Novel phosphate derivatives as scaffolds for the preparation of synthetic phosphoserine-based peptides using the Fmoc/ t -Bu solid-phase strategy

Article abstract of DOI:10.1055/s-0031-1290119

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. Georg Thieme Verlag Stuttgart - New York.

Full text of DOI:10.1055/s-0031-1290119

290
LETTER
Novel Phosphate Derivatives as Scaffolds for the Preparation of Synthetic
Phosphoserine-Based Peptides Using the Fmoc/t-Bu Solid-Phase Strategy
Phosphate
D
eriva
t
g
ive
s
as Scaff
o
olds for Phos
s
phoserine
t
-Base
d
i
Peptid
n
es o Cilibrizzi, Albert Isidro-Llobet, Natalia Mateu, Warren R. J. D. Galloway, David R. Spring*
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK
Fax +44(1223)336362; E-mail: spring@ch.cam.ac.uk
Received 20 October 2011
(AIP-III), a naturally occurring molecule involved in the
Abstract: Synthetic peptides incorporating analogues of phospho-
serine are valuable tools for the study of protein kinases and phos-
phatases. In addition, derivatives of naturally occurring peptides
incorporating phosphate groups may have interesting biological
regulation of quorum sensing in the Gram positive bacte-
rium Staphylococcus aureus (Figure 1).10 Non-natural an-
alogues of AIPs hold significant value as chemical probes
properties. Herein we describe a new Fmoc/t-Bu solid-phase pep- for studies on this intercellular signaling process and the
tide synthesis (SPPS) strategy for the convenient generation of
delineation of structure-activity relationships may allow
phosphoserine-based peptides. A proof-of-concept synthesis that
the rational development of antagonists which could po-
demonstrates the feasibility of this approach is presented.
tentially be used in a therapeutic context.10a
Key words: phosphopeptide, serine, phosphorus, solid-phase syn-
thesis, amino acids
O
Protein phosphorylation and dephosphorylation reactions
are reversible processes, catalysed by kinases and phos-
O
N
H
NH2
O
S
HN
O
phatases respectively, which play a key role in the regula-
tion of cell function, growth and homeostasis.1–3 Many
diseases, including cancer, result from perturbations in the
activity, or level of expression, of these enzymes.4 Conse-
quently the study of how nature orchestrates these oppos-
ing processes is a subject of considerable interest.1,3
Synthetic peptides incorporating phosphorylated amino
acid residues and their analogues (so-called phosphopep-
tides) have proven to be valuable tools for such investiga-
tions.1,5–7 Furthermore derivatives of naturally occurring
peptides incorporating phosphorylated amino acid resi-
dues (including serines) may have other interesting, po-
tentially useful, biological properties.8
O
O
H
N
N
H
N
H
O
NH
O
O
HO
H2N
auto-inducing peptide III (AIP-III)
O
HN
OH
O
O
P
O
H2N
H2N
O
O
O
In most cells, a large proportion of total protein phospho-
rylation occurs on serine residues; as such, there has been
significant effort directed towards the development of
methods for the incorporation of phosphoserine-based
residues into artificial peptide chains.3,9 In this context,
the 9-fluorenylmethoxycarbonyl/tert-butyl (Fmoc/t-Bu)
solid-phase peptide synthesis (SPPS) strategy has proven
to be especially valuable.3,5,6,9 However, there are chal-
lenges associated with phosphopeptide synthesis using
Fmoc/t-Bu SPPS and improvements in speed and efficien-
cy as well as the complexity and diversity of the resulting
phosphopeptides are desirable.3 Towards this end we re-
port here the development of a novel Fmoc/t-Bu SPPS
strategy suitable for the efficient generation of structurally
diverse phosphoserine-based peptides. As a proof-of-
concept this approach was applied to the synthesis of 1, a
phosphorylated derivative of auto-inducing peptide III
NH2
N
H
O
NH
HN
HO
OH
N
H
O
O
phosphoserine-based
derivative of AIP-III
O
1
Figure 1 Structure of AIP-III and the phosphoserine-based deriva-
tive 1
The SPPS of serine phosphopeptides via the on-resin
phosphorylation of a protected peptide prepared using the
standard Fmoc/t-Bu-strategy is known to suffer from a
number of drawbacks.11 As such it has been argued that
Fmoc/t-Bu SPPS methods which directly incorporate a
serine phosphate equivalent are more desirable.9 Towards
this end we decided to adopt a synthetic strategy involving
novel tetra-protected phosphoserine analogues of the gen-
eral form 2, which present three levels of orthogonality
(Scheme 1).12 We envisaged compounds of this sort could
SYNLETT 2012, 23, 290–294
x
x.
x
x.
