Pseudoproline & Isoacyl dipeptides
Pseudoproline and Isoacyl dipeptides offered by Bachem
The synthesis of very long peptides and “difficult sequences” by chemical means still poses a challenge to the peptide chemist. The difficulties encountered during such syntheses usually are due to the aggregation of the resin-bound peptide. Proline residues disrupt such ordered structures efficiently.
Temporary Pro mimics can be readily obtained from Ser and Thr by oxazolidine or from Cys by thiazolidine (“pseudoproline”) formation. Both 2,2 dimethyloxazolidines and -thiazolidines are smoothly cleaved by trifluoroacetic acid and thus suitable for Fmoc-SPPS. Disruption of Ser- and / or Thr containing aggregates is achieved as well by introducing depsipeptide (“O-acyl isopeptide”) bonds into the peptide backbone. The resulting isopeptide is rearranged yielding the desired sequence in slightly basic solution.
Preformed Dipeptides
For facilitating the introduction of oxazolidine or thiazolidine moieties or isopeptide bonds during solid-phase synthesis, protected pseudoproline dipeptides and O-acyl dipeptides have been developed. We offer a broad choice of these versatile building blocks for preventing aggregation during Fmoc-SPPS.
Pseudoproline Dipeptides
Stepwise Fmoc-SPPS may become very difficult or even fail if the resin-bound peptide aggregates. Unfortunately, the predictions of “difficult sequences” based on an assumed propensity of the sequence to form β-sheets are not very reliable, with the exception that such aggregates cannot be formed in the vicinity of proline. The induction of a cis-amide bond by Pro disrupts β-sheets as well as α-helices.
The aggregation of Ser-, Thr- or Cys-containing peptides during Fmoc-SPPS can thus be efficiently prevented by introducing the Fmoc-protected oxazolidines or thiazolidines, which can be obtained from these amino acids and aldehydes or ketones (Fig. 1). These heterocycles have been appropriately termed pseudoprolines. The pseudoproline approach, originally developed by Manfred Mutter et al. at EPFL Lausanne12, usually employed the Ser and Thr derivatives, as the oxazolidines were cleaved more readily by acids than the thiazolidines3.
Fig. 1
In the meantime, thiazolidine cleavage conditions suitable for standard Fmoc-SPPS have been developed 4. The incorporation of cysteine pseudoproline derivatives facilitated the synthesis of Cys-rich peptides5, which will also benefit from milder deprotection conditions. The acid lability of both types of heterocycle can be fine-tuned by the choice of substituents at the 2-position.
The isopropylidene group of the oxazolidine (X = O, R’, R’’ = CH3) proved to be the best choice for Fmoc-SPPS 3, as the cycle is opened readily yielding Ser (R = H) or Thr (R = CH3) during the final cleavage with TFA. Both isopropylidene (X = S, R’, R’’ = CH3) and benzylidene (X = S, R’= 2,4-Dimethoxyphenyl, R’’ = H) derivatives have been used for incorporating cysteine, as the rings can be cleaved by TFA6.
Fig. 2
The main drawback of this approach is the difficult coupling of the following amino acid to the hindered heterocycle. To circumvent this, Fmoc pseudoproline dipeptides were introduced7(Fig. 2). The coupling of these building blocks represents the most straightforward method to incorporate pseudoprolines. The ”preventive” insertion of such moieties is highly recommended when synthesizing long peptides lacking prolines (Fig. 3).
The incorporation of pseudoprolines facilitates cyclization of peptides
The repeated inclusion of pseudoproline units during the elongation of the peptide will improve the overall coupling efficiency, even if aggregation does not pose a severe problem. A considerable number of syntheses of long or “inaccessible” peptides, which succeeded only due to the insertion of pseudoprolines in appropriate positions, has been published since the introduction of these derivatives 8910. Difficult peptides as IAPP 1112 or RANTES 13could be synthesized following standard Fmoc-SPPS protocols after evaluation of the required number and optimal position of the oxazolidine moieties to be inserted.
Fig. 3
As with Fmoc amino acid derivatives, couplings can be accelerated by microwave irradiation 141516. Pseudoproline dipeptides show their high versatility as building blocks not only during the SPPS of long and difficult peptides. When synthesizing short peptides, a distinctly purer crude product may be obtained by incorporating merely a single pseudoproline unit 15.
