2:6) results in protectedpeptides (available for fragment condensation).

ClTrt resin prevents the diketopiperazine formation !Attachment of Cys and His derivatives to the resin is free from

enantiomerisation!

1 g ClTrt-resin + 2 mmol Fmoc-Aaa(X)-OH+ 8 mmol DIEA in 3-5 mL DCM, for 1.5 h then 0.8 mL MeOH to block the unreacted groupswashing with DCM, iPrOH, MeOH, ether

(Calculation of the resin capacity)

Determination of loading

10-20 mg of dried resin are weighted exactly into a 100 mL measuring flask (for a load of ca. 0.5 meq/g 20 mg is sufficient);

Piperidine/DMF (1:4, V/V) is added to the mark;

The mixture is shaken thoroughly and left for 25-30 min;

The resin is filtered off and the absorbance of the filtrate is measured at 301 nm ( = 7800).

NH2(mol/g) = [A301.V(ml)/301.m(mg)].106

1.

2.ca. 4-6 mg Fmoc-Aaa-resin ca. 2 mg Fmoc-Gly-OH+400 L 50% piperidine/DMF +400 L 50% piperidine/DMF30 min at RT, then filtration 30 min at RTdilute with MeOH to 25 mL dilute with MeOH to 25 mL

Capacity of the resin (mmol/g) = 1000.mgly

.Aresin301

Mgly.mresin

.Agly301

Mgly =297

Rink Amide Resin: synthesis of peptides with CONH2 C-terminus

CHNH2 P

CH3

CHNFmoc-H OCH2-P

OCH3

COH3

Cleavage with high concentration ofTFA can lead to the break down of the linker byproducts.Use low TFA concentration and/ortrialkylsilanes in the cleavage mixture.Peptide-resin bond can be detached with 5% TFA. Removal of protecting groups needs a separate step.

CHNFmoc-H OCH2-CO-Nle-R

OCH3

COH3

Rink Amide-AM and Rink Amide-MBHA resins:

CHNH2 P2

Aminomethyl-PS-DVB

4-methylbenzhydrylamine-PS-DVB

Peptide cleavage with 90-95% TFA solution.Nle is a reference for quantitation.

Pegylated resins: composition of polyethylene glycol (Mw:3000-4000)and low-cross linker polystyrene gel-type resins.

Advantages: excellent pressure stability (continuous flow synthesis)excellent swelling properties (also in water)high diffusion ratesavailable with many types of functional groupslow capacity (0.2-0.6 mmol/g), suitable for the synthesisof aggregating peptides, for on resin cyclisation andfragment condansation.

The basic polymer support is aminomethyl PEG-PS-DVB (NovaSyn R TG)

PEGNH2

CH2OCH2HO C

O

OH

4-hydroxymethylphenoxyacetic acid linker

NovaSyn R TGA resin

Similar to Wang resin

HO C

O

OH

4-carboxytrityl linkerNovaSyn R TGT alcohol resin

Before use the resin must be converted to the chloride form by heating with

AcCl or SOCl2 in toluene.Similar to ClTrt resin.

CHNH2 OCH2

OCH3

COH3

2,4-dimethoxy-benzhydryl linkerNovaSyn R TGR resin

Similar to Rink Amide MBHA resin

C

O

OH

Applied side chain protecting groups in Fmoc-chemistry

-OH (Ser, Thr, Tyr)

Side chain functional group protecting group name (abbreviation)

CH3 C

CH3

CH3

tert-butyl (tBu)

Trt group can be used if on-resin derivatization is required (glycosylation, phosphorylation). Trt can be cleaved with diluted TFA, while tBu needs 90% TFA solution for effective removal.

