3-Mercaptopropionic acid-mediated synthesis of peptide and protein thioesters

Article abstract of DOI:10.1039/b815888f

Peptides and proteins fragment sequence-specifically in the presence of 3-mercaptopropionic acid to afford thioesters which can be used in native chemical ligation reactions. The Royal Society of Chemistry.

Full text of DOI:10.1039/b815888f

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3-Mercaptopropionic acid-mediated synthesis of peptide and
protein thioestersw
Jaskiranjit Kang, Jonathan P. Richardson and Derek Macmillan*
Received (in Cambridge, UK) 10th September 2008, Accepted 10th October 2008
First published as an Advance Article on the web 5th November 2008
DOI: 10.1039/b815888f
Peptides and proteins fragment sequence-specifically in the
presence of 3-mercaptopropionic acid to afford thioesters which
can be used in native chemical ligation reactions.
Native chemical ligation (NCL) is an extremely useful method
1
for the production of synthetic and semi-synthetic proteins.
Since it is widely accepted that approximately fifty amino acid
residues represents the limit for efficient automated solid phase
peptide synthesis (SPPS) the use of recombinant methods for
Scheme 1 (a) NCL, and (b) acid mediated N - S thioester formation
the production of the required thioester and cysteine-containing
2
components has been widely exploited. However, while the
1
compared. R is usually an acyl-transfer facilitating group such as
5
alkyl, benzyl, or d-mercaptomethyl prolyl.
number of available synthetic methods for the production of
3
the thioester component has risen dramatically, micro-
indeed formed it was isolated by semi-preparative HPLC and
8
successfully ligated to model peptide 2 (Scheme 2(b)).
organism-derived thioesters are most frequently generated by
4
the commercially available intein-fusion expression system.
Recently, several variations on a central theme of thioester
To determine whether or not MPA could effect this trans-
formation more widely we investigated the MPA mediated
fragmentation of a recombinant 21 kDa protein sample,
His10-WT human erythropoietin (EPO). This 166 residue pro-
production via an N to S acyl shift, that are mechanistically
more similar to the intein system, have been reported
5
(
Scheme 1).
7
tein contains four cysteine residues in the arrangement I C,
29
5
Following the work of Kawakami and Aimoto on the use
d
33
161
G C, H C and A C and was treated with 20% MPA over
time periods of 1–48 h and at temperatures ranging from 40 to
of cysteinylprolyl esters (CPEs) for the transient production of
thioesters which can participate in NCL reactions, we had
attempted to utilise CPEs in our studies geared towards the
semi-synthesis of erythropoietin (EPO) and had managed to
8
0 1C. The protein appeared to fragment in 20% v/v MPA at
temperatures as low as 40 1C (Fig. 1(a)) and surprisingly there
appeared to be an accumulation of the His10-EPO(1-28)-
Gly-MPA thioester and His -EPO(1-32)His-MPA thioester
6
apply them with limited success (Scheme 2(a)). Consequently,
we attempted to ‘‘rescue’’ CPE 1 employing 3-mercapto-
propionic acid (MPA) to produce thioesters via a N - S acyl
10
5b
shift in aqueous MPA as reported by Hojo and co-workers.
In doing so we observed formation of an undesired glycine
thioester 3 at the expense of our desired alanine thioester.
Interestingly the ‘‘isolated’’ cysteine residue appeared to facili-
tate conversion to a glycine-MPA thioester in the absence of
any additional stimulus such as a CPE, a mercaptomethyl
5b
5e
proline residue, or cysteine N-alkylation and led us to
predict that some selectivity may be achievable in peptide
and protein fragmentation leading to thioesters in a single
7
step, using MPA as the sole thioesterification reagent. The
desired alanine thioester was observed in trace quantities only
and although it was possible that any Ala thioester could be
further processed to the Gly-thioester, it was not yet clear if
this was the case. To validate that the glycine thioester 3 had
Department of Chemistry, University College London, 20 Gordon
Street, London, UK WC1H 0AJ. E-mail: d.macmillan@ucl.ac.uk;
Tel: 020-7679 4684
w Electronic supplementary information (ESI) available: Experimental
procedures for the production of 1, all model peptides and proteins
and their reactions with MPA including HPLC, LC-MS and NMR
characterisation of selected products. See DOI: 10.1039/b815888f
Scheme 2 (a) Attempted CPE-mediated ligation between peptide 1
complete sequence: AENITTGCAEA-CPE) and peptide 2 (sequence:
(
6
CSLNENIT) and subsequent MPA treatment. (b) Successful ligation
8
reaction employing the Gly-thioester 3.