The heterocycles are left intact when cleaving fully protected peptide fragments from SASRIN or 2-chlorotrityl resin with diluted TFA 1717 and their presence markedly increases the solubility of the cleavage products. Accordingly, the purification and coupling of the fragments as well as the modification of partially protected peptides in solution are facilitated by insertion of pseudoproline moieties. As fragments containing a C-terminal proline, fragments with a C-terminal pseudoproline can be coupled with minimal concomitant racemization.
Hence pseudoproline dipeptides may establish additional possibilities in convergent peptide synthesis 18. The incorporation of an oxazolidine moiety greatly facilitates cyclizations of Ser- or Thr-containing peptides, disulfide bridge formations 19, as well as headto-tail cyclizations2021. The increased tendency to cyclize is due to the presence of a temporary cis-amide bond in the molecule 2223.
Fig. 4
Even though Cys occurs only rarely in peptides and proteins, far less often than Ser and Thr, the incorporation of cysteine pseudoproline is highly attractive due to the peculiar properties of the amino acid. Postma and Albericio showed that on-resin macrocyclization of Cys-containing peptides proceeds more smoothly if Cys(Trt) is replaced by a cysteine pseudoproline24.
They also observed that cysteine 2,2-dimethylthiazolidines are opened during the final cleavage with TFA/TIS/H2 O (95:2.5:2.5), lows avoiding this risk, an advantage worth considering when synthesizing multiple disulfide bridge-containing peptides or anchoring cysteine to carriers. If its thiazolidine ring is left intact, 2,2-dimethylthiazolidin-4-carboxylic acid (Me2 Thz), conveniently introduced as pseudoproline dipeptide, acts as a highly effective cis-proline mimic [24,25] 172526. An analog of the cyclopeptide phakellistatin 19 containing three Me2 Thz residues replacing proline, all of them incorporated by coupling pseudoproline dipeptides, showed enhanced activity 27. For synthesizing peptides containing this proline surrogate the standard TFA-labile lateral protecting groups may have to be replaced by highly acid-labile moieties such as Mtt (His), Trt (Ser) or OPp (Glu)29.
Fig. 5
Isoacyl Dipeptides
A major disadvantage of the pseudoproline approach cannot be left unmentioned: The solubilizing effect of the oxazolidine moiety is lost when deblocking the peptide with TFA. Albeit the quality of the crude product may be vastly improved, further purification will be tedious due to its low solubility. The N-O shift, a notorious side reaction during HF cleavage, turned out to be the key to the solution of this dilemma. This acid-catalyzed rearrangement involving the hydroxyl moiety of Ser or Thr residues can be smoothly reversed by keeping the peptide in a slightly basic medium (Fig. 4).
As the N-O shift, i.e. O-isoacyl peptide formation, causes a disruption of the secondary structure, it is accompanied by an increase in solubility3031. At first, this type of depsipeptide was obtained by FmocSPPS involving on-resin esterification of the subsequent Fmoc-amino acid to the free hydroxyl moiety of an N-terminal Boc-Ser/Thr followed by elongation of the peptide 32. Low conversions, concomitant racemization, and diketopiperazine formation during the subsequent SPPS cycle 33 are the main drawbacks of this straightforward approach.
Fig. 6
The recently introduced Fmoc O-acyl dipeptides help to overcome these problems 3435(Fig. 5), though diketopiperazine formation is not affected by the method chosen for introducing the ester bond. It is best combated by the use of the more labile Bsmoc protecting group3637 or a less nucleophilic base. 38 β-elimination during the activation step has been described by Coin et al. as a side reaction of isoacyl dipeptides 39. To combat this. the coupling can be performed in non-polar solvents, if solubility is not an issue 40. Otherwise, a base-free activation procedure can be used as well. 39
As pseudoproline dipeptides, O-acyl dipeptides turned out to be versatile building blocks for the synthesis of difficult peptides such as β-amyloid (1-42)41, which indeed showed an improved solubility and reduced propensity for fibril formation, and insulin, where isoacyl dipeptides were inserted in both A- and B-chain42. Peptides containing O-acyl bonds to Ser or Thr may act as soluble prodrugs, hence they have also been termed “switch peptides”43 (Fig. 6).
