Dual kinetically controlled native chemical ligation using a combination of sulfanylproline and sulfanylethylanilide peptide

Article abstract of DOI:10.1021/ol202316v

Dual kinetically controlled native chemical ligation using a newly developed sulfanylproline-mediated reaction in combination with an N-sulfanylethylanilide peptide was successfully applied to a previously unreported sequential coupling of peptide fragments added simultaneously to the reaction.

Full text of DOI:10.1021/ol202316v

ORGANIC
LETTERS
2011
Vol. 13, No. 20
5588–5591
Dual Kinetically Controlled Native
Chemical Ligation Using a Combination of
Sulfanylproline and Sulfanylethylanilide
Peptide
Hao Ding, Akira Shigenaga, Kohei Sato, Ko Morishita, and Akira Otaka*
Institute of Health Biosciences and Graduate School of Pharmaceutical Sciences, The
University of Tokushima, Shomachi, Tokushima 770-8505, Japan
aotaka@ph.tokushima-u.ac.jp
Received August 25, 2011
ABSTRACT
Dual kinetically controlled native chemical ligation using a newly developed sulfanylproline-mediated reaction in combination with an
N-sulfanylethylanilide peptide was successfully applied to a previously unreported sequential coupling of peptide fragments added
simultaneously to the reaction.
Native chemical ligation (NCL) has had a significant
impact on protein/peptide chemistry.1 The original NCL
protocol requires two peptide segments: one is a thioester
peptide and the other is an N-terminal cysteinyl peptide.
Chemoselective reactions between the thioester and
N-terminal cysteine, including successive SꢀS and SꢀN
acyl transfers, yield the corresponding ligated product.
One potential limitation of the original NCL is that the
ligation site requires cysteine. The feasibility of NCLs at
noncysteinyl sites has been extensively explored, including
NCLs at alanine,2,3 valine,4 phenylalanine,5 lysine,6 leucine,7,8
and threonine sites.9 Essentially, such NCLs feature the
original NCL-like reaction of β- or γ-sulfanylamino acid
(4) (a) Haase, C.; Rohde, H.; Seitz, O. Angew. Chem., Int. Ed. 2008,
47, 6807. (b) Chen, J.; Wan, Q.; Yuan, Y.; Zhu, J.; Danishefsky, S. J.
Angew. Chem., Int. Ed. 2008, 47, 8521.
(5) Crich, D.; Banerjee, A. J. Am. Chem. Soc. 2007, 129, 10064.
(6) (a) Yang, R.; Pasunooti, K. K.; Li, F.; Liu, X.-W.; Liu, C.-F.