2
0
1
1
Advanced online publication: 22.12.2012
DOI: 10.1055/s-0031-1290119; Art ID: D57911ST
© Georg Thieme Verlag Stuttgart · New York

LETTER
Phosphate Derivatives as Scaffolds for Phosphoserine-Based Peptides
291
be prepared efficiently using solution-phase methods and quired. However, Fmoc/t-Bu SPPS methods employing
then loaded onto the solid phase, whereupon they could fully protected serine phosphotriester residues are known
serve as scaffolds around which amino acid residues could to be troublesome due to the propensity of the phosphate
be installed using standard Fmoc/t-Bu SPPS techniques, functionality to undergo b-elimination under the basic
thus providing a convenient method for the generation of conditions required for Fmoc group removal.8,13 Thus we
phosphoserine-based peptides.
decided to include a third base-labile cyanoethyl protect-
ing group on the phosphoserine analogues 2 which would
only be lost after based-induced Fmoc removal, allowing
the solid-phase synthesis of the remaining phosphoserine
peptide using Fmoc-based chemistry.13
It was anticipated that 2 could be loaded onto a solid-
phase resin after removal of the t-Bu group and reaction
of the carboxylic acid group so liberated. Standard remov-
al of the Fmoc protecting group under basic conditions
should reveal the amino group thus allowing the peptide We envisaged that 2a and 2b could each be accessed from
chain to be extended at one end of the phosphate group.12 three building blocks; commercially available 2-cyanoet-
The presence of the Alloc-protected amino alcohol would hyl-N,N-diisopropyl-chlorophosphoramidite (3), Fmoc-L-
provide a synthetic handle for further derivatisation of the Ser-O-t-Bu (4) and either Alloc-ethanolamine (5) or
peptide (after the peptide chain had been ‘capped’ with an Alloc-L-leucinol (6; Scheme 2).
orthogonally protected Boc-amino acid) potentially al-
lowing access to a diverse range of products. For example,
amino acids could be selectively introduced at the other
side of the phosphate group to generate novel ‘branched’
phosphoserine-based peptides. In order to facilitate the so-
lution-phase synthesis of the phosphoserine analogues 2
(vide infra) it was expected that the presence of an addi-
tional protecting group on the phosphorous would be re-
Alcohols 5 and 6 were readily obtained in one and two
steps, respectively, from commercial sources (see Sup-
porting Information). Subsequent coupling with 3 fur-
nished intermediates 7a and 7b in high yields (Scheme 3).
Reaction with Fmoc-L-Ser-Ot-Bu ester 4 (readily ob-
tained in quantitative yield from Fmoc-L-Ser-OH, see
Supporting Information) was performed in the presence of
CN
O
CN
H
O
P
P
N
1. deprotection
2. loading onto
solid support
O
O
Alloc
H
N
H
H
N
O
N
R
Fmoc
O
O
Alloc
Fmoc
O
R
H2N AA (AA)x
O
O
O
N
AA (AA)x
H
R = H, 2a; i-Bu, 2b
deprotection
OH
OH
H
N
H2N
(AA)y
AA
O
P
H
N
O
O
Alloc
P
O
O
O
Alloc
HN
O
O
R
chain extension
H2N
R
(AA)x
N
AA
O
H
N
AA
(AA)x
H
1. capping
2. deprotection
OH
OH
H
BocHN
(AA)y+1
AA
NH2
(AA)z
O
P
N
AA
BocHN
(AA)y+1
AA
O
P
NH2
O
O
O
O
O
O
HN
O
O
R
chain extension
HN
O
R
(AA)x
N
AA
(AA)x
N
AA
H
H
1. resin cleavage
2. deprotection
advantages
OH
P
• rapid approach using Fmoc/t-Bu-SPPS
H
N
H
H2N
(AA)y+1
NH2
R = H or i-Bu
AA
(AA)z
AA
N
O
O
• amino acids can be installed selectively
at either side of phosphate group
O
O
R
O
HO
O
HN
AA
(AA)x
• potential for diverse range of products
including linear and branched variants
O
phosphoserine-based peptides
Scheme 1 Overview of the strategy for the generation of phosphoserine-based peptides using phosphoserine analogues 2a and 2b as scaffolds
around which amino acid residues can be installed using Fmoc/t-Bu SPPS. AA = amino acid (unspecified); x and y refer to an unspecified num-
ber of amino acid residues. Alloc = allyloxycarbonyl. The phosphoserine portion of the final product is highlighted in red.
