Phosphate-assisted peptide ligation

Article abstract of DOI:10.1039/b906492c

A novel ligation method for the synthesis of phosphopeptides and peptides is described, which utilises the inherent reactivity of a peptide bearing an N-terminal phosphoserine or phosphothreonine residue to facilitate amide bond formation with a range of

Full text of DOI:10.1039/b906492c

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Phosphate-assisted peptide ligationw
Gemma L. Thomas and Richard J. Payne*
Received (in Cambridge, UK) 1st April 2009, Accepted 28th April 2009
First published as an Advance Article on the web 26th May 2009
DOI: 10.1039/b906492c
A novel ligation method for the synthesis of phosphopeptides and
We report herein the development of an efficient new ligation
peptides is described, which utilises the inherent reactivity
of a peptide bearing an N-terminal phosphoserine or phospho-
threonine residue to facilitate amide bond formation with a range
of C-terminal peptide thioesters.
method for the synthesis of peptides and phosphopeptides,
namely phosphate-assisted ligation. Our design strategy aimed
to harness the nucleophilicity of a phosphate group on the
N-terminus of a peptide, which we envisaged would play an
analogous role to the thiolate utilised in native chemical ligation.
Specifically, we speculated a reaction process whereby a phos-
phate moiety on the N-terminus of a peptide could react with a
peptide thioester to generate an acyl phosphate intermediate
(cf. the thioester intermediate in native chemical ligation). A
subsequent O - N acyl shift involving a 7-membered ring
transition state could then generate the desired peptide bond
(cf. an S - N acyl shift via a 5-membered ring in native chemical
ligation). We were encouraged that such a reaction process
would prove to be viable by the common occurrence of acyl
phosphate intermediates in acylation reactions in nature. For
example, aminoacylation of tRNA is promoted by aminoacyl
tRNA synthetases which are known to utilise an acyl phosphate
intermediate.24,25 In this process ATP activates the carboxylate
of amino acids generating an aminoacyl adenylate (an acyl
phosphate of AMP). The aminoacyl moiety is subsequently
transferred to the 30-OH of the cognate tRNA generating the
‘‘charged’’ tRNA with the amino acid linked via an ester. In
addition, there have been a number of recent reports from
Kluger and co-workers, who have utilised acyl phosphates as
biomimetic donors for the formation of esters25,26 and amides27
in an intermolecular fashion. The same group has also utilised
acyl phosphates to crosslink proteins such as haemoglobin by
amide bond formation with lysine side chains.28
Phosphorylation is a ubiquitous post-translational modification
reported to occur on 30–50% of human proteins.1 This reversible
process is known to be intimately involved in signal transduction
and the control of a host of biological processes that are critical
for normal cellular function.2 Unlike protein synthesis, phospho-
rylation events are not under template control, but rather
are dictated by the combined activities of a range of kinase
(phosphorylation) and phosphatase (dephosphorylation) enzymes.
To date, the study of phosphoproteins has been significantly
hampered by the fact that they exist as heterogeneous mixtures of
differentially phosphorylated species. It is currently accepted that
chemical synthesis may provide a viable avenue for the construc-
tion of phosphopeptides3,4 and other post-translationally
modified peptides and proteins in a homogeneous fashion and
thus has been the focus of intense research efforts.5,6 To date, the
most efficient strategy for the generation of these biomolecules
has relied on chemoselective ligation methods.7,8 Native chemical
ligation, initially introduced by Wieland et al.9 and later exploited
by Kent and co-workers for the convergent synthesis of peptides
and proteins,10,11 represents the most useful approach. The
method involves the condensation of a C-terminal peptide
thioester and a peptide bearing an N-terminal cysteine residue
to afford a native peptide bond in a rapid and chemoselective
manner.10 A number of alternative approaches have been
developed to overcome the requirement for an N-terminal
cysteine residue, with a view to expanding the number of ligation
sites that can be accessed by peptide ligation chemistry. Recent
examples include the traceless Staudinger ligation,12,13 native
chemical ligation at phenylalanine14 and valine residues,15,16
native chemical ligation followed by conversion of cysteine to
serine,17 sugar-assisted ligation18–20 and thiol free ‘‘direct amino-
lysis’’ methods.21–23 However, there is still significant demand
for the development of new ligation strategies that can be
implemented in the synthesis of underivatised and post-
translationally modified peptides and proteins for biological study.