-SH (Cys) trityl (Trt)

CH2 NH C

O

CH3 acetamidomethyl(Acm)

For selective deprotection

Racemisation during the attachment of Cys derivatives to the resins in the presence of DMAP: Fmoc-Cys(Trt)-OH > Fmoc-Cys(Acm)-OH

However, Fmoc-Cys(Acm) at the C-terminal resultes in side reaction:

CH2

O PO CH2CCHNH

CH2Acm-S

CH2

O PO CH2

O

CCNH

CH2O

piperidine

piperidine

CCHNH

CH2ON

H2O

CCHNH

CH2OHO

Cys Mcalc

Ala Mcalc – 34

DL-Ala(Pip) Mcalc+ 41

DL-Ser Mcalc – 16


tert-butyloxycarbonyl(Boc)

NH2 (Lys)

O

O CC CH3

CH3

CH3

Selectively removable protecting groups for preparation of modified peptides (labeled, functionalised, branched or cyclic peptides):

NH2 (Lys)CH3

4-methytrityl(Mtt)

Mtt can be removed selectively with 1%TFA/DCM solution in the presence of 3-5% TES (triethylsilane) at RT in 15-30 min.Trt groups may be not stable enough under this condition.


NH2 (Lys)

O

CCH3

CH3

OR

R = metil1, isopropyl2

1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde)1

1-(4,4-dimethyl-2,6-dioxocyclohex-1-

ylidene)-3-methylbutyl(ivDde)2ivDde is more stable in basic cleavage mixture

applied for Fmoc removal than Dde.

Both protecting groups can be removed with 2% NH2-NH2 in DMF

NH2 (Lys)O

OC CH2CHCH2

allyloxycarbonyl(Aloc)

Aloc protecting group is compatible with Boc as well as Fmoc-chemistry. It is stable in acids and bases. It can be removed in P(Ph)3 by Pd(0)catalysis. To prevent addition on double bond under other cleavage conditions application of allyl alcohol in cleavage mixtures is recommended.

COOH (Asp, Glu)


O C CH3

CH3

CH3

tert-butyl ester (OtBu)

Selectively removable protecting groups for preparation of cyclic peptides:

(pairs of amino and carboxyl protecting groups: Dde-ODmab, Aloc-OAll)

CCH3

CH3

O

O

CH3

CH3 CH

NHO CH2

4-{N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]-amino}

Benzyl ester(ODmab)

Similarly to Dde and ivDde, the Dmab protecting group can be removed with 2% hydrazine in DMF.

O CH2CHCH2 allyl ester (OAll)

It can be removed in P(Ph)3 by Pd(0) catalysis.

Succinimide ring formation (Asp):

Acid catalised reaction results in or -Asp-peptides, however, piperidinecatalised side reaction under Fmoc cleavage procedure results in piperidide:

-Asp-Gly-

NH CH C

O

NH CH2 C

O

CH2

COtBu

O

-Asu-Gly-

NH CH N

CH2

C

C

O

CH2

O

C

O

NH- tBuOH

NH CH C

O

NH CH2 C

O

CH2

C

O

NH CH

CH2

C

C

O

O

NH

N CH2 C

O

piperidine

piperidine

N

N

M = Mcalc+ 57

Application of other cleavage reagents (DBU, TBAF, DEA, morpholine)eliminate the piperidide formation, but not the succinimide formation.Addition of HOBt to the cleavage mixture can reduce the succinimidering closure. But the best results may get with the use of Fmoc-(Hmb)-amino acid derivatives:

Hmb: 2-hydroxy-4-methoxybenzyl (removable with TFA)

NH CH C

O

N CH2 C

O

CH2

C OtBu

O

CH2

CH3OHO (Hmb)amino acid derivatives are

secundary amines: Removal of Fmoc group and the attachement of the next Aspderivative is difficult, needs longer time. Ninhydrin test can’t detect the efficacy of the coupling.

Fmoc-(Fmoc-Hmb)Gly-OH1g = 370 EUR (NovaBiochem)

The increasing of the solubility of protected peptide fragments as well as preventing of aggregation of ”difficult” sequences can be reach by the application of Hmb groups.


CONH2 (Asn, Gln) trityl (Trt)

The solubility of Fmoc-Asn-OH and Fmoc-Gln-OH is extremely bad. The Trt protecting group increases the solubility and prevents thedehydratation as well as ring closure side reactions during the synthesis. N-terminal Gln or Asn-Gly (Arg, Ser, Ala, Asn) sequence may causeproblems after the cleavage of the protecting group.

N NH

(His)

imidazol group

tert-butyloxymethyl(Bum) ()CH2 O C CH3

CH3

CH3

The same problem as in case of Bom in Boc startegy. Don’t use it for thesynthesis of peptides containing Cys at the N-terminal !


