Planning a Peptide Synthesis HEADING_TITLE

N- and C-Terminal Functionality

Synthetic peptides are usually prepared to mimic naturally occurring peptides or segments of peptides or proteins.  If the synthetic peptide is based on a naturally occurring peptide, then the N- and C-terminals probably will not require modification.  If the synthetic peptide is mimicking a segment of a larger peptide or protein, then the basic N-terminal and the acidic C-terminal may need to be modified to approximate the neutral amide functions of the native peptide or protein.

  • If the peptide sequence is from the N-terminal, then the C-terminal should be an amide.
  • If the peptide sequence is from the C-terminal, the N-terminal may need to be acylated.
  • If the peptide sequence is from the interior portion of the model, both ends of the peptide may need to be modified.

C-terminal amides are most conveniently prepared on an amide-forming resin such as MBHA, Rink or Sieber resins.  C-terminal amides can also be formed by cleaving the peptide from the resin by amminolysis.  While amminolysis can be performed on many standard resins such as Merrifield and Wang resins, Oxime and HMBA resins are preferred.

N-terminal acetylation is easily achieved by adding a final capping step to the peptide synthesis protocol.

Boc vs. Fmoc

The first step in planning a successful peptide synthesis is to choose the protection scheme.  Boc/Bzl protection, when utilized with in situ neutralization, can provide superior results for long or difficult peptide sequences.  Cleaving the peptide product from the resin requires strong acids such as TFMSA or HF.  HF is toxic and reacts with ordinary laboratory glassware.  It requires special apparatus, which can make scaling up difficult if a laboratory is not specially equipped for large-scale peptide production.

Fmoc/tBu protection typically does not require reagents stronger than 50% TFA to remove side-chain protecting groups and cleave the peptide from the resin support, hence it can be scaled up easily in the laboratory.  The side chains can be deprotected while the N-terminal Fmoc remains in place, allowing side chain modification.  In addition, there are a variety of other side chain protecting groups available which allow selective deprotection at a specific site.

Aggregation tends to be more of a problem when Fmoc/tBu protection is utilized, since the peptide-resin is always in a neutral state.  This is not significant with short sequences, but can become problematical when large peptides are synthesized.  Fmoc deprotection, which is rather rapid in small peptides, often becomes slower in longer peptides due to aggregation.

In general, Boc/Bzl protection is best for long or difficult sequences and base sensitive peptides while Fmoc/tBu protection is best for acid sensitive peptides and peptides with sidechain modifications.

 

Boc/Bzl

Fmoc/tBu

N-alpha Deprotection

50% TFA

40% Piperidine

Final Cleavage

HF, special equipment

50% TFA, plain glassware

Synthetic Steps

deprotect, wash, neutralize, wash, couple, wash

deprotect, wash, couple, wash

Scale

Limited by HF cleavage apparatus

Any scale

Recommended for:

Difficult sequences prone to aggregation, base sensitive peptides

Acid sensitive peptides, peptides labeled or modified on the sidechains

 

Selecting a Resin for Peptide Synthesis

Before choosing a resin to use in a peptide synthesis, you must answer several questions first:

1)     Will Boc-amino acids or Fmoc-amino acids be used to synthesize the peptide?

2)     Will the product be a peptide acid or a peptide amide?

3)     How large is the peptide?

4)     Will the product peptide be unprotected (all protecting groups removed) or protected for further applications (fragment condensation, ligation, etc.)?

There are additional questions that may need to be considered, but these are the main questions that must be answered before choosing a resin.

Will Boc-amino acids or Fmoc-amino acids be used to synthesize the peptide?

In synthesizing peptides, the N-protecting group of the previous amino acid has to be removed before the next amino acid is coupled.  The Boc protecting group is usually removed with 50% trifluoroacetic acid (TFA) in dichloromethane (DCM), so the peptide-linker bond has to be stable in these conditions.  Merrifield, Hydroxymethyl polystyrene, PAM and MBHA resins are the resins most commonly used for peptide synthesis with Boc-amino acids.

Using Fmoc-amino acids, you can prepare peptides under neutral or basic conditions, so most of resins used in Fmoc-peptide synthesis can be cleaved under relatively mild acid conditions.  Wang, 2-chlorotrityl, Rink amide and Sieber amide resins are some of the resins commonly used with Fmoc-amino acids.