© Thieme Stuttgart · New York
Synlett 2012, 23, 290–294

292
A. Cilibrizzi et al.
LETTER
benzotriazol-1-yloxy)tripyrrolidinophosphonium hexa-
fluorophosphate (PyAOP) and DIPEA furnished the
desired adduct 12. Base-induced Fmoc removal yielded
the free amine derivative 13 with concomitant cleavage of
the cyanoethyl protecting group observed as desired.19
Subsequent chain extension using Fmoc-L-Asn-OH and
Boc-L-IIe-OH was achieved by a standard Fmoc/t-Bu
SPPS approach to furnish the phosphopeptide analogue
14.17 With this derivative in hand we next investigated the
introduction of the additional amino acids at the other side
of the phosphate group. The Alloc protecting group was
readily removed using Pd(0) and the resulting free amine
15 was coupled with Alloc-L-Leu-OH to give 16. Alloc
group removal followed by chemoselective acidolysis of
the 2-chlorotrityl ester resin linkage using a TFA–Et3SiH
mix then afforded the partially protected hexapeptide 17.
Partial purification was accomplished by filtration of the
acidolytic resin suspension into pyridine–MeOH and
evaporation of the filtrate to dryness under reduced pres-
sure. The desired phosphopeptide-based derivative 1 was
obtained by treatment of 17 with HCl–dioxane to remove
the remaining t-Bu and BOC protecting groups.20
CN
R
O
H
H
N
P
N
Fmoc
O
O
Alloc
O
t-BuO
O
R = H, 2a; i-Bu, 2b
CN
O
O
P
H
H
N
N
Fmoc
Ot-Bu Alloc
OH
N
Cl
R
HO
3
Fmoc-L-Ser-Ot-Bu, 4
R = H, 5; i-Bu, 6
Scheme 2 Three building blocks used in the synthesis of 2a and 2b
tetrazole. The obtained phosphite intermediates 8a and 8b
were oxidised in situ with tert-butyl hydroperoxide (t-
BuOOH) to furnish the desired fully protected phospho-
serine analogues 2a and 2b. Subsequent removal of the t-
Bu group under acidic conditions proceeded smoothly to
generate the free acid derivatives 9a and 9b. We were next
interested in exploring the suitability of compounds of this
sort for the preparation of synthetic phosphopeptides us-
ing Fmoc/t-Bu SPPS. In line with our groups continued
interest in quorum sensing14 we targeted the synthesis of
1, a phosphorylated derivative of naturally occurring AIP-
III (Scheme 4). This molecule is known to be involved in
the regulation of intercellular communication in the Gram
positive bacterium S. aureus and non-natural analogues
are of value for chemical biology studies on this system.
In conclusion, we have described a novel strategy for the
synthesis of synthetic phosphoserine-based peptides using
phosphate derivatives 2a and 2b as molecular scaffolds
around which amino acid residues can be installed by
Fmoc/t-Bu SPPS. Proof-of-concept work demonstrated
the feasibility of this approach; compound 1, a novel
phosphoserine-based derivative of the quorum sensing
signaling molecule AIP-III was accessed in an efficient
and straightforward fashion from readily accessible start-
ing materials. The ability of 1 and related derivatives to
modulate bacterial quorum sensing systems is currently
being investigated and results will be presented in due
course.21 Overall the strategy described in this report
potentially offers a general and convenient means of
accessing a structurally diverse range of synthetic phos-
phoserine-based peptides, including both linear and
branched derivatives. As such, it represents an attractive
alternative method for the synthesis of this important class
of biological tool compounds, with potentially broad ap-
plications in both target-oriented and diversity-oriented
synthesis.22
SPPS started with the limited incorporation of Fmoc-L-
Phe-OH on 2-chlorotrityl chloride polystyrene resin (2-
CTC; 10, Scheme 4) in order to have a loading of 0.7
mmol/g.15 The remaining resin chloride functions were
capped with methanol to prevent the formation of tertiary
amines after the piperidine treatment used to remove the
Fmoc group. Fmoc-L-Asp(t-Bu)-OH was then coupled
onto the solid phase using N,N-diisopropylcarbodiimide
(DIC) and ethylcyanoglyoxylate-2-oxime to minimise
epimerisation16 generating derivative 11.17,18 Attempts to
append phosphoserine analogue 9a using these coupling
conditions failed (Scheme 2); instead the use of (7-aza-
CN
O
CN
CN
5 or 6
DIPEA
4
H
N
H
O
P
O
P
P
N
tetrazole
H
Fmoc
O
O
Alloc
N
CH2Cl2, r.t.
