Novel Phosphate Derivatives as Scaffolds for the Preparation of Synthetic Phosphoserine-Based Peptides Using the Fmoc/t-Bu Solid-Phase Strategy

Abstract

Synthetic peptides incorporating analogues of phosphoserine are valuable tools for the study of protein kinases and phosphatases. In addition, derivatives of naturally occurring peptides incorporating phosphate groups may have interesting biological properties. Herein we describe a new Fmoc/t-Bu solid-phase peptide synthesis (SPPS) strategy for the convenient generation of phosphoserine-based peptides. A proof-of-concept synthesis that demonstrates the feasibility of this approach is presented.

Key words

phosphopeptide - serine - phosphorus - solid-phase synthesis - amino acids

References and Notes

• 1 de bont DBA. Moree WJ. Vanboom JH. Liskamp RMJ. J. Org. Chem.  1993,  58:  1309 
• 2 Wiemann A. Frank R. Tegge W. Tetrahedron  2000,  56:  1331 
• 3 For example phosphoserine residues are known to be sensitive to the strong acidic and basic conditions typically employed in Fmoc SPPS. See: Perich JW. Synthesis of Phosphopeptides, In Synthesis of Peptides and Peptidomimetics, Houben-Weyl Methods in Organic Chemistry   Volume E22b:  Thieme; Stuttgart: 2004.  p.375-424  
• 4 Singer D. Hoffman R. In Peptide-Based Drug Design, Methods in Molecular Biology   Vol. 494:  Otvos L. Humana Press; Totowa (NJ / USA): 2008.  p.209 
• 5 Shapiro G. Buechler D. Enz A. Pombovillar E. Tetrahedron Lett.  1994,  35:  1173 
• 6 Wakamiya T. Nishida T. Togashi R. Saruta K. Yasuoka J. Kusumoto S. Bull. Chem. Soc. Jpn.  1996,  69:  465 
• 7 Wakamiya T. Saruta K. Yasuoka J. Kusumoto S. Chem. Lett.  1994,  24:  1099 
• 8a For a recent discussion of the use of peptides and peptide derivative as therapeutic agents, see: Mason JM. Future Med. Chem.  2010,  2:  1813 
• 8b For a recent general review of the use of small molecules as tools to study biological systems, see: O’Connor CJ. Laraia L. Spring DR. Chem. Soc. Rev.  2011,  40:  4332 
• 9 Shapiro G. Büchler D. Dalvit C. Frey P. Fernandez MdC. Gomez-Lor B. Pombo-Villar E. Stauss U. Swoboda R. Waridel C. Bioorg. Med. Chem.  1997,  5:  147 
• 10a Gorske BC. Blackwell HE. Org. Biomol. Chem.  2006,  4:  1441 
• 10b Lyon GJ. Wright JS. Muir TW. Novick RP. Biochemistry  2002,  41:  10095 
• 12a Barany G. Albericio F. J. Am. Chem. Soc.  1985,  107:  4936 
• 12b Isidro-Llobet A. Alvarez M. Albericio F. Chem. Rev.  2009,  109:  2455 
• 13a It is known that the stability of the phosphate moiety is increased when the phosphate triester is replaced with the diester. In addition, cyanoethyl group removal is generally quicker than serine β-elimination, thus preventing it. See: McMurray JS. Coleman DR. Wang W. Campbell ML. Biopolymers (Pept. Sci.)  2001,  60:  3 
• 13b

  Fmoc/t-Bu SPPS using derivatives of this sort as starting materials is superior to that employing the analogous compounds containing a free phosphoric acid group (see ref. 7).

• 14a Galloway WRJD. Hodgkinson JT. Bowden SD. Welch M. Spring DR. Chem. Rev.  2011,  11:  28 
• 14b Hodgkinson JT. Galloway WRJD. Casoli M. Keane H. Su X. Welch M. Spring DR. Tetrahedron Lett.  2011,  52:  3291 
• 14c Hodgkinson JT. Galloway WRJD. Saraf S. Baxendale IR. Ley SV. Ladlow M. Welch M. Spring DR. Org. Biomol. Chem.  2011,  9:  57 
• 14d Hodgkinson JT. Bowden SD. Galloway WRJD. Spring DR. Welch M. J. Bacteriol.  2010,  192:  3833 
• The use of 2-CTC resin allows the cleavage of peptide from the solid phase with 1-2% of TFA without removing any side-chain protecting groups that may be present within the main peptide, which could be important from the perspective of any post-cleavage synthetic manipulations. See:
• 15a Benz H. Synthesis  1994,  337 
• 15b Barlos K. Chatzi O. Gatos D. Stavropoulos G. Int. J. Pept. Protein Res.  1991,  37:  513 
• 15c An additional advantage of the 2-CTC resin is that its hindered structure minimises the formation of diketopiperazines during removal of the temporary protecting group of the second amino acid on the solid phase (Scheme 4, 10 to 11, step iv); see: Rovero P. Vigano S. Pegoraro S. Quartara L. Lett. Pept. Sci.  1996,  2:  319 
• 16 Subirós-Funosas R. Prohens R. Barbas R. El-Faham A. Albericio F. Chemistry  2009,  37:  9394 
• 17a The chloranil test for detection of primary and secondary amines was performed after each solid-phase coupling reaction to confirm that it had proceeded to completion. See: Vojkovsky T. Peptide Res.  1995,  8:  236 
• 17b

  When the test was negative the Fmoc group was removed with piperidine-DMF (1:4); otherwise the Fmoc-amino acid was re-coupled under the same conditions. See Supporting Information for full details.

• For recent reviews on diversity-oriented synthesis, see:
• 22a Galloway WRJD. Isidro-Llobet A. Spring DR. Nat. Commun.  2010,  1:  80 
• 22b Dandapani S. Marcaurelle LA. Curr. Opin. Chem. Biol.  2010,  14:  362 
• 22c Schreiber SL. Nature (London)  2009,  457:  153 
• 22d Nielsen E. Schreiber SL. Angew. Chem. Int. Ed.  2008,  47:  48 
• 22e Galloway WRJD. Bender A. Welch M. Spring DR. Chem. Commun.  2009,  2446 

11

Drawbacks of typical on-resin phosphorylation methods include: difficulties with the incorporation of oxidation-sensitive residues, capriciousness and a general need for large excess of phosphorylation reagents and long reaction times (see ref. 9).

18

t-Bu and Boc were chosen for either carboxylic (11) or amino (14) protection as they were stable to piperidine treatment, used to remove the Fmoc group, and to low concentrations of TFA, used to cleave the peptide from the 2-CTC resin (Scheme  [4] ).

19

It is noteworthy that compounds 2a and 2b, as well as the corresponding free carboxylic acids 9a and 9b, were obtained as diastereomeric mixtures. However, once the cyanoethyl group undergoes β-elimination during the basic treatment to remove the Fmoc group in SPPS (Scheme  [4] ), the phosphorus centre is no longer chiral leading to only one stereoisomer of the desired phosphopeptide.

20

HPLC analysis of the crude material obtained after workup revealed that the peptide was approx. 70% pure.

21

The cyclised analogue of 1 is of interest; comparison of the biological effects of the acyclic and cyclic variants would provide useful information on the importance of the macro-cyclic ring structure for the biological activity of these types of compounds. Preliminary attempts at cyclising 1
via intramolecular amide formation have provided some evidence for the formation of the desired product. Studies towards the isolation and biological testing of the cyclised analogue of 1 are ongoing and will be reported in due course.