Will the product be a peptide acid or a peptide amide?

Most peptides have either a carboxylic acid group (–COOH) or an amide group (–CONH2) at the C-terminal.  The common resins used for preparing acid peptides are Merrifield, hydroxymethyl polystyrene, Wang, and 2-chlorotrityl resins.  The common resins for preparing peptide amides are MBHA, Rink amide and Sieber amide resins.

A few peptides and peptide fragments have other functional groups at the C-terminal.  Alcohols (–CH2OH), methyl amides (–CONHCH3) and ethyl amides (–CONHCH2CH3) are the most common alternatives.  There are several methods, and resins, for preparing these peptides.  A common method for preparing these products is to synthesize the peptide on an appropriate resin, then to displace the peptide from the resin with an amine or amino acid (Scheme 1).  Some of the resins used in this application are

Displacement from resin

Scheme 1

phenol resin and oxime resin, used for Boc peptide synthesis; HMBA resin, used for Fmoc peptide synthesis; and Aliphatic Safety Catch resin, also used for Fmoc peptide synthesis.

Another resin that you can use to prepare peptides with non-standard C-terminals is BAL resin.  With this resin, the peptide is attached to the resin linker at one of the “backbone” amide groups instead of the C-terminal.  The C-terminal is protected by an ester as the peptide is synthesized, then is deprotected and modified (Scheme 2).

 

BAL Resin

Scheme 2

How large is the peptide?

In preparing large peptides, aggregation of the growing peptide chains can cause difficulty during coupling reactions.  Generally, using a low-substitution resin will reduce this difficulty.  If the peptide you will synthesize is very large (30 to 50 amino acids), then a resin with a low substitution (0.1 to 0.4 mmol/g) is best.  For peptides of 10 to 20 amino acids, you can usually use a resin with standard substitution (0.5 to 1.2 mmol/g).  To prepare a short peptide (<10 amino acids), a higher substitution resin (1.3-2.0 mmol/g) may be utilized.

Resins that provide a more polar, peptide-like environment than polystyrene are also helpful for reducing aggregation.  TentaGel is one example.  TentaGel resins incorporate polyethylene glycol chains between the polystyrene bead and the linker.

Surface resins are another option for preparing large peptides.  The reactive sites are confined to a thin layer on the surface of the beads so reagents do not have to diffuse to the core of the beads.  In addition, the peptide chains attached to resin have conformational freedom and are not constrained by the volume of the pores within the resin.  The N-terminal of the peptides attached to the resin remain readily accessible for deprotection and coupling, thus improving the yield in each coupling step and minimizing deletions.

Will the product peptide be unprotected or protected for further applications?

In standard solid-phase synthesis protocols, the peptide is cleaved from the resin and at the same time the side-chain protecting groups are removed.  If you are preparing small protected peptides to couple together to form large peptides or small proteins, though, you want to cleave the peptides from the resin without removing the side-chain protecting groups.  2-Chlorotrityl chloride resin is an acid labile resin commonly used for this purpose. 

Oxime resin and phenol resin may also be used to prepare short, protected peptide segments for fragment condensation.  The protected peptide is cleaved from these resins with hydrazine, producing the peptide hydrazide which, in turn, can couple to the unprotected N-terminal of another peptide.

The choice of resin can have a large effect in the success of the peptide synthesis.  By considering these questions when planning a synthesis, the chemist will be able to choose a resin that will best suit the requirements and yield good results.

Chemistry Peptide Product Suitable Resins Coupling Methods Cleavage Reagents
Boc Peptide acid Merrifield resin

Cesium salt of amino acid;

Potassium fluoride method

HF; 

TFMSA;

TFSOTf;

HBr/AcOH

Hydroxymethyl polystyrene

DIC/HOBt/DMAP catalyst;

Mitsunobu Coupling

HF; 

TFMSA;

TFSOTf;

HBr/AcOH

Pre-loaded Merrifield resins -

HF; 

TFMSA;

TFSOTf;

HBr/AcOH

PAM resin DIC/HOBt/DMAP catalyst; HF
Pre-loaded PAM resins - HF
Peptide amides MBHA resin Desalt then DIC/HOBt or HBTU/DIEA

HF; 

TFMSA;

TFSOTf;

