Orthogonal ligation: A three piece assembly of a PNA-peptide-PNA conjugate

Article abstract of DOI:10.1039/b801242c

A PNA-peptide-PNA conjugate was assembled from three fragments using a combination of native chemical ligation and an orthogonal, auxiliary-mediated ligation. The Royal Society of Chemistry.

Full text of DOI:10.1039/b801242c

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Orthogonal ligation: a three piece assembly of a PNA–peptide–PNA
conjugatew
a
b
b
a
´
Fabienne Burlina,* David D. Dixson, Robert P. Doyle, Gerard Chassaing,
Christopher N. Boddy,b Philip Dawsonc and John Offer*d
Received (in Cambridge, UK) 23rd January 2008, Accepted 17th March 2008
First published as an Advance Article on the web 14th April 2008
DOI: 10.1039/b801242c
A PNA–peptide–PNA conjugate was assembled from three
fragments using a combination of native chemical ligation and
an orthogonal, auxiliary-mediated ligation.
bioconjugate construction. The advantages of ligation over
stepwise synthesis on a solid phase or protected fragment
coupling are well known for protein synthesis. These include
greater ease of handling, purification and characterization of
the fragments and producing a homogeneous final material in
a single step that does not require further modification.
Additionally, purification of the final product is simplified
since impurities differ from the product by the deletion of a
whole fragment rather than a single residue. Lastly, no pro-
tecting group manipulations are involved, simplifying synth-
esis and removing the possibility of deprotection artifacts.8
The PNA–peptide–PNA conjugate was a challenging target
for a ligation assembly as it required a strategy for the regio-
controlled assembly of the three fragments. The principal
problem was how to steer each of the PNAs to exclusively
one of the two nucleophilic ligation sites without reintroducing
protecting groups.
We have applied two complementary ligation strategies to
assemble three unprotected fragments (peptide and peptide
nucleic acids (PNAs)). This novel strategy for regioselective
bioconjugate construction will have broad scope in the synth-
esis of defined three component conjugates, as well as libraries
of these bioconjugates.1
The target chosen for the development of this synthetic
approach was a branched PNA–peptide–PNA conjugate
(Fig. 1a). PNAs are oligonucleotide mimics in which the
nucleobases are attached to a polyamide backbone composed
of N-(2-aminoethylglycine) repeats.2 PNAs are promising as
antigene therapeutics. They bind dsDNA with high affinity
and specificity but their utility has been limited by their poor
solubility and cellular uptake and the slow kinetics of dsDNA
strand invasion.2,3 Conjugating PNAs to cell-penetrating
peptides including Penetratin (Pen) has been shown to im-
prove cellular uptake.4,5 We want to explore a novel dsDNA
binding construct for inhibition of gene transcription, a three
component PNA–peptide–PNA conjugate. The major groove
binding peptide Pen,6 which corresponds to helix 3 of the
Antennapedia homeodomain,7 was selected as a platform.
PNAs were appended via amide bonds to the side chains of
lysines located on the N- and C-terminus of Pen, providing a
construct as shown in Fig. 1a. This structure is expected to
favour binding to the major groove without effecting strand
invasion as proposed in Fig. 1b. To improve DNA binding of
the construct, libraries of conjugates will have to be screened.
We thus needed a rapid construction method for accessing
these libraries. This necessitated the development of a regio-
selective convergent ligation approach for installing the PNAs
onto the peptide scaffold.
A three piece ligation strategy based on the consecutive use of
two amide bond ligations, native chemical ligation (NCL)8 and
an auxiliary-mediated ligation (Scheme 1), was envisioned to
regioselectively attach the PNA sequences to the Pen peptide.