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Chem. Commun., 2009, 407–409 | 407

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and contained two possible positions at which fragmentation
could take place, though in each case the amino acid adjacent
to the cysteine residue had been altered such that the outcome
of the cleavage reaction could be predicted. In keeping with
the observed reactivity profile of peptide thioesters in native
chemical ligation reactions we allowed Gly-Cys, Cys-Cys,
Ile-Cys and His-Cys ‘‘pairs’’ to compete with each other.
Gly, Cys, and His thioesters are known to participate in native
chemical ligation reactions with particularly fast kinetics
1
0
whereas Ile is among the slower thioesters to react. Con-
sequently, we expected that peptides which contained exclu-
sively GC junctions or a GC junction combined with CC or
HC junctions to yield mixtures of thioesters whereas peptides
that contained IC or protected GC(S-Acm) to yield singly-
cleaved compounds and this was indeed found to be the case.
Thioester formation at HC junctions appeared to be the most
rapid and most extensive followed by cleavage at GC and CC
junctions. Interestingly the peptide containing the GC and HC
pair (entry 1) appeared to fragment in a very similar manner to
the recombinant protein fragment providing a mixture of His
and Gly thioesters.
Fig. 1 (a) MPA-mediated fragmentation of His10-WT human EPO
with 20% MPA for 16 h at 60 1C gives thioesters corresponding to
cleavage between GC and HC junctions but not between IC or AC.
(b) A G28A mutation appears sufficient to suppress mixed thioester
formation.
peptides, with the His-thioester peptide appearing the most
7
abundant. The peptides corresponding to cleavage at the I C
161
junction or between A C were not observed. While it proved
difficult to separate this pair of thioesters, the mutation G28A
was sufficient to suppress thioesterification at this site (Fig. 1(b))
and the His EPO-1-32(G28A)-MPA thioester was isolated in
Having demonstrated that the thioesterification reaction
appeared to be selective, we conducted another small screen
of test peptides to probe for particularly favourable or un-
8
favourable thioesterification sites. Ten short peptides of
sequence H-AENITTXC-NH (where X = A, D, E, F, G,
1
0
2
8% yield.
The presumed peptide thioesters were isolated by reverse-
2
K, P, S, V, W) were cleaved from the solid support. LC-MS of
the crude products indicated that the peptides were highly pure
though in some cases (with the exception of FC, PC, KC, WC
and VC containing peptides) two resolved peaks with identical
masses were observed which were tentatively assigned as the
phase HPLC and also successfully ligated to model peptide 2
in the presence of 6 M guanidineÁHCl, 300 mM Na phosphate
9
buffer; pH 7.4, 25 mM 4-mercaptophenylacetic acid (MPAA),
2
8
0 mM TCEP to afford the expected NCL products. This
5f
experiment further suggested that MPA facilitated protein
fragmentation at cysteine residues and that the relative degree
of fragmentation may vary depending on the nature of the
amino acid adjacent to the cysteine residue. Combining these
results led us to postulate an approximate reactivity ranking of
H Z G 4 A 4 I, a trend similar to that observed in the
reactivity of thioesters in regular NCL reactions, the reverse
N- and the S- peptide. Each crude sample was isolated in
near quantitative yield so each peptide was used directly in
MPA-mediated thioesterification reactions. The peptides were
exposed to forcing conditions of 20% MPA at 80 1C for 24 h,
anticipating that Gly-thioester formation should be near
complete and Val-thioester formation negligible. Analysis of
the reaction mixtures after 20 h revealed that, as expected,
Gly-thioester formation had progressed to near completion
whereas Pro and Val showed negligible, though detectable,
thioester formation. Notably the Asp-Cys containing peptide
had been completely consumed yet did not give rise to a
thioester product, the major species corresponding to a product
which had lost both the Asp and Cys residues. In contrast
1
0
process. Consequently we predicted that useful thioesterifi-
cation may be limited to NCL’s favoured three residues
(
Gly, His, Cys).
Seven peptides (Table 1) were prepared to probe this
hypothesis in more detail. Each peptide was related to the
recombinant EPO sample that had undergone fragmentation
Table 1 Peptides designed to test for selectivity in MPA-mediated fragmentation
8
X C cleavage
b
12
X C cleavage
b
a
12 8
C : X C
c
d
Isolated yield (%)
Entry
Peptide sequence
Calc. m/z
Obs. m/z
Calc. m/z
Obs. m/z
X
1
2
3
4
5
6
7
H-AENITTGCAEHC-NH
2
793.3
793.3
849.4
849.4
793.3
793.3
793.3
793.5
n/o
n/o
1233.5
1304.5
1289.5
1209.5
1153.4
1199.4
1209.5
1233.6
1304.6
1289.7
1209.7
1153.6
1199.6
n/o
E1 : 1
49 : 1
49 : 1
49 : 1
E1 : 1
E1 : 1
1 : 9
28
39
60
n.d.