HBr/AcOH

Fmoc Peptide acids Wang resin

DIC/HOBt/DMAP catalyst;

Mitsunobu Coupling

50% TFA + scavengers
Pre-loaded Wang resin - 50% TFA + scavengers
Peptide amides Rink amide resin Remove Fmoc then DIC/HOBt or HBTU/DIEA 50% TFA + scavengers
Protected peptide acids 2-Cl-Trt chloride resin Fmoc-amino acid/DIEA

1% TFA;

TFE/AcOH

Pre-loaded 2-Cl-Trt resins -

1% TFA;

TFE/AcOH

Protected peptide amides Sieber amide resin Remove Fmoc then DIC/HOBt or HBTU/DIEA 1 - 2% TFA

Peptide acids;

Peptide amides;

Peptide esters;

Cyclic peptides

HMBA resin

DIC/HOBt/DMAP catalyst;

Mitsunobu Coupling

NaOEt then NaOH: acids;

Amine: amides;

NaOR: esters;

N-deprotect: cyclic peptides

Peptide aldehydes Weinreb resin Remove Fmoc then DIC/HOBt or HBTU/DIEA LiAH4
Peptide ketones Weinreb resin Remove Fmoc then DIC/HOBt or HBTU/DIEA Grignard reagents

 

Selecting Amino Acid Derivatives

Careful selection of the amino acid derivatives used in the synthesis of a long peptide sequence can minimize the formation of by-products, increase the yield of peptide and ease the purification of the isolated product.  In preparing short peptides, the selection of side chain protecting groups usually isn’t critical, they just need to prevent major side reactions.  On the other hand, the choice of protecting groups is more nuanced when a long peptide is synthesized.  The amino acid residues near the C-terminal, since they are incorporated first, are exposed to many deprotection and coupling cycles.  Even if there is a very small percentage of side reaction in each cycle, the cumulative total can become significant.  Side chain protecting groups with greater stability should be considered for the amino acids in the early portion of the peptide synthesis.

Problems can arise when the peptide is deprotected and cleaved from the resin.  Reactive intermediates formed during the cleavage of the protecting groups can react with vulnerable moieties in the peptide.  Again, this usually in not a big problem with short peptide sequences, as short sequences are less probable to have two interacting species.  Long peptide sequences are more likely to have at least one vulnerable residue and choosing appropriate protecting groups and cleavage additives is becomes critical. The following tables list some of the common amino acid side chain protecting groups, the standard conditions used to remove them, and comments about their applications.

Boc Amino Acid Derivatives 

Amino Acid

Protecting Groups

Deprotection

Comments

Arg

NO2

HF or hydrogeolysis

Prone to side reactions,

seldom used

Tos

HF

Can react with Trp residues during peptide cleavage, use Trp(For) and add thioanisole during peptide cleavage.

Asp/Glu

OBzl

HF

Standard protection, acid catalyzed cyclization byproducts possible

OcHx

HF

Suppresses aspartimide and pyroglutamate formation, recommended in long peptide sequences

Asn/Gln

Xan

HF

Suppresses dehydration and increases solubility of the protected amino acid

Cys

Bzl

HF

Standard

MeOBzl

HF, TFMSA

Standard

MeBzl

HF, HBr/HOAc

Standard

Acm

Hg(II), I2

Iodine forms S-S bond during deprotection. Stable to cleavage conditions, useful for preparing protected peptides

Trt

TFA, HF, I2

Can be used with Cys(Acm) for on resin cyclization

His

Boc

TFA

Temporary protection

Bom

HF

Suppresses racemization

Dnp

Thiolysis

Remove protecting group before peptide cleavage

Tos

HOBt

Suppresses racemization

Trt

TFA, HF

Temporary protection

Lys/Orn

Boc

TFA

Temporary protection

2-Cl-Z

HF, TFMSOTf, TFSMA, HBr/HOAc

Standard

Fmoc

Piperidine

Used for preparing protected peptide fragments and on-resin derivatization of the side chain

Z

HF, Hydrogenolysis

Standard

Met

Sulfoxide

Mercaptoethanol

Oxidized form of methionine, simplifies purification of crude peptide when a mixture of oxidized and reduced forms occur.