NCL is best known for its application to the synthesis of
proteins but it has also been applied to biopolymers including
oligonucleotides9 and PNAs.10 Earlier we disclosed an auxiliary-
mediated peptide ligation effective for the synthesis of proteins
termed extended ligation (EL).11 This ligation uses the 4,5,6-
trimethoxy-2-mercaptobenzyl (Tmb) auxiliary, which functions
analogously to cysteine in NCL, combining thioester exchange
with efficient intramolecular acyl transfer to the Na-terminus of
the peptide. The Tmb group and closely related auxiliaries have
been successfully applied to the synthesis of peptides and
glycopeptides.11–13 We previously reported that cysteine (NCL)
and Tmb-mediated ligation (EL) of two peptides require differ-
ent buffer conditions to proceed efficiently. In particular, EL is
In addition to enabling rapid library construction, a con-
vergent ligation approach provides numerous advantages for
a UPMC Univ Paris 06, CNRS UMR 7613, 4 place Jussieu, F-75005
Paris, France. E-mail: fabienne.burlina@upmc.fr
b Department of Chemistry, Syracuse University, Syracuse, NY 13244,
USA
c The Skaggs Institute for Chemical Biology, The Scripps Research
Institute, 10550 North Torrey Pines Road, La Jolla, California
92037, USA
d Department of Biochemistry, University of Oxford, South Parks
Road, Oxford, UK OX1 3QU. E-mail: john.offer@bioch.ox.ac.uk
w Electronic supplementary information (ESI) available: Experimental
protocols and product characterization. See DOI: 10.1039/b801242c
Fig. 1 (a) Structure of the conjugate. (b) Targeted binding to DNA.
Chem. Commun., 2008, 2785–2787 | 2785
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Scheme 2
tion (C4 column). When the PNA thioester was mixed with the
TmbPen peptide under identical conditions, no conjugate was
detected, even overnight. This observation confirms that EL
can be completely suppressed under standard NCL conditions.
The conditions for coupling of PNA thioester to Tmb-
containing peptides were then investigated. No peptide–PNA
conjugate could be detected under conditions known to give
fast and complete ligation of peptide–MPAL thioesters with
peptides containing a N-terminal Tmb (i.e. phosphate buffer pH
7 containing 6 M GdÁHCl and tris(2-carboxyethyl)phosphine
(TCEP)).11 Ligation only occurred when the pH was raised to
7.5 and thiophenol was added to form the more reactive PNA
thiophenol thioester species in situ.16 Using these reaction
conditions and with peptide and PNA concentrations of 7.5
mM, the reaction was completed within 36 h, giving the
expected PNA–peptide conjugate (see ESIw). The product was
isolated with 45% yield after HPLC purification. To better
define the parameters that control the Tmb-mediated ligation of
PNA thioester, we repeated the experiment in presence of
benzylmercaptan. This resulted in a decrease in the ligation
rate. This result is in contrast to peptide–peptide extended
ligations where benzylmercaptan alone completely suppressed
the ligation reaction.11 Thus, the combination of both alkylthiol
addition and lower pH is necessary to suppress the ligation of
PNA thioesters to N-terminal Tmb-containing peptides.
Scheme 1 Proposed mechanism of NCL and EL.
suppressed by addition of alkylthiols.11,14 This initial observa-
tion suggested that conditions for the regio-controlled ligation of
a PNA thioester to the Cys of a peptide also containing Tmb
would be possible in the presence of alkylthiols, as the nucleo-
philicity of the Tmb piece would be masked.
To investigate conditions for NCL and EL of PNAs to the
Pen peptide, three Pen derivatives (Pen peptide is Penetratin
without modifications) and two model PNA thioesters were
synthesized (Table 1). The Pen peptides were constructed using
solid phase stepwise synthesis and the Tmb group was installed
by coupling a Boc derivatized Tmb-Gly monomer to the lysine
side chain. The synthesis of the monomer is shown in
Scheme 2. The PNA thioesters were synthesized using Boc
protected PNA monomers on solid phase with in situ neutra-
lization on a mercaptopropionic acid leucine (MPAL) resin15
and cleaved with HF to give the MPAL thioester derivatives.
All components were characterized by electrospray or
MALDI-TOF mass spectrometry (see ESIw).
Ligation of T4 PNA to PenCys was efficient under standard
conditions for NCL (i.e. 1 mM of both fragments in phosphate
buffer pH 7 containing 6 M guanidine hydrochloride (GdÁ
HCl), 2% thiophenol and 2% benzylmercaptan).8 The half-
life for the reaction was estimated at 3 h, with no starting
material observed after overnight reaction (see ESIw). The
conjugate was isolated with 58% yield after HPLC purifica-
With orthogonal conditions identified for the attachment of
PNA thioesters to either a Cys or Tmb, the construction of the
target PNA–peptide–PNA conjugate was attempted (Fig. 2).