J. Am. Chem. Soc. 2009, 131, 13592. (b) Pasunooti, K. K.; Yang, R.;
Vedachalam, S.; Gorityala, B. K.; Liu, C.-F.; Liu, X.-W. Bioorg. Med.
Chem. Lett. 2009, 19, 6268.
(7) (a) Harpaz, Z.; Siman, P.; Kumar, K. S. A.; Brik, A. ChemBio-
Chem 2010, 11, 1232. (b) Tan, Z.; Shang, S.; Danishefsky, S. J. Angew.
Chem., Int. Ed. 2010, 49, 9500.
(8) Combination of ligations at Ala, Val, and Leu sites followed by
desulfurization; see: Shang, S.; Tan, Z.; Danishefsky, S. J. Proc. Natl.
Acad. Sci. U.S.A. 2011, 108, 5986.
(1) (a) Dawson, P.; Muir, T.; Clark-Lewis, I.; Kent, S. Science 1994,
266, 776. (b) Dawson, P. E.; Kent, S. B. H. Annu. Rev. Biochem. 2000, 69,
923. (c) Hackenberger, C. P. R.; Schwarzer, D. Angew. Chem., Int. Ed.
2008, 47, 10030.
(2) For a review of desulfurization protocol, see: Rohde, H.; Seitz, O.
Peptide Sci. 2010, 94, 551.
(9) Chen, J.; Wang, P.; Zhu, J.; Wan, Q.; Danishefsky, S. J. Tetra-
hedron 2010, 66, 2277.
(3) (a) Yan, L. Z.; Dawson, P. E. J. Am. Chem. Soc. 2001, 123, 526.
(b) Bang, D.; Makhatadze, G. I.; Tereshko, V.; Kossiakoff, A. A.; Kent,
S. B. Angew. Chem., Int. Ed. 2005, 44, 3852. (c) Pentelute, B. L.; Kent,
S. B. H. Org. Lett. 2007, 9, 687. (d) Wan, Q.; Danishefsky, S. J. Angew.
Chem., Int. Ed. 2007, 46, 9248. The use of selenocysteine followed by
deselenization; see:(e) Gieselman, M. D.; Xie, L.; van der Donk, W. A.
Org. Lett. 2001, 3, 1331. (f) Gieselman, M. D.; Zhu, Y.; Zhou, H.;
Galonic, D.; van der Donk, W. A. ChemBioChem 2002, 3, 709.
(g) Metanis, N.; Keinan, E.; Dawson, P. E. Angew. Chem., Int. Ed.
2010, 49, 7049.
(10) For other examples, see: (a) Roelfes, G.; Hilvert, D. Angew.
Chem., Int. Ed. 2003, 42, 2275. (b) Brik, A.; Yang, Y.-Y.; Ficht, S.;
Wong, C.-H. J. Am. Chem. Soc. 2006, 128, 5626. (c) Okamoto, R.;
Kajihara, Y. Angew. Chem., Int. Ed. 2008, 47, 5402. (d) Thomas, G. L.;
Payne, R. J. Chem. Commun. 2009, 4260. (e) Hojo, H.; Ozawa, C.;
Katayama, H.; Ueki, A.; Nakahara, Y.; Nakahara, Y. Angew. Chem.,
Int. Ed. 2010, 49, 5318. (f) Ackrill, T.; Anderson, D. W.; Macmillan, D.
Peptide Sci. 2010, 94, 495. (g) Thomas, G. L.; Hsieh, Y. S. Y.; Chun,
C. K. Y.; Cai, Z.-L.; Reimers, J. R.; Payne, R. J. Org. Lett. 2011, 13,
4770.
r
10.1021/ol202316v
Published on Web 09/14/2011
2011 American Chemical Society