Ser/Thr

Bzl

HF

Standard

tBu

TFA, HF

Temporary protection

Trp

For

HF

Standard

Tyr

tBu

TFA

Temporary protection

Bzl

HF

Can for side products during cleavage

2-Br-Z

HF

Standard

2,6-Cl2Bzl

HF

Standard

 

Fmoc Amino Acid Derivatives

Amino Acid

Protecting Groups

Deprotection

Comments

Arg

Mtr

TFA at 35°C

Best in small peptides with only one Arg residue

Pmc

TFA

Standard

Pbf

TFA

Standard, Less likely to react with Trp residues during cleave than Pmc

Asp/Glu

OtBu

TFA

Standard

OBzl

H2/Pd, HF

Seldom used

OcHx

HF

Seldom used

Asn/Gln

Trt

TFA

Standard, suppresses dehydration and increases solubility of the protected amino acid

Cys

Acm

Hg (II), I2

Iodine forms S-S bond during deprotection. Stable to cleavage conditions, useful for preparing protected peptides

tBu

TFSMA, Hg(II), TFA/DMSO/Anisole

Useful in selective formation of multiple disulfide bridges

pMeOBzl

TFMSA

Useful in selective formation of multiple disulfide bridges

Mmt

1% TFA

On resin modification

Trt

TFA, I2

Standard

His

Fmoc

Piperidine

Temporary

Trt

90% TFA/DCM

Standard

Mtt

15% TFA/DCM

Standard

Lys/Orn

Boc

TFA

Standard

tfa

TFA

Does not form side products during cleavage

Trt

TFA

Fewer side reactions during cleavage

Mtt

1% TFA/DCM

On-resin modification

Dde, ivDde

Hydrazine

Hydrazine removes Fmoc-protecting groups, too.

Fmoc

Piperidine

Temporary

Ser/Thr tBu

TFA

Standard

Trt

1% TFA/DCM

On resin modification

Bzl

H2/Pd, HF

Seldom used

Trp Boc

TFA

Greatly reduces by-products formed during cleavage

Tyr tBu

TFA

Standard

 

Planning the Synthesis of Peptides Containing Multiple Disulfide Bridges

Peptides that contain multiple disulfide bridges are especially challenging synthetically.  In some instances, all of the cysteine residues can be deprotected and simple air oxidation will form primarily the correctly bridged peptide.[1]  When substantial mispairing makes this approach impractical, selective deprotection and stepwise bridge formation are required. 

A peptide containing two disulfide bridges requires two pairs of differentially protected cysteines.  One pair can have protecting groups that are removed at the same time as the peptide is cleaved from the resin while the other pair are protected with groups, such as Acm, that remain in place during cleavage.  After the peptide has been synthesized and cleaved from the resin, the first disulfide bridge is formed, then the remaining pair of cysteine residues are deprotected and the second disulfide bridge is formed. 

Preparing a peptide with three disulfide bonds requires three pairs of cysteine residues that can be independently deprotected.  In this case, Fmoc chemistry is preferred over Boc chemistry because there is a greater selection of independently deprotected cysteine derivatives available.  The set of Cys(Trt), Cys(Acm) and Cys(pMeOBzl) is example that has been employed successfully in the synthesis of several peptides, including defensins, relaxin, and sapecin.[2]  The preparative sequence is outlined in the table below.

Cycle

Cysteine Protection

Deprotection

Cyclization

Cycle 1

Cys(Trt)

TFA/H2O/Scavengers

Air oxidation

Cycle 2

Cys(Acm)

Cleavage and cyclization with I2

Cycle 3

Cys(pMeOBzl)

TFSMA/TFA/anisole

Air oxidation or I2

 


[1] Moroder, L.; Besse, D.; Musiol, H. J.;, Rudolph-Böhmer, s.; Siedler, F. Biopolymers, 1996, 40, 207-234.

[2] Durieux, J. P.; Nyfeler, R., in “Peptides 1994, Proceedings of the 23rd European Peptide Synposium, Braga 1994”, Maia, H. L. S. Ed., ESCOM Publishers, Leiden 1995, 165.; Büllesbach, E. E.; Schwabe, C. J. Biol. Chem., 1991, 266, 10754-10761.; Mergler, M.; Nyfeler, R., in “Proceedings of the 4th international Symposium on Innovation and Perspectives in SPPS, Edinburgh 1995”, Epton, R. Ed., Mayflower Scientific Ltd, Birmingham 1996, 42.