Simplifying native chemical ligation with an N-acylsulfonamide linker

Article abstract of DOI:10.1039/c2cc15911b

We report a simplified procedure for the chemical ligation of peptides by using the sulfamylbutyryl linker as a mildly activating group capable of participating in ligation. When the peptidyl N-methylsulfonamide is directly added with excess thiols to lig

Full text of DOI:10.1039/c2cc15911b

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Simplifying native chemical ligation with an N-acylsulfonamide linkerw
Fabienne Burlina,a Caroline Morris,b Raymond Behrendt,c Peter White*d and John Offer*b
Received 22nd September 2011, Accepted 11th January 2012
DOI: 10.1039/c2cc15911b
We report a simplified procedure for the chemical ligation of
peptides by using the sulfamylbutyryl linker as a mildly activating
group capable of participating in ligation. When the peptidyl
N-methylsulfonamide is directly added with excess thiols to ligation
reactions, the speed of reaction is comparable to native chemical
ligation.
thioesters10 glycoproteins11 and phosphoproteins.6,12,13 However,
the thiolysis step can sometimes be problematic because of poor
solvation of the resin bound peptide and difficulties with peptide
recovery from DMF.
We wanted to explore the ligation properties of N-peptidyl-
N-alkylsulfonamides because, although poorly reactive with
nucleophiles14 we considered that for both stability during
purification and chemoselectivity a weakly activating group
was desirable. The reactivity could theoretically be modulated
by the addition of thiols to the ligation milieu. The possibility
of unprotected N-peptidyl-N-alkylsulfonamides participating
directly in ligation would expand the applicability of NCL and
remove the thioester conversion steps (Scheme 1). Unprotected
N-peptidyl-N-alkylsulfonamides are easy to access by Fmoc
SPPS by scalable, robust procedures and they are very stable
compounds.
The use of native chemical ligation (NCL) has revolutionized
the synthesis and semi-synthesis of small proteins.1 NCL is
the coupling of two unprotected peptides, one having an
N-terminal cysteine and the other a C-terminal thioester to
give an amide bond at the ligation site. The mildly activated
peptide thioesters are generally synthesized by the Boc/benzyl
method using in situ neutralization protocols.2,3 However, the
requirement for anhydrous HF and the increased difficulties in
obtaining HF have driven chemists to look for alternatives.4,5
Furthermore, demand for phosphorylated or glycosylated
peptide thioesters or other acid-sensitive modifications that
preclude the use of HF has led to increased activity in this
search.6 The problem of developing a robust Fmoc thioester
method is part of the broader problem of synthesising a
C-terminal active ester peptide using Fmoc SPPS. The depro-
tection of the Fmoc group with base at each cycle is not
compatible with an active ester at the C-terminus. Many
ingenious approaches have been developed to generate the
required thioester peptide.4,5,7 However, the most popular
has been to use an N-acylsulfonamide as a base and acid
stable (safety-catch) linker for peptide synthesis. Alkylation
of the sulfonamide after peptide assembly makes the linker
labile to cleavage with nucleophiles.8 The first application of
this approach to peptide thioester preparation was described
by Pessi and co-workers,9 wherein, following activation, the
fully-protected peptide was cleaved from the resin by sodium
thiophenolate in DMF. The resulting protected peptide
thioester was subsequently treated with acid to remove side-chain
protection. The use of the sulfonamide linker has allowed the
synthesis of several impressive targets, including long peptide
To prepare peptide N-alkylsulfonamides the sulfamylbutyryl
linker was attached to an acid-labile resin, in this case
Sieber amide resin. Acid cleavage after alkylation would give
the N-alkylsulfonamide derivatised peptide (Scheme 2). An
additional advantage, previously reported, is that it allows
the monitoring of the alkylation state of the sulfonamide by
LC-MS on a small portion of resin.15 In our case, however,
the principle consideration for using a double linker strategy was
a UPMC Univ Paris 06, CNRS, ENS, UMR 7203 Laboratoire des
´
Biomolecules, 4 place Jussieu, 75005 Paris, France
b Division of Physical Biochemistry, MRC National Institute for
Medical Research, Mill Hill, London, NW7 1AA, UK.
E-mail: joffer@nimr.mrc.ac.uk; Tel: +44 2088162082
c Merck & Cie, Im Laternenacker 5, 8200 Schaffhausen, Switzerland
d Merck Chemicals, Padge Road, Beeston, Notts, NG9 2JR UK.
E-mail: peter.white@merckgroup.com
Scheme 1 Peptide ligation with an N-acylsulfonamide component.
w Electronic supplementary information (ESI) available: See DOI:
10.1039/c2cc15911b
Pep 1 and 2 correspond to unprotected peptides.
c
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resin and analysing the peptides by HPLC and MALDI-TOF
mass spectrometry (ESI). Where the reaction was incomplete it
was continued and monitored with further sample cleavage
until completion.15 Standard cleavage of the bulk resins gave
peptides 1 and 2.
