Extending Pseudoprolines Beyond Ser & Thr


Introducing Fmoc-Xaa-Cys(ΨDmp,Hpro) -OH dipeptides


Fmoc-Ala-Cys
Fmoc-Leu-Cys
Fmoc-Ala-Cys (ΨDmp, Hpro) -OH Fmoc-Leu-Cys (ΨDmp,Hpro) -OH
Fmoc-Lys(Boc)-Cys
Fmoc-Val-Cys
Fmoc-Lys(Boc)-Cys (ΨDmp, Hpro) -OH Fmoc-Val-Cys (ΨDmp, Hpro) -OH

Mutter's pseudoproline dipeptides [1] are powerful tools for enhancing synthetic efficiency in Fmoc SPPS. Their use leads to better and more predictable acylation and deprotection kinetics, which results in higher purities and solubilities of crude products, easier HPLC purification and improved yields, with less need to repeat failed syntheses. They have proved particularly effective in the synthesis of intractable peptides [2 - 5], long peptides/small proteins [6 - 13], and cyclic peptides [14, 15], enabling in many cases the production of peptides that otherwise could not be made. Pseudoproline derivatives can be derived from Ser, Thr or Cys, however, until now only those based on Ser and Thr have been commerically available. Novabiochem's new cysteine-based pseudoproline dipeptides expand the scope of the structure breaking building blocks available for Fmoc SPPS.

Merck:/Freestyle/LE-Lab-Essentials/Learning Center/Peptides/AA-cysteinyl-pseudoproline-dipeptides-700x457-12152015.jpg
Figure 1: Principles of using cysteinyl pseudoproline dipeptides.

Cys-based pseudoproline dipeptides are used in exactly the same manner as those derived from Ser or Thr. They can be coupled using any standard coupling method, such as PyBOP/DIPEA or DIPCDI/Oxyma Pure, substituting a Cys residue together with the preceding amino acid residue in the peptide sequence with the appropriate pseudoproline dipeptide (figure 1). The thiazolidine ring is labile to TFA, so the native sequence cysteinyl-containg peptide is regenerated on cleavage and deprotection.

The cysteine pseudoprolines can be used in combination with standard pseudoprolines and Dmb-dipeptides. Positioning of these structure-breaking derivatives approximately 6 residues apart in the peptide sequence at regular intervals has proven to be an extremely effective approach for the synthesis of long and amyloidogenic peptides.

Prevention of Epimerization During Coupling

Trityl-protected cysteine is known to undergo racemization during coupling, particularly if base-mediated activated methods are used. Cysteine-derived pseudoprolines, in constrast, have excellent chiral stability, as illustrated by the results shown in figure 2. H-Lys-Cys-Phe-Pro-Glu-Tyr-Thr-Pro-Asn-Phe-OH (EGF (36-45)) prepared with TBTU/DIPEA activation using Fmoc-Cys(Trt)-OH contained 3.7% D-Cys Table 1, A), whereas using Fmoc-Lys(Boc)-Ser(ΨDmp,Hpro)-OH only 0.4% D-Cys ( (table 1, B) was generated.

EGF
Peptide
purity
(% area)
% D-Cys
A 81 3.9
B. TFA/H2O/TIPS 47 0.4
B. TFA/H2O/EDT 76 0.4
B. TFA/H2O/TIPS/EDT 81 0.4

Table 1:
Purity and D-Cys content of EGF (36-45) prepared with Fmoc-Cys(Trt)-OH A and Fmoc-Lys(Boc)-Cys(ΨDmp,Hpro)-OH B.
HPLC profile of crude H-Lys-Cys-Phe-Pro-Glu-Tyr-Thr-Pro-Asn-Phe-OH
Figure 2: HPLC profile of crude H-Lys-Cys-Phe-Pro-Glu-Tyr-Thr-Pro-Asn-Phe-OH prepared with TBTU/DIPEA activation (top) using Fmoc-Cys(Trt)-OH and (bottom) using Fmoc-Lys(Boc)-Cys(ΨDmp,Hpro)-OH.

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Overcoming Aggregation

The ability of pseudoprolines-derived from Ser and Thr to disrupt aggregation during peptide assembly is well demonstrated. It is thought that the dimethyloxazolidine ring of the pseudoproline imposes a kink on the peptide chain due to it favouring a cis-amide bond conformation. Pseudoprolines derived from cysteine and dimethoxybenzaldehyde are know to be less effective at promoting a cis-amide conformation and, therefore, might be expected to be less efficient at preventing aggregation.