26
28
n.d.
H-AENITTGC(Acm)AEHC-NH
H-AENITTICAEHC-NH
H-AENITTICAEGC-NH
H-AENITTGCAEGC-NH
H-AENITTGCAECC-NH
H-AENITTGCAEIC-NH
2
2
2
n/o
2
793.5
793.5
793.5
2
2
a
b
Peptides prepared on Rink amide MBHA resin. Each 50 mL reaction contained approx. 8 mM peptide and was analysed by LC-MS after 36 h
c
d
at 50 1C (unoptimised) in 20% MPA. The calculated mass corresponds to the MPA thioester. Determined by LC-MS. Unoptimised yield of the
full length (11 mer) thioester. n.d. = not determined and applies to peptides where isolation was hindered by overlapping MPA derived peaks.
4
08 | Chem. Commun., 2009, 407–409
This journal is ꢀc The Royal Society of Chemistry 2009

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of the reaction conditions and exploration of alternative
experimental protocols, e.g. use of microwave irradiation, in
combination with site-directed mutagenesis, may ultimately
yield a direct, small molecule-mediated, method for the pro-
duction of bacterially expressed protein thioesters to compli-
ment the intein-fusion system.
The authors acknowledge financial support from The
Wellcome Trust and the EPSRC.
Notes and references
1
2
(a) P. E. Dawson, T. W. Muir, I. Clark-Lewis and S. B. Kent,
Science, 1994, 266(5186), 776; (b) P. E. Dawson and S. B. H. Kent,
Annu. Rev. Biochem., 2000, 69, 923; (c) D. Macmillan, Angew.
Chem., Int. Ed., 2006, 45(46), 7668; (d) C. Haase and O. Seitz,
Angew. Chem., Int. Ed., 2008, 47(9), 1553.
1
Fig. 2 H NMR analysis of test peptide H-AENITTHC-NH
a) before and (b) after MPA-mediated thioesterification.
2
(a) V. Muralidharan and T. W. Muir, Nat. Methods, 2006, 3(6),
4
(
29; (b) T. W. Muir, D. Sondhi and P. A. Cole, Proc. Natl. Acad.
Sci. USA, 1998, 95(12), 6705; (c) C. J. Noren, J. M. Wang and
F. B. Perler, Angew. Chem., Int. Ed., 2000, 39(3), 451;
(d) R. K. McGinty, J. Kim, C. Chatterjee, R. G. Roeder and
T. W. Muir, Nature, 2008, 453(7196), 812.
(a) Y. Shin, K. A. Winans, B. J. Backes, S. B. H. Kent,
J. A. Ellman and C. R. Bertozzi, J. Am. Chem. Soc., 1999,
the Glu-Cys containing peptide (like the Ala-Cys, Ser-Cys, and
Phe-Cys containing peptides) gave rise to an approximately
3
5
0 : 50 mix of starting material and product. The Ser-Cys
containing peptide (both the starting material and the
product) appeared to have further condensed with MPA under
the reaction conditions, presumably on the primary hydroxyl
group of serine, to afford an MPA ester. This modification of
serine, along with Ser-MPA thioester formation, was neglig-
able when the reaction was repeated at 60 1C for 16 h with the
vast majority of the starting material remaining unchanged.
Besides Val and Pro, Lys and Trp appeared to have performed
least efficiently in thioester formation at 80 1C. From these
results we concluded that, of the nine additional fragmentation
junctions tested, only Gly-Cys junctions and His-Cys are likely
to give rise to thioesters under more desirable conditions
1
21(50), 11684; (b) R. Ingenito, E. Bianchi, D. Fattori and A.
Pessi, J. Am. Chem. Soc., 1999, 121(49), 11369; (c) D. Swinnen and
D. Hilvert, Org. Lett., 2000, 2(16), 2439; (d) H. Hojo, E. Haginoya,
Y. Matsumoto, Y. Nakahara, K. Nabeshima, B. P. Toole and
Y. Watanabe, Tetrahedron Lett., 2003, 44(14), 2961; (e) J. A.
Camarero, B. J. Hackel, J. J. D. Yoreo and A. R. Mitchell,
J. Org. Chem., 2004, 69(12), 4145; (f) O. S. Franziska Mende,
Angew. Chem., Int. Ed., 2007, 46(24), 4577; (g) S. Manabe,
T. Sugioka and Y. Ito, Tetrahedron Lett., 2007, 48(5), 849;
(
h) T. J. Hogenauer, Q. Wang, A. K. Sanki, A. J. Gammon,
C. H. L. Chu, C. M. Kaneshiro, Y. Kajihara and K. Michael, Org.
Biomol. Chem., 2007, 5, 759; (i) S. Ficht, R. J. Payne, R. T. Guy
and C.-H. Wong, Chem. Eur. J., 2008, 14(12), 3620;
(
j) J. B. Blanco-Canosa and P. E. Dawson, Angew. Chem., Int.