To gauge the efficiency of the proposed ligation reaction we
first subjected model peptide 1, bearing an N-terminal phospho-
serine residue, and peptide thioester 5, possessing a C-terminal
glycine, to a mixed solvent buffer comprising 4 : 1 v/v
N-methylpyrrolidinone : 1.25 M GnꢀHCl, 0.2 M HEPES,
pH 7.5 in the presence of thiophenol at 37 1C (see ESIw). After
48 h, we were delighted to observe that the reaction was
complete, with the desired phosphopeptide product isolated in
91% yield (entry 1, Table 1). Gratifyingly, peptide 2, bearing an
N-terminal phosphothreonine residue, also reacted with thio-
ester 5 in a facile manner to afford the desired phosphopeptide in
95% yield (entry 2, Table 1). In both cases, amide bond
formation was confirmed by stability of the resulting product
to extended hydrazine treatment (see ESIw). The importance of
the phosphate moiety for the efficiency of this ligation reaction
was verified by conducting control ligations with peptides 3 and
4, bearing N-terminal serine and threonine residues, respectively.
These were reacted with 5 under the same mixed solvent system
to afford ligated peptide products in only 45 and 36% yields,
respectively, after a substantially increased reaction time of 72 h
(entries 3 and 4, Table 1). Given the increased steric bulk of the
School of Chemistry, The University of Sydney, Sydney, Australia.
E-mail: payne@chem.usyd.edu.au; Fax: +61 2 9351 3329;
Tel: +61 2 9351 5877
w Electronic supplementary information (ESI) available: Detailed
procedures for the synthesis of peptides, phosphopeptides, peptide
thioesters, ligation reactions and dephosphorylation reactions. Crude
traces and analytical data for peptides, phosphopeptides, peptide
thioesters, ligation products and dephosphorylated peptides. See
DOI: 10.1039/b906492c
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4260 | Chem. Commun., 2009, 4260–4262

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Table 1 Scope of the phosphate-assisted ligation reaction. Reaction
times: a48 h; b72 h; c14 d. Reaction yield error ꢂ 5%. R3
phosphate moiety in 1 and 2 confers a six-fold rate acceleration
when compared to unphosphorylated peptides 3 and 4.
=
(CH2)2CO2Et; NMP = N-methylpyrrolidinone; GnꢀHCl = guanidine
Having demonstrated that N-terminal phosphorylamino acids
enhance the rates of ligation reactions, the next goal was to
investigate the scope of the phosphate-assisted ligation reactions.
To this end, we synthesised peptide thioesters bearing a range of
amino acids at the C-terminus (see ESIw).29 Gly, Ala, Met, Phe,
Tyr, Ser and Val were selected as a representative range of the 20
proteinogenic amino acids to incorporate into the C-terminus of
peptide thioesters (5–11). These were reacted with phospho-
peptides 1 and 2 under the previously described conditions
(Table 1). We were delighted to find that the phosphate-assisted
ligation reactions with 1 were high yielding in almost all cases
(entries 5–10, Table 1). Indeed, ligations with thioesters 5–10
bearing C-terminal Gly, Ala, Met, Phe, Tyr and Ser residues gave
the desired phosphopeptides in yields ranging from 62 to 91%. In
line with reactivity patterns observed in native chemical ligation,
the ligation with C-terminal valine thioester 11 resulted in a much
slower reaction, requiring 14 days to reach completion. Fortu-
nately, the stability of valine thioester 11 under the ligation
conditions led to slow hydrolysis and allowed for a satisfactory
ligation yield (47%, entry 10, Table 1). Ligations with phospho-
peptide 2 containing an N-terminal phosphothreonine residue
provided lower yields, presumably due to the increased steric
bulk of the threonine residue. Nonetheless, isolated reac-
tion yields were moderate to high in all cases and represent
synthetically useful reactions (entries 11–16, Table 1).