To determine if this is indeed the case, analogs of the difficult peptide influenza virus hemagglutinin were prepared using either Fmoc-Ser(tBu)-OH, Fmoc-Ala-Ser(ΨMe,Mepro)-OH or Fmoc-Ala-Cys(ΨDmp,Hpro)-OH. Figure 3 shows the HPLC profiles of the crude peptides obtained from these syntheses. As expected, the peptide prepared using Fmoc-Ser(tBu)-OH was highly heterogeneous. In constrast, the purities of the analogs prepared using both pseudoproline building blocks were excellent, indicating the Cys-derived pseudoproline dipeptides to be equally as effective as those derived from Ser or Thr at inhibiting aggregation (figure 3).

HPLC profiles of crude H-Met-Glu-Asp-Ser-Thr-Tyr-Tyr-Lys-Ala-Ser-Lys-Gly-Cys-NH2
Figure 3: HPLC profiles of crude H-Met-Glu-Asp-Ser-Thr-Tyr-Tyr-Lys-Ala-Ser-Lys-Gly-Cys-NH2prepared with (top) Fmoc-Ser(tBu)-OH and (middle) Fmoc-Ala-Ser(ΨMe,Mepro)-OH, and (bottom) crude H-Met-Glu-Asp-Ser-Thr-Tyr-Tyr-Lys-Ala-Cys-Lys-Gly-Cys-NH2 prepared with Fmoc-Ala-Cys(ΨDmb,Hpro)-OH.

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Prevention of Alkylation During Cleavage

Ring opening of Cys(ΨDmp,Hpro) residues with TFA releases reactive dimethoxybenzalehyde. Cleavage of the EGF model peptide with the standard TFA/TIPS/water cocktail gave two major by-products: dimeric peptide derived from dimethoxybenzaldehyde and a dimethoxybenzaldehyde adduct.

The addition of EDT to the cocktail eliminated both by-products and led to a clean product. Omission of TIPS from the cocktail afforded products containing free dimethoxybenzaldehyde. Therefore, peptides containing Cys(Dmb,Hpro) residues should be cleaved with TFA/TIPS/water/EDT (table 1).

H-Lys(Boc)-Cys(Dmb,Hpro)-Phe-Pro-Glu(OtBu)-Tyr(tBu)-Thr(tBu)-Pro-Asn(Trt)-Phe-Wang cleaved with (top) TFA/TIPS/water
Figure 4: HPLC profiles of crude H-Lys(Boc)-Cys(ΨDmb,Hpro)-Phe-Pro-Glu(OtBu)-Tyr(tBu)-Thr(tBu)-Pro-Asn(Trt)-Phe-Wang cleaved with (top) TFA/TIPS/water (95:2.5/2.5), (middle) TFA/EDT/water (95:2.5/2.5) and (bottom) TFA/TIPS/water/EDT (92.5:2.5/2.5/2.5).

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Products

Pseudoproline Dipeptide
Catalog Number
Fmoc-Ala-Cys(ΨDmp,Hpro) -OH 852381
Fmoc-Leu-Cys(ΨDmp,Hpro) -OH 852382
Fmoc-Val-Cys(ΨDmp,Hpro) -OH 852383
Fmoc-Lys(Boc)-Cys(ΨDmp,Hpro)-OH 852384

References

      1. a) T. Haack & M. Mutter (1992) Tetrahedron Lett., 33, 1589; b) M. Mutter, et al. (1995) Pept. Res., 8, 145.
      2. P. White, et al. in “Peptides 1998, Proc. of 25th European Peptide Symposium”, Budapest, Akadémiai Kiadó, 1998, pp. 120.
      3. C. Hyde, et al. (1994) Int. J. Peptide Protein Res., 43, 431.
      4. R. von Eggelkraut-Gottanka, et al. (2003) ChemBioChem, 4, 425.
      5. F. Shabanpoor, et al. (2007) J. Pept. Sci., 13, 113.
      6. a) P. White, et al. (2004) J. Pept. Sci., 10, 18; b) P. White, et al. (2003) Biopolymers, 71, 338.
      7. F. García-Martín, et al. (2006) Bipolymers, 84, 566.
      8. S. Abu-Baker & G. A. Lorigan (2006) Biochemistry, 45, 13312.
      9. V. Goncalves, et al. (2009) J. Pept. Sci., 15, 417.
      10. F. El Oualid, et al. (2010) Angew. Chem. Int. Ed., 49, 10149.
      11. S. N. Bavikar, et al. (2012) Angew. Chem. Int. Ed., 51, 758.
      12. P. Nagorny, et al. (2012) Angew. Chem. Int. Ed., 51, 975.
      13. C. Yves-Marie, et al. (2010) J. Pept. Sci., 16, 98.
      14. A. Ehrlich, et al. (1996) J. Org. Chem., 61, 8831.
      15. N. Schmiedeberg & H. Kessler (2002) Org. Lett., 4, 59.

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