R = H, 83%
R = i-Bu, 90%
N
O
Alloc
THF, r.t.
N
Cl
R
O
O
R
3
R = H, 7a; i-Bu, 7b
R = H, 8a; i-Bu, 8b
CN
CN
O
O
TFA
t-BuOOH
H
N
H
H
H
P
N
N
P
N
THF, r.t.
R = H, 57%
R = i-Bu, 20%
Fmoc
O
O
Alloc
Fmoc
O
O
Alloc
CH2Cl2
O
O
R = H, 97%
R = i-Bu, 97%
R
R
O
O
HO
O
R = H, 2a; i-Bu, 2b
R = H, 9a; i-Bu, 9b
Scheme 3 Synthesis of phosphoserine analogues 2a and 2b and the free acid analogues 9a and 9b
Synlett 2012, 23, 290–294
© Thieme Stuttgart · New York

LETTER
Phosphate Derivatives as Scaffolds for Phosphoserine-Based Peptides
293
i) Fmoc-L-Phe-OH,
DIPEA,CH2Cl2
ii) piperidine–DMF(1:4, v/v)
9a
PyAOP
DIPEA
poly-
styrene
Fmoc-Ser-Asp(t-Bu)-Phe
O
Cl
H-Asp(t-Bu)-Phe O
Cl
Cl
iii) Fmoc-L-Asp-(t-Bu)-OH
ethylcyanoglyoxylate-2-
oxime, DIC, DMF
DMF
O
P
O
11
12
O
iv) piperidine–DMF (1:4, v/v)
NC
N
Alloc
2-CTC (10)
H
i) Fmoc-L-Asn-OH
ethylcyanoglyoxylate-2-oxime
DIC, DMF
ii) piperidine–DMF (1:4, v/v)
H-Ser-Asp(t-Bu)-Phe
O
Pd(PPh3)4
PhSiH3
Boc-Ile-Asn-Ser-Asp(t-Bu)-Phe O
piperidine–DMF
(1:4, v/v)
HO
P
O
HO
P
O
13
14
iii) Boc-L-Ile-OH
ethylcyanoglyoxylate-2-oxime
DIC, DMF
CH2Cl2
O
O
N
Alloc
N
H
Alloc
H
i) Pd(PPh3)4
PhSiH3, CH2Cl2
Boc-Ile-Asn-Ser-Asp(t-Bu)-Phe O
Boc-Ile-Asn-Ser-Asp(t-Bu)-Phe O
Alloc-L-Leu-OH
HO
P
O
HO
P
O
15
16
ethylcyanoglyoxylate-2-oxime
DIC, DMF
ii) TFA, Et3SiH, CH2Cl2
O
O
N
Leu Alloc
NH2
H
O
O
HO
O
Boc-Ile-Asn-Ser-Asp(t-Bu)-Phe OH
H-Ile-Asn-Ser-Asp-Phe OH
H
HCl
N
HO
P
O
HO
P
O
NH
HN
OH
17
H
N
dioxane
NH2
O
O
O
O
N
Leu H
N Leu H
H
H
O
O
H2N
O
O
P
O
H2N
HN
1
OH
O
Scheme 4 Synthesis of branched phosphoserine-based peptide 1. The phosphoserine potion of the final compound is highlighted in red.