Ligation in the most favoured case between peptide 1
(glycine terminal) and H-CFALRGWR-NH2 (3) at high
concentration (10 mM each) was conducted in neutral aqueous
buffer and in the presence of 4-mercaptophenylacetic acid
(MPAA). Gratifyingly, ligation was found to occur rapidly
(approx. t1/2 = 30 min). The efficacy of our approach was further
tested using peptide 2 that contains the more sterically challenging
C-terminal residue, alanine. The reaction was complete in
2 h (Fig. 1), indicating that N-peptidyl-N-methylsulfonamides
are effective substitutes for pre-formed thioesters in NCL
reactions.
The ligation mechanism probably occurs via the thioester
formed by exchange of the N-peptidyl-N-methylsulfonamide
with MPAA. This thioester was observed in the absence of
N-teminal cysteine peptides but not in their presence, giving
very clean HPLC profiles for the ligation reaction (Fig. 1 and 2).
This is in contrast to standard NCL with preformed alkyl
thioesters, where the monitoring of the reaction by HPLC
is often complicated by the presence of multiple thioester
exchange products. In the sulfonamide ligation, the MPAA
thioester formed is instantly captured by cysteine, a better
nucleophile than MPAA. A similar observation was made by
Melnyk and co-workers, who performed ligation reactions at
high MPAA concentration with mildly activated peptides.7
Small, model peptides are often imperfect guides for how
larger peptides ligate because of their low molecular weight
and limited functionality. Model ligations are typically per-
formed at high concentrations of approximately 10 mM;
however most syntheses of small proteins are carried out at
around 1 mM because of the higher molecular weights of the
peptides used. Therefore we undertook the synthesis of a
classic target for chemical synthesis, BPTI.18,19 This 58 residue,
small protein is a stringent test of the acylsulfonamide approach
with its full range of side chain functional groups. The length is
more typical of peptides used for NCL. We adopted the strategy
of Lu and co-workers and prepared BPTI by the ligation of
Scheme 2 Preparation of the peptidyl-N-methylsulfonamide.
to derivatise peptides with the sulfonamide so it acts as a mildly
activated functional group.
A complication with the use of the sulfamylbutyryl linker is
incomplete acylation on solid phase of the poorly nucleophilic
sulfonamide by the first amino acid. This has been overcome
here by derivatisation of the linker with the C-terminal residue
prior to its attachment to the solid phase (ESI, Scheme S1).
Another key consideration for this approach was the choice
of alkylating agent. The alkylation reaction has to be quantitative
to prevent complicating HPLC purification after cleavage. In
addition, the alkyl group has an effect on the reactivity of the
sulfonamide,14 the less reactive the sulfonamide, the smaller the
risk of self-condensation during purification and of unwanted side
reactions during ligation. Therefore methylation was selected.
Trimethylsilyl diazomethane (TMS-CHN2) after Pessi and
co-workers9,16,17 was used for methylation with the solvent mix
hexane/DCM (1 : 1) identified by Mezzato et al.15
To evaluate our approach, we prepared model N-peptidyl
N-methylsulfonamides 1 (H-AYRAG- N(CH3)SO2(CH2)3-
CONH2) and 2 (H-AYRAA- N(CH3)SO2(CH2)3CONH2) to
explore their ligation properties with an N-terminal cysteine
peptide. Fmoc-Gly-sulfamylbutyric acid and Fmoc-Ala-
sulfamylbutyric acid were synthesised (ESI, Scheme S1) and
coupled to the Sieber Amide resin. Peptide chain extension
was carried out with standard Fmoc SPPS protocols, except
the N-terminal residue, was incorporated as a Boc-protected
amino acid. Alkylation, typically performed overnight with
TMS-CHN2 was checked by cleaving a small sample of each
Fig. 1 Analytical HPLC time course of the ligation reaction of
AYRAA N-methylsulfonamide to CFALRGWR. Ligation condi-
tions: peptide (10 mM each), 200 mM sodium phosphate buffer pH
7.5, 2 mM EDTA, 1 M Guanidine.HCl, 50 mM TCEP, 60 mM
MPAA, 40 1C. HPLC conditions: Chromolith column, 0–30% B
(0.1% TFA, 90% CH3CN) in 10 min, 3 ml.minÀ1. A = H-AYRAA-
N(CH3)SO2(CH2)3CONH2, B = H-CFALRGWR-NH2 C = ligation
product H-AYRAACFALRGWR-NH2, * MPAA.
c
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latent thioester. The notoriously poor reactivity of N-peptidyl-
N-methylsulfonamides is in this context advantageous as it
minimises unwanted side reactions during purification and
ligation and additionally its stability is promising for long
term storage.14 This development, of a proven methodology
that is compatible with the multiple functionalities and con-
ditions used in peptide synthesis should prove to be valuable
for the synthesis of the chemically defined post-translationally
modified proteins currently demanded by biological research.