The first PNA fragment (T4) was directed to the Cys ligation
site of the TmbPenCys peptide by performing the reaction at
pH 7 in presence of benzylmercaptan (2%). After 12 h, more
than 90% of the peptide had been converted to the expected
PNA–peptide conjugate (Fig. 2b, compound 6). No trace of
double conjugation of the PNA on the peptide was detected by
HPLC or mass spectrometry. After thiol removal by single
pass preparative HPLC (compound 6 isolated with 55%
yield), the second PNA thioester (CT4) was ligated onto the
N-terminal Tmb-Gly of the conjugate in the absence of
benzylmercaptan at pH 7.5. The reaction was completed
within 2 days (greater than 90% conversion of compound 6
into product 7*, Fig. 2b). The conjugate was purified by
HPLC (41% isolated) and the Tmb auxiliary was quantita-
tively removed under acidic conditions (see ESIw).11
Table 1 Peptide and PNA sequences
Name
Sequence
PenCys
TmbPen
TmbPenCys
T4
KRQIKIWFQNRRMKWKK[Ne-C]a
K[Ne-TmbG]RQIKIWFQNRRMKWKKa
K[Ne-TmbG]RQIKIWFQNRRMKWKK[Ne-C]a
(aeg.T)4-MPALb
C T4
aeg.C(aeg.T)4-MPALb
a
Cys and/or Tmb-Gly are linked via an isopeptide bond to the side-
b
chain of the lysine residue. N-(2-aminoethyl)-glycine PNA unit
functionalized by thymine (aeg.T) or cytosine (aeg.C).
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position of choice. This will now be used to define the best
orientation of the PNAs within the conjugate for binding to
DNA. Modified residues can also be introduced in the peptide
and PNA domains to improve binding. We are currently
studying extension of the three piece ligation strategy to the
synthesis of proteins and glycoproteins.
We would like to thank the Groupe Franc¸ ais des Peptides et
Proteines for financial support and Prof. O. Lequin for his
´
assistance in the design of the conjugate.
Notes and references
1. D. W. Romanini and M. B. Francis, Bioconjugate Chem., 2008, 19,
153.
´
2. A. Ray and B. Norden, FASEB J., 2000, 14, 1041; G. Wang and
X. S. Xu, Cell Res., 2004, 14, 111.
3. B. A. Armitage, Drug Discovery Today, 2003, 8, 222.
´ ´ ´ ´
4. J. J. Dıaz-Mochon, L. Bialy, J. Watson, R. M. Sanchez-Martın
and M. Bradley, Chem. Commun., 2005, 3316.
5. A. Joliot and A. Prochiantz, Adv. Drug Delivery Rev., 2008, 60,
608.
6. The affinity of Pen for a DNA duplex containing the target
TAATNN sequence is about 2 orders of magnitude lower than
that of the Antennapedia homeodomain (Y. H. Shim,
P. B. Arimondo, A. Laigle, A. Garbesi and S. Lavielle, Org.
Biomol. Chem., 2004, 2, 915). Stabilizing the structure of peptides
based on helix 3 of Engrailed for the design of miniature home-
odomains greatly increased affinity for DNA (J. K. Montclare and
A. Shepartz, J. Am. Chem. Soc., 2003, 125, 3416; L. Guerrero,
O. S. Smart, A. Woolley and R. K. Allemann, J. Am. Chem. Soc.,
2005, 127, 15624).
7. E. Fraenkel and C. O. Pabo, Nat. Struct. Biol., 1998, 5, 692.
8. P. E. Dawson, T. W. Muir, I. Clark-Lewis and S. B. H. Kent,
Science, 1994, 266, 776; P. E. Dawson, M. Churchill,
M. R. Ghadiri and S. B. H. Kent, J. Am. Chem. Soc., 1997, 119,
4325.
Fig. 2 (a) Assembly of the PNA–peptide–PNA conjugate using
orthogonal ligations. (b) Analytical HPLC of the reaction time course.