N NH

(His)

imidazol group

trityl (Trt) ()

Trt group protects the N. However, its application prevents both epimerisaton (not in case of attachment to resins) and alkylation.

-NH-C-NH2

NHguanidino group

CH3S

O

O

CH3

CH3CH3

O4-methoxy-2,3,6-trimethylbenzene-sulfonyl (Mtr)

(Arg)

Mtr is too stable in TFA. Elevated temperature (30oC) and/or increasedtime (4-6 hrs) is necessary for effective cleavage. 1M TMSOBr-thioanisol/TFA mixture is an alternative cleavage mixturethat can remove Mtr more effectively.


-NH-C-NH2

NHguanidino group

(Arg)

2,2,5,7,8-pentamethyl-chroman-5-sulfonyl

(Pmc)

2,2,4,6,7-pentamethyl-dihydrobenzofurane-

6-sulfonyl (Pbf)

S

O

O

CH3

CH3CH3

OCH3

CH3

S

O

O

CH3

CH3CH3

OCH3

CH3

Pmc can be cleaved with TFA in 2-3 hrs, but Pbf protecting group can be removed 1.5-2 times faster than Pmc. Pbf also gives rise to less

sulfonated Trp byproduct than Pmc or Mtr.Use Fmoc-Arg(Pbf)-OH for the synthesis of oligo-Arg as a

cell penetrating peptide !


indole

NH

(Trp) tert-butyloxycarbonyl(Boc)

O

O CC CH3

CH3

CH3

The protection of indole side chain of Trp is not necessary, but the application of Boc group is recommended. Under TFA cleavage theappearance of inN-carboxy indole protects Trp vs alkylation and sulfonation.inN-carboxy group is removed under aqueous condition in working upprocedure.

Protection of the side chain of Met is not needed in Fmoc-strategy.

Fmoc/Bzl (benzyl type protecting groups for blocking of side chains) strategy is applied for the synthesis of protected peptide fragments,because of the better solubility of benzyl protected fragments over tert-butyl and trityl protected fragments.

1) Wash the resin 3x with DMF; 0.5-1.0 min each2) Cleavage of Fmoc protection with 2% piperidine + 2%DBU/DMF; 2+2+5+10 min*3) Wash the resin 8x with DMF; 0.5-1.0 min each**4) Coupling: Fmoc-amino acid derivative-DIC-HOBt in DMF*** (3 equiv each calculated to the resin capacity); 60 min5) Wash the resin 2x with DMF; 0.5-1.0 min each6) Wash the resin 2x with DCM; 0.5-1.0 min each7) Ninhydrin monitoring

Synthetic protocol of Fmoc-strategy

(-) yellow

(+) blue

* DBU is the cleavage reagent, piperidine is for the capture of dibenzofulvene 20% or 50% piperidine in DMF, 50% morpholine or DEA in DMF and 20mM TBAF in DMF are also used as cleavage mixture.

** After 4 DMF washing, IPA washing may be applied for shrinking the resin. An unefficient removal of base from the resin may cause Fmoc cleavage in the next coupling step.

*** DIC is used instead of DCC in this method, because of the limited solubility of DCU in the applied solvents.

N,N’-diisopropylcarbodiimide (DIC, DIPCDI))

Coupling agents

N NC

N,N’-dicyclohexylcarbodiimide (DCC)

N NC CHHC

CH3

CH3H3C

H3C

N NHC

OC

OCHX-NH

RX: Boc, Fmoc

O-acyl-isourea derivatives

N NHC

O

CO

CHX-NH

R

N-acyl-urea derivatives

O-N acyl shift

NH NHC

Ourea derivatives: DCU, DIU

CO

CHX-NH

R

OBtHOBt

in situ active ester

+

N

N

N

OH1-hydroxybenzotriazole

(HOBt)

N

N

N

OH

N

1-hydroxy-7-aza-benzotriazole(HOAt)

N

N

N

O

P+

(CH3)2NN(CH3)2

N(CH3)2

PF6-

benzotriazol-1-yl-oxy-tris(dimethylamino)-phosphonium hexafluoro phosphate (BOP)

N

N

N

O

P+

PF6-

NNN

benzotriazol-1-yl-oxy-tris(pyrrolidino)phosphonium hexafluoro phosphate (PyBOP)

They don’t needDCC or DIC forpreparation of

in situ active ester

Hexamethylphosphoramide (carcinogen)!