hydrochloride; HEPES
= 4-(2-hydroxyethyl)piperazine-1-ethane-
sulfonic acid. 5: X = AsnGly; 6: X = AsnAla; 7: X = AsnMet; 8:
X = AsnPhe; 9: X = Tyr; 10: X = Ser; 11: X = Val
Isolated
ligation
yield (%)
Ligation
junction
Entry
X
R1
R2
1
2
3
4
5
6
7
8
AsnGly
AsnGly
AsnGly
AsnGly
AsnAla
AsnMet
AsnPhe
Tyr
PO3H
PO3H
H
H
CH3
H
CH3
H
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
Gly-pSer
Gly-pThr
Gly-Ser
91a
95a
45b
36b
90a
71a
82b
82b
62b
47c
61b
50b
52b
90b
62b
47c
H
Gly-Thr
Ala-pSer
Met-pSer
Phe-pSer
Tyr-pSer
Ser-pSer
Val-pSer
Ala-pThr
Met-pThr
Phe-pThr
Tyr-pThr
Ser-pThr
Val-pThr
PO3H
PO3H
PO3H
PO3H
PO3H
PO3H
PO3H
PO3H
PO3H
PO3H
PO3H
PO3H
9
Ser
10
11
12
13
14
15
16
AsnVal
AsnAla
AsnMet
AsnPhe
Tyr
Having established that the phosphate moiety enhanced the
rate of peptide ligations, we next investigated its specific role in the
reaction. This began by attempting to detect the acyl phosphate
intermediate that would be formed if the reaction were operating
via the mechanism depicted in Scheme 1. Unfortunately, all
attempts at identifying the proposed acyl phosphate intermediate
by HPLC, LC-MS, 31P NMR and FTIR proved unsuccessful.
We therefore prepared a model of the proposed intermediate,
peptide 12 containing an N-terminal serine residue, bearing an
acyl phosphate moiety (see Scheme 2 and ESIw). Acyl phosphate
12 proved to be an extremely reactive intermediate which, upon
removal of the N-terminal Cbz-protecting group by hydro-
genolysis, rearranged spontaneously via an intramolecular
O - N acyl shift to afford the N-acetylated phosphopeptide
13. Phosphopeptide 13 was produced in relatively low yield due to
the rapid hydrolysis of 12 under the reaction conditions.
Although not conclusive evidence for the intramolecular mecha-
nism proposed in Scheme 1, this study does provide initial
evidence that such an intermediate can undergo an intramolecular
O - N acyl shift to afford an amide bond in a rapid manner.
Finally, as an additional application of the phosphate-
assisted ligation, we set upon dephosphorylation reactions of
these ligation products to afford unmodified peptides. To this
end, a selection of phosphopeptide ligation products 14–17 were
treated with alkaline phosphatase in 50 mM tris buffer at pH 8.6
to provide dephosphorylated peptide products 18–21 in high
yields in all cases (74–98% isolated yields, Scheme 3). Notably,
the successful implementation of dephosphorylation reactions
significantly expands the utility of the phosphate-assisted
ligation, whereby the phosphate moiety can be introduced
as a traceless ligation auxiliary with a view to incorporating native
serine and threonine residues into target peptides or proteins.
Ser
AsnVal
phosphate group, this study provided initial evidence that the
phosphate moiety was crucial to the efficiency of the ligation
reactions, consistent with our experimental design. Kinetic
studies were subsequently conducted to quantify this increase
in reaction rate (Fig. 1). After 10 h, reactions with 1 and 2
provided a 50% yield of the desired ligation product and
reactions were complete within 48 h. In contrast, unphospho-
rylated peptides 3 and 4 underwent sluggish ligation reactions,
corroborating that the phosphate moiety in 1 and 2 was assisting
the formation of the peptide bond and that these reactions
were not proceeding through a direct aminolysis reaction
alone. Indeed, sampling the reactions at 12 h revealed that the
Fig. 1 Kinetics of the phosphate-assisted peptide ligation between
peptide thioester 5 and peptides 1–4; = 1, = 2, = 3, = 4.
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bond. The method displayed impressive scope for a range of
amino acids at the ligation junction, and, as such, should serve
as a useful tool for the construction of biologically relevant
peptides, phosphopeptides, proteins and phosphoproteins in
future studies. We have conducted preliminary experiments
which suggest that acyl phosphates can undergo rapid acyl
migration to the N-terminal amine of a peptide however, it is
not yet clear that such an intermediate is formed during the
ligation reactions. Further delineation of the mechanism of the
phosphate-assisted ligation will be the focus of future research
in our laboratory. In addition, the phosphate-assisted ligation is
currently being employed in the total synthesis of biologically
relevant phospho- and glycoproteins in our research group.
We thank the Australian Research Council for funding and
the Endeavour Research Fellowship for financial support
(GLT). We would also like to thank Dr Kelvin Picker for
his technical assistance with HPLC and LC-MS analysis.
Notes and references
1 D. E. Kalume, H. Molina and A. Pandey, Curr. Opin. Chem. Biol.,
2003, 7, 64–69.