(8) (a) For a recent discussion of the use of peptides and peptide
derivative as therapeutic agents, see: Mason, J. M. Future
Med. Chem. 2010, 2, 1813. (b) For a recent general review
of the use of small molecules as tools to study biological
systems, see: O’Connor, C. J.; Laraia, L.; Spring, D. R.
Chem. Soc. Rev. 2011, 40, 4332.
(9) Shapiro, G.; Büchler, D.; Dalvit, C.; Frey, P.; Fernandez,
Md. C.; Gomez-Lor, B.; Pombo-Villar, E.; Stauss, U.;
Swoboda, R.; Waridel, C. Bioorg. Med. Chem. 1997, 5, 147.
(10) (a) Gorske, B. C.; Blackwell, H. E. Org. Biomol. Chem.
2006, 4, 1441. (b) Lyon, G. J.; Wright, J. S.; Muir, T. W.;
Novick, R. P. Biochemistry 2002, 41, 10095.
(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).
(12) (a) Barany, G.; Albericio, F. J. Am. Chem. Soc. 1985, 107,
4936. (b) Isidro-Llobet, A.; Alvarez, M.; Albericio, F.
Chem. Rev. 2009, 109, 2455.
(13) (a) 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 b-elimination, thus preventing it. See:
McMurray, J. S.; Coleman, D. R. IV; Wang, W.; Campbell,
M. L. Biopolymers (Pept. Sci.) 2001, 60, 3. (b) 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).
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synlett.
References and Notes
(1) de bont, D. B. A.; Moree, W. J.; Vanboom, J. H.; Liskamp,
R. M. J. 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, J. W. Synthesis of
Phosphopeptides, In Synthesis of Peptides and
Peptidomimetics, Houben-Weyl Methods in Organic
Chemistry, Volume E22b; Thieme: Stuttgart, 2004, 375–
424.
(4) Singer, D.; Hoffman, R. In Peptide-Based Drug Design,
Methods in Molecular Biology, Vol. 494; Otvos, L., Ed.;
Humana Press: Totowa (NJ / USA), 2008, 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.
© Thieme Stuttgart · New York
Synlett 2012, 23, 290–294

294
A. Cilibrizzi et al.
LETTER
(18) t-Bu and Boc were chosen for either carboxylic (11) or
(14) (a) Galloway, W. R. J. D.; Hodgkinson, J. T.; Bowden, S. D.;
Welch, M.; Spring, D. R. Chem. Rev. 2011, 11, 28.
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).
(b) Hodgkinson, J. T.; Galloway, W. R. J. D.; Casoli, M.;
Keane, H.; Su, X.; Welch, M.; Spring, D. R. Tetrahedron
Lett. 2011, 52, 3291. (c) Hodgkinson, J. T.; Galloway, W. R.
J. D.; Saraf, S.; Baxendale, I. R.; Ley, S. V.; Ladlow, M.;
Welch, M.; Spring, D. R. Org. Biomol. Chem. 2011, 9, 57.
(d) Hodgkinson, J. T.; Bowden, S. D.; Galloway, W. R. J. D.;
Spring, D. R.; Welch, M. J. Bacteriol. 2010, 192, 3833.
(15) 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:
(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 b-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.
(a) Benz, H. Synthesis 1994, 337. (b) Barlos, K.; Chatzi, O.;
Gatos, D.; Stavropoulos, G. Int. J. Pept. Protein Res. 1991,
37, 513. (c) An additional advantage of the 2-CTC resin is
that its hindered structure minimises the formation of
diketopiperazines during removal of the temporary
(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
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.
(17) (a) 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. (b) 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.
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.
(22) For recent reviews on diversity-oriented synthesis, see:
(a) Galloway, W. R. J. D.; Isidro-Llobet, A.; Spring, D. R.
Nat. Commun. 2010, 1, 80. (b) Dandapani, S.; Marcaurelle,
L. A. Curr. Opin. Chem. Biol. 2010, 14, 362. (c) Schreiber,
S. L. Nature (London) 2009, 457, 153. (d) Nielsen, E.;
Schreiber, S. L. Angew. Chem. Int. Ed. 2008, 47, 48.
(e) Galloway, W. R. J. D.; Bender, A.; Welch, M.; Spring,
D. R. Chem. Commun. 2009, 2446.
Synlett 2012, 23, 290–294
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