residues with a thioester, followed by desulfurization.
Other options without the desulfurization reaction have
also been reported.10 Our efforts to synthesize a high-
proline-content GM2-activator protein (GM2-AP)11 have
prompted us to examine NCL at the proline site. Very
recently, Danishefsky and co-workers publishedthe results
of their research on a proline ligation involving an NCL-
like reaction between trans-4-sulfanylproline (trans-
HSPro) and a thioester precursor bearing a 2-ethyldithio-
phenyl ester moiety.12 We were interested in studying
ligation chemistry at a proline surrogate possessing a
sulfanylated tether on 4-hydroxyproline (HOPro). In the
course of this study, we prepared an ethoxythiocarbonyl-
ated HOPro as a requisite synthetic intermediate and
fortuitously found that the O-ethoxythiocarbonyl group
was smoothly transferred to the amino group in aqueous
solution whereas such migration hardly occurred in an
organic solvent (DMF in the presence of diisopropylethyl-
amine (DIPEA)) (Figure 1). This finding encouraged us
to attempt NCL at the proline site using HSPro. In this
report, we will place special emphasis upon the utility of
HSPro-mediated ligation with application to an unprece-
dented dual kinetically controlled reaction.
Scheme 1
Figure 1. Unexpected OꢀN ethoxythiocarbonyl transfer.
The first step was the syntheses of the protected forms
(6 and 7) of both possible diastereomers, i.e., the (4R)-
sulfanylpyrrolidine-(2S)-carboxylic acid (H-trans-HSPro-
OH) and its 4S diastereomer (H-cis-HSPro-OH), as pro-
line surrogates, from Boc-trans-HOPro-OH (1), as shown
in Scheme 1.13 An intramolecular Mitsunobu reaction of 1,
followed by saponification and subsequent protection of
the carboxy group, gave cis-hydroxyproline derivative 3.
The secondary hydroxyl group was subsequently con-
verted to the S-acetyl group, with inversion of the config-
uration, by the action of methanesulfonyl chloride (MsCl)
in the presence of Et3N, followed by treatment with
potassium thioacetate (AcSK). Alkaline hydrolysis of the
ester 4, followed by protection of the resulting sulfanyl
group with triphenylmethyl chloride (Trt-Cl) in pyridine,
Figure 2. HPLC monitoring of trans-sulfanylproline (trans-
HSPro)-mediated ligation for the synthesis of NMU. (A) Liga-
tion (t = 0 min): thioester 8 (1.5 mM) and trans-HSPro peptide 9
(1.0 mM) were ligated. (B) Ligation (t = 14 h). HPLC condi-
tions are described in the Supporting Information.
€
(11) Klima, H.; Tanaka, A.; Schnabel, D.; Nakano, T.; Schroder, M.;
Suzuki, K.; Sandhoff, K. FEBS Lett. 1991, 289, 260.
(12) Shang, S.; Tan, Z.; Dong, S.; Danishefsky, S. J. J. Am. Chem.
Soc. 2011, 133, 10784.
(13) Although Danishefsky’s group utilized commercially available
materials, we carried out the synthesis according to a patent. Raines,
R. T.; Shoulders, M. D.; Hodges, J. A. Collagen mimics. U.S. Patent
Application 20070275897, November 29, 2007. URL: http://ip.com/pa-
tapp/US20070275897.
produced the desired trans-HSPro derivative 6. An identical
sequence of reactions to that used for conversion of 3 to 6
was applied to 1 to synthesize the cis-HSPro derivative 7.
Org. Lett., Vol. 13, No. 20, 2011
5589