>

AOP

PyAOP

2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate

HBTU

N

N

N

O

C+

(CH3)2NN(CH3)2

PF6-

N

N

N

O

C+

(CH3)2NN(CH3)2

BF4-

2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium tetrafluoroborate

TBTU

N

N

N

O

C+

N(CH3)2

PF6-

N(CH3)2

+

-

N-[(1H-benzotriazol-1-yl)(dimethylamino)-methylene]-N-methanaminiumhexafluorophosphate N-oxide

According to NMR and röntgendiffraction studies a new structure was suggested:

HATU, TATU, HBPyU, HAPyU, etc.

Guanylation with uronium type coupling reagents

N

N

N

O

C+

(CH3)2NN(CH3)2

PF6-

CO

CHHNH

R

NH-PEPTIDE+

CO

CHNH

R

NH-PEPTIDE

C(CH3)2N

N+(CH3)2

+ HOBt

PF6-

Don’t use excess of coupling agent (cyclisation, fragment condensation);

Make preactivation of the incoming amino acid;

Apply: X-Aaa-OH: HBTU: DIEA = 3:2.9:3 (equiv) to the resin capacity.

Fmoc cleavage flow chartDoes the peptide

contain N-terminal Fmoc group?yes

no

Remove Fmoc Does the peptide contain Arg, Met, Trp or Trt?

yes no

Does the peptide contain Arg, Met?

Use cleavage mixture A

yes no

Use cleavage mixture B Does the peptide contain Trp or Trt?

noyes

Use cleavage mixture C

A: 0.5 mL d.i. water 9.5 mL TFA

C: 0.25 mL EDT 0.25 mL d.i. Water 9.50 mL TFA

B: 0.75 g cryst. phenol 0.25 mL EDT 0.50 mL thioanisole 0.50 mL d.i. water 10 mL TFA

Boc/Bzl or Fmoc/tBu strategy

Amino acid derivatives and resins for Boc-strategy is still cheaper:

Boc-Ala-OH (Mw: 189) 5g 11 EUR, 1mmol 0.416 EUR

Fmoc-Ala-OH (Mw: 311) 5g 11 EUR, 1mmol 0.684 EUR

Boc-Arg(Tos)-OH (Mw: 429) 5g 32 EUR, 1mmol 2.746 EUR

Fmoc-Arg(Pbf)-OH (Mw: 649) 5g 90 EUR, 1mmol 11.682 EUR

MBHA resin (0.4-1.2 mmol/g) 5g 49 EUR

Rink Amide MBHA resin (0.4-0.8 mmol/g) 5g 168 EUR

Cleavage of protecting groups (decapeptide): 15 EUR (Boc), 5 EUR (Fmoc)

DCM (for peptide synthesis) 49 EUR/L

DMF (for peptide synthesis) 111 EUR/L

However, application of Boc-strategy needs a special HF cleavage apparatus!

Many synthesizers are designed for Fmoc chemistry. They are TFA sensitive.

Ordering of piperidine might need allowance, because it is the starting materialin the synthesis of morphine.

Boc Fmoc

It is better for avoiding DKP formation;

There is no problem with the Boc cleavage, so it is better in case of peptides that aggregate easily. Aggregates are destroyed in every TFA cleavage step;

Because of the extra neutralisation step, the synthetic cycle takes longer time;

Resins for Boc-strategy are available for Fmoc-chemistry, too. Two steps cleavage procedure may results in better crude product. First step TFA cleavage (side chain protecting groups) then HF (peptide-resin bond). More suitable for preparation of branched peptides.

ClTrt resin must be used to prevent DKP formation;

Incomplete Fmoc deprotection in case of aggregating peptides;

It is better for acid sensitive peptides (Trp, Met), oxidation, alkylation can be avoided. Asp-Pro bond is highly acid sensitive.

especially recommended for O-glycosylated or sulfated peptides;

Because of the orthogonality of N and side chain protecting groups fully protected sequences can be prepared.

Documents

Solid phase peptide synthesis Part II Application of Fmoc/ t Bu strategy Gábor Mező Research Group of Peptide Chemistry Hungarian Academy of Sciences Eötvös