This work was supported by a grant-in-aid from the MRC
number U117592730.
Notes and references
1 S. B. Kent, Chem. Soc. Rev., 2009, 38, 338–351.
2 T. M. Hackeng, J. H. Griffin and P. E. Dawson, Proc. Natl. Acad.
Sci. U. S. A., 1999, 96, 10068–10073.
3 M. Schnolzer, P. Alewood, A. Jones, D. Alewood and S. B. Kent,
Int. J. Pept. Protein Res., 1992, 40, 180–193.
4 J. Kang and D. Macmillan, Org. Biomol. Chem., 2010, 8, 1993–2002.
5 F. Mende and O. Seitz, Angew. Chem., Int. Ed., 2011, 50, 1232–1240.
6 J. Li, I. A. Taylor, J. Lloyd, J. A. Clapperton, S. Howell,
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36019–36030.
7 N. Ollivier, J. Dheur, R. Mhidia, A. Blanpain and O. Melnyk, Org.
Lett., 2010, 12, 5238–5241.
8 G. W. Kenner, J. R. McDermott and R. C. Sheppard, J. Chem.
Soc., Chem. Commun., 1971, 636–637.
Fig. 2 BPTI ligation (a) MALDI-TOF mass spectrum of the ligation
product. (b) Analytical HPLC time course of the ligation. Ligation
conditions: [BPTI(1–37)N(CH3)SO2(CH2)3CONH2]
=
0.65 mM,
[BPTI(38–58)] = 1 mM in 200 mM phosphate buffer pH 7.5, 2 mM
EDTA, 6 M Guanidine.HCl, 50 mM TCEP, 60 mM MPAA, 40 1C.
HPLC conditions: RP-C18, 0–70% B in 25 min, 1 ml.minÀ1. A =
BPTI(1–37)N(CH3)SO2(CH2)3CONH2, B = BPTI(38–58), C = ligation
product, * MPAA.
9 R. Ingenito, E. Bianchi, D. Fattori and A. Pessi, J. Am. Chem.
Soc., 1999, 121, 11369–11374.
10 F. Mende, M. Beisswenger and O. Seitz, J. Am. Chem. Soc., 2010,
132, 11110–11118.
11 D. Macmillan and C. R. Bertozzi, Angew. Chem., Int. Ed., 2004,
43, 1355–1359.
12 M. Huse, M. N. Holford, J. Kuriyan and T. W. Muir, J. Am.
Chem. Soc., 2000, 122, 8337–8338.
13 R. R. Flavell, M. Huse, M. Goger, M. Trester-Zedlitz, J. Kuriyan
and T. W. Muir, Org. Lett., 2002, 4, 165–168.
14 B. J. Backes, A. A. Virgilio and J. A. Ellman, J. Am. Chem. Soc.,
1996, 118, 3055–3056.
15 S. Mezzato, M. Schaffrath and C. Unverzagt, Angew. Chem., Int.
Ed., 2005, 44, 1650–1654.
two fragments: BPTI(1–37) and BPTI (38–58).19 BPTI(38–58)
was assembled on Fmoc-Ala-Wang resin using standard HOBt/
DIC activation. BPTI(1–37) was assembled in a similar manner
on Fmoc-Gly-sulfamylbutyryl Sieber amide resin (ESI). The
16
linker was methylated with TMS-CHN2 monitoring with
HPLC till completion and cleaved with TFA to afford depro-
tected BPTI(1–37)-N-methylsulfonamide. Following purifi-
cation by RP-HPLC, the two fragments were ligated under
denaturing conditions (Fig. 2) using low millimolar fragment
concentration. The reaction was almost complete in 8 h and was
comparable to the classical NCL using preformed thioester of
Lu and co-workers.19
16 Caution, trimethylsilyl diazomethane is extremely toxic.
N. G. Murphy, S. M. Varney, J. M. Tallon, J. R. Thompson and
P. D. Blanc, Clin. Toxicol., 2009, 47, 712.
17 This reagent does, however, methylate phosphate diesters, such as the
monobenzyl esters employed for the introduction of phosphoamino
acids during Fmoc-based SPPS. Fortunately, this side reaction can be
reversed by using TMSBr/TFA for the cleavage of the peptide from
the solid phase. E. A. Kitas, J. W. Perich, R. B. Johns and
G. W. Tregear, Tetrahedron Lett., 1988, 29, 3591–3592.
18 M. Ferrer, C. Woodward and G. Barany, Int. J. Pept. Protein Res.,
1992, 40, 194–207.
In conclusion, we have demonstrated a variation on the
sulfamylbutyryl linker approach that overcomes many of the
current limitations, and provides a simpler method for peptide
ligation via Fmoc SPPS. The sulfamylbutyryl group acts as a
19 W. Lu, M. A. Starovasnik and S. B. Kent, FEBS Lett., 1998, 429,
31–35.
c
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