Ed., 2008, 47, 6851.
4 www.neb.com.
(
ments with test peptides also provided access to multimilli-
temperatures ranging from 50 to 60 1C). The model experi-
5
(a) N. Ollivier, J.-B. Behr, Q. El-Mahdi, A. Blanpain and
O. Melnyk, Org. Lett., 2005, 7(13), 2647; (b) F. Nagaike,
Y. Onuma, C. Kanazawa, H. Hojo, A. Ueki, Y. Nakahara and
Y. Nakahara, Org. Lett., 2006, 8(20), 4465; (c) H. Hojo,
Y. Onuma, Y. Akimoto, Y. Nakahara and Y. Nakahara, Tetra-
hedron Lett., 2007, 48(1), 25; (d) T. Kawakami and S. Aimoto,
Tetrahedron Lett., 2007, 48(11), 1903; (e) H. Hojo, Y. Murasawa,
H. Katayama, T. Ohira, Y. Nakahara and Y. Nakahara, Org.
Biomol. Chem., 2008, 6, 1808; (f) K. Nakamura, M. Sumida,
T. Kawakami, T. Vorherr and S. Aimoto, Bull. Chem. Soc. Jpn.,
1
gram quantities of products allowing H and C NMR
13
1
analysis of the proposed thioester products. The H NMR
spectrum of H-AENITTHC-NH2 and H-AENITTGC-NH2
before and after exposure to MPA clearly indicate the loss of
cysteine, the incorporation of MPA (Fig. 2) along with the
associated downfield shift in the resonance of the a-hydrogens
8
of the C-terminal residue. Thioester formation was further
supported by the appearance of a signal at 205 ppm, typical
2
006, 79, 1773; (g) B. Wu, J. Chen, J. D. Warren, G. Chen, Z. Hua
and S. J. Danishefsky, Angew. Chem., Int. Ed., 2006, 45(25), 4116.
1
3
for the carbonyl carbon of a thioester, in the C NMR
spectrum of H-AENITTHC-NH after treatment with MPA.
C labelling studies are currently underway to further confirm
6 Although the reaction was not successful, when the CPE peptide
was later re-prepared with the cysteine residue adjacent to glycine
protected with an S-Acm group the desired CPE ligation product
was observed and the Ala-MPA thioester could be isolated (after
treatment of the CPE peptide with MPA).
2
1
3
thioester formation.
In summary, through our investigations employing CPE
terminated peptides, we observed an interesting one-step
thioesterification reaction in which peptide and protein
samples containing GC, CC and HC junctions preferentially
7 N-S acyl transfer has been observed in a synthetic peptide
containing a Gly-Cys junction, in ref. 5f, upon exposure to 71%
TFA in CDCl as part of a two-step thioesterification procedure.
3
The glycine thioester so produced was not isolated or used in
ligation reactions due to difficulties encountered as a consequence
of the two-step procedure adopted, (the S-peptide spontaneously
reverted to the N-peptide upon attempted isolation). Neither the
potential selectivity of the thioesterification, nor application to
recombinant samples was subsequently explored.
1
1
fragment at these positions in the presence of MPA. While
fragmentation apparently occurs most readily at histidine the
extent of racemisation needs detailed investigation though a
short thioester, Ac-Ala-His-SCH
peptide Ac-Ala-(L-)His-Cys-NH
2
CH CO H derived from
upon exposure to 20%
2
2
8 See ESIw for experimental details.
9
E. C. B. Johnson and S. B. H. Kent, J. Am. Chem. Soc., 2006,
1
2
28(20), 6640.
MPA for 48 h at 55 1C surprisingly exhibits undetectable
racemisation when compared (by HPLC) with Ac-Ala-(D-)-
1
0 T. M. Hackeng, J. H. Griffin and P. E. Dawson, Proc. Natl. Acad.
Sci. USA, 1999, 96(18), 10068.
11 L. Zhang and J. P. Tam, Tetrahedron Lett., 1997, 38, 3.
8
His-Cys-NH exposed to identical conditions. Optimisation
2
This journal is ꢀc The Royal Society of Chemistry 2009
Chem. Commun., 2009, 407–409 | 409