With both diastereomers 6 and 7 available, we evaluated
the applicability of the proline surrogates to NCL followed
by desulfurization in the synthesis of rat neuromedin U14
(NMU: H-YKVNEYQGPVAPSGGFFLFRPRN-NH2),
which consists of 23 amino acid residues. Initially, ligations
were performed between the Gly8-Pro9 site by reactions of
alkylthioester peptide 8, corresponding to NMU (1ꢀ8), with
N-terminal trans-HSPro peptide 9 or cis-HSPro peptide 10,
covering NMU (9ꢀ23). The alkylthioester (-SCH2CH2CO-
A-NH2) 8 was synthesized by Boc solid phase peptide
synthesis (SPPS).15 The HSPro-containing peptides 9 and
10 were prepared from Fmoc SPPS using 6 and 7, respec-
tively. Ligation of 1.5 equiv of 8 with trans-HSPro peptide 9
under standard NCL conditions (6 M guanidine hydro-
chloride (Gd HCl)/0.1 M phosphate buffer (pH 7.4) in the
3
presence of 30 mM tris(2-carboxyethyl)phosphine hydro-
chloride (TCEP) and 30 mM 4-(carboxymethyl)thiophenol
(MPAA)16 at 37 °C) unambiguously proceeded to yield the
corresponding ligation product 11 in 54% isolated yield
along with a hydrolyzed product, cyclized product, and
MPAA ester of 8 (Figure 2); this is because ligation of the
trans-HSPro peptide with 8 proceeds more slowly than
ligation of the N-terminal cysteinyl peptide.17 The use of
the cis-HSPro peptide 10 did not afford a detectable amount
of the desired ligation product.12 Compound 11 was then
Figure 3. Examination of the difference in reactivities of the
trans-HSPro and Cys-containing peptide (9 and 12) to al-
kylthioester peptide 8. aThe fraction ligated was determined
by HPLC separation and integration of ligated product (integ.
product) detected at 220 nm as a fraction of the sum of the
unreacted sulfanyl peptide (integ. 9 or 12) þ integ. product. Top:
reaction time range 0ꢀ1 h; bottom: reaction time range 0ꢀ24 h.
subjected to desulfurization3d with VA-044 in 6 M Gd HCl/
3
0.1 M phosphate buffer (pH 7.0) in the presence of 40 mM
TCEP and 10 mM reduced-form glutathione (GSH) at
37 °C. Reaction for 20 h, followed by HPLC purification,
gave the desired NMU in 70% isolated yield. These results
clearly indicated that trans-HSPro-mediated ligation has a
firmly established place among NCL protocols.
Next, we examined the difference in reactivities of 9 and
its cysteinyl counterpart peptide 12 to the peptide thioester
8. Reactions of 8 with 1 equiv of 9 or 12 in 6 M Gd HCl/0.1
3
M phosphate buffer (pH 7.4) in the presence of 30 mM
TCEP and 30 mM MPAA (or 50 mM sodium ascorbate18)
at 37 °C were performed (Figure 3). Ligation of the
cysteinyl peptide 12 in the presence of MPAA proceeded
essentially to completion in 30 min, whereas the same
ligation using trans-HSPro required more than 360 min
to reach a plateau. Addition of MPAA to the reaction with
9 accelerated the ligation, even though the reaction was
promoted to a lesser extent than in the case of the cysteinyl
peptide.
Figure 4. Requirements for the successful coupling of three
peptide fragments mixed simultaneously.
Recently, we developed a kinetically controlled NCL19
using N-sulfanylethylanilide (SEAlide) peptides,20 which
function as cryptic thioester peptides,20b,21 in which ex-
cellent selectivity can be achieved. Based on the use of such
SEAlide peptides, we expected that ordered couplings of
three fragments added in one pot simultaneously should be
possible. In this protocol, three peptide fragments (N-
terminal fragment (Fr): N-Fr; middle Fr: M-Fr; C-term-
inal Fr: C-Fr) simultaneously present in the reaction are
sequentially ligated in an N-to-C-directed manner. The
(14) Minamino, N.; Kangawa, K.; Honzawa, M.; Matsuo, H. Bio-
chem. Biophys. Res. Commun. 1988, 156, 355.
(15) Aimoto, S. Peptide Sci. 1999, 51, 247–265.
(16) Johnson, E. C. B.; Kent, S. B. H. J. Am. Chem. Soc. 2006, 128,
6640.
(17) Linkage between Ala11 and Pro12 is another potential ligation
site. Attempted ligation of the Ala thioester with a trans-HSPro peptide
proceeded with lesser efficacy to yield the desired ligation product with
an accompanying Ala-epimerizing peptide (ca. 5%).
(18) Rohde, H.; Schmalisch, J.; Harpaz, Z.; Diezmann, F.; Seitz, O.
ChemBioChem 2011, 12, 1396.
(19) (a) Bang, D.; Pentelute, B. L.; Kent, S. B. H. Angew. Chem., Int.
Ed. 2006, 45, 3985. (b) Zheng, J.-S.; Cui, H.-K.; Fang, G.-M.; Xi, W.-X.;
Liu, L. ChemBioChem 2010, 11, 511.
(20) (a) Tsuda, S.; Shigenaga, A.; Bando, K.; Otaka, A. Org. Lett.
2009, 11, 823. (b) Sato, K.; Shigenaga, A.; Tsuji, K.; Tsuda, S.;
Sumikawa, Y.; Sakamoto, K.; Otaka, A. ChemBioChem 2011, 12, 1840.
(21) Dheur, J.; Ollivier, N.; Vallin, A.; Melnyk, O. J. Org. Chem.
2011, 76, 3194.
(22) The addition of phosphate is critically important for the sequen-
tial ligation. The SEAlide moiety efficiently functions as a thioester in
the presence of a phosphate salt, whereas this unit did not work as an
efficient thioester precursor in the presence of another salt.
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Org. Lett., Vol. 13, No. 20, 2011

in the presence of 30 mM TCEP and 30 mM MPAA at
37 °C. The reaction proceeded with an efficiency compar-
able to that observed in the NCL of 8 with 