Top: Cys-mediated ligation (RP-C4 column). Bottom: Tmb-mediated
ligation (RP-C18 column), 7* corresponds to product 7 before Tmb
removal.
In the present study we have documented the unexpectedly
low reactivity of PNA thioesters toward Cys and Tmb-
mediated ligation reactions. The rate limiting step of ligation
is thioester exchange.8 The reaction rate is slowed considerably
as the C-terminal thioester becomes more sterically
hindered.11–13,15 While the PNA thioester possesses the steri-
cally accessible C-terminal glycine residue, its reactivity is
closer to sterically hindered C-terminal b-branched residue
thioesters. The slow rate of PNA thioester exchange may be
due to the constraints induced by the presence of a tertiary
amide bond in the PNA backbone.
9. D. A. Stetsenko and M. J. Gait, J. Org. Chem., 2000, 65, 4900.
10. M. C. de Koning, D. V. Filippov, N. Meeuwenoord,
M. Overhand, G. A. van der Marel and J. H. van Boom, Synlett,
2001, 10, 1516; A. Mattes and O. Steiz, Chem. Commun., 2001,
2050.
11. J. Offer and P. E. Dawson, Org. Lett., 2000, 2, 23; J. Offer, C. N.
C. Boddy and P. E. Dawson, J. Am. Chem. Soc., 2002, 124, 4642.
12. D. Macmillan and D. W. Anderson, Org. Lett., 2004, 6, 4659;
B. Wu, J. Chen, J. D. Warren, G. Chen, Z. Hua and
S. J. Danishefsky, Angew. Chem., Int. Ed., 2006, 45, 4116;
J. Chen, G. Chen, B. Wu, Q. Wan, Z. Tan, Z. Hua and
S. J. Danishefsky, Tetrahedron Lett., 2006, 47, 8013;
T. Kawakami, K. Akaji and S. Aimoto, Org. Lett., 2001, 9,
1403; C. Haase and O. Seitz, Angew. Chem., Int. Ed., 2008, 47,
1553.
13. S. Tchertchian, O. Hartley and P. Botti, J. Org. Chem., 2004, 69,
9208; V. M. Cardona, O. Hartley and P. Botti, J. Pept. Res., 2003,
61, 152; D. W. Low, M. G. Hill, M. R. Carrasco, S. B. Kent and
P. Botti, Proc. Natl. Acad. Sci. U. S. A., 2001, 98, 6554.
14. NCL is also suppressed by alkyl thiols but a much higher
concentration is required to abolish ligation.
The central tenet of chemical ligation, which gives it such
great practical value, is the omission of protecting groups.
Their absence increases peptide solubility in aqueous solution,
eliminates deprotection steps, the requirement of the target to
be stable to deprotection and simplifies characterisation. How-
ever, when multiple fragment couplings by NCL are required
the reintroduction of protecting groups has proved neces-
sary.17 Combining NCL with an orthogonal EL obviates
cysteine protection and confers greater synthetic flexibility.
This study demonstrates that by minor alterations to the
conditions (pH, thiol composition and fragment concentra-
tion) a thioester can be steered selectively towards a cysteine in
the presence of Tmb. This was used to realize the sequential
assembly of three unprotected fragments through amide
bonds, obviating the need for ligation compatible protecting
groups. Products showed excellent purity by HPLC and mass
spectrometry. The three piece ligation strategy reported here
appears to be particularly well adapted to the constitution of a
small library of PNA–peptide–PNA conjugates; different C- or
N-terminal PNA thioesters can simply be introduced at the
15. T. M. Hackeng, J. H. Griffin and P. E. Dawson, Proc. Natl. Acad.
Sci. U. S. A., 1999, 96, 10068.
16. Above pH 7.5 the hydrolysis of thiophenol thioesters becomes a
serious competing reaction. Alkyl thioesters are moderately stable
up to pH 8.5 over the lifetime of a ligation reaction (24 h).
However ligations are usually performed at pH 7.0 to prevent
complications such as epimerization of the C-terminal residue.
17. D. Bang and S. B. Kent, Angew. Chem., Int. Ed., 2004, 43, 2534;
D. Bang, B. L. Pentelute and S. B. Kent, Angew. Chem., Int. Ed.,
2006, 45, 3985.
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