2 G. Manning, D. B. Whyte, R. Martinez, T. Hunter and
S. Sudarsanam, Science, 2002, 298, 1912–1934.
3 G. K. Toth, Z. Kele and G. Varadi, Curr. Org. Chem., 2007, 11, 409–426.
4 T. J. Attard, N. O’Brien-Simpson and E. C. Reynolds, Int. J. Pept.
Res. Ther., 2007, 13, 447–468.
Scheme 1 Proposed mechanism of phosphate-assisted ligation.
5 B. G. Davis, Chem. Rev., 2002, 102, 579–601.
6 D. P. Gamblin, E. M. Scanlan and B. G. Davis, Chem. Rev., 2009,
109, 131–163.
7 A. Dirksen and P. E. Dawson, Curr. Opin. Chem. Biol., 2008, 12,
760–766.
8 D. Macmillan, Angew. Chem., Int. Ed., 2006, 45, 7668–7672.
9 T. Wieland, E. Bokelmann, L. Bauer, H. U. Lang and H. Lau,
Justus Liebigs Ann. Chem., 1953, 583, 129–149.
10 P. E. Dawson, T. W. Muir, I. Clark-Lewis and S. B. H. Kent,
Science, 1994, 266, 776–779.
11 P. E. Dawson and S. B. H. Kent, Annu. Rev. Biochem., 2000, 69, 923–960.
12 B. L. Nilsson, L. L. Kiessling and R. T. Raines, Org. Lett., 2000, 2,
1939–1941.
Scheme 2 Intramolecular O - N acyl shift of acyl phosphate.
13 E. Saxon, J. I. Armstrong and C. R. Bertozzi, Org. Lett., 2000, 2,
2141–2143.
14 D. Crich and A. Banerjee, J. Am. Chem. Soc., 2007, 129,
10064–10065.
15 C. Haase, H. Rohde and O. Seitz, Angew. Chem., Int. Ed., 2008, 47,
6807–6810.
16 J. Chen, Q. Wan, Y. Yuan and S. J. Danishefsky, Angew. Chem.,
Int. Ed., 2008, 47, 8521–8524.
17 R. Okamoto and Y. Kajihara, Angew. Chem., Int. Ed., 2008, 47,
5402–5406.
18 A. Brik, Y. Y. Yang, S. Ficht and C. H. Wong, J. Am. Chem. Soc.,
2006, 128, 5626–5627.
19 S. Ficht, R. J. Payne, A. Brik and C. H. Wong, Angew. Chem., Int.
Ed., 2007, 46, 5975–5979.
20 R. J. Payne, S. Ficht, S. Tang, A. Brik, Y. Y. Yang, D. A. Case and
C. H. Wong, J. Am. Chem. Soc., 2007, 44, 13527–13536.
21 S. Aimoto, Biopolymers, 1999, 51, 247–265.
22 G. Chen, Q. Wan, Z. Tan, C. Kan, Z. Hua, K. Ranganathan and
S. J. Danishefsky, Angew. Chem., Int. Ed., 2007, 46, 7383–7387.
23 R. J. Payne, S. Ficht, W. A. Greenberg and C.-H. Wong, Angew.
Chem., Int. Ed., 2008, 47, 4411–4415.
24 R. S. Mulvey and A. R. Fersht, Biochemistry, 1978, 17, 5591–5597.
25 L. L. Cameron, S. C. Wang and R. Kluger, J. Am. Chem. Soc.,
2004, 126, 10721–10726.
26 S. Tzvetkova and R. Kluger, J. Am. Chem. Soc., 2007, 129,
15848–15854.
27 J. Wodzinska and R. Kluger, J. Org. Chem., 2008, 73, 4753–4754.
28 D. Hu and R. Kluger, Org. Biomol. Chem., 2008, 6, 151–156.
29 S. Ficht, R. J. Payne, R. T. Guy and C. H. Wong, Chem.–Eur. J.,
2008, 14, 3620–3629.
Scheme 3 Enzymatic dephosphorylation of ligation products.
The generation of unmodified peptides by enzymatic dephos-
phorylation reactions post-ligation therefore provides a direct
disconnection at serine and threonine residues, a novel addition
to the ligation chemistry toolbox.
In summary, we have developed an efficient new method for
the construction of phosphopeptides and peptides. The reaction
utilises the reactivity of an N-terminal phosphoserine or phospho-
threonine residue to facilitate the formation of a native peptide
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This journal is The Royal Society of Chemistry 2009
4262 | Chem. Commun., 2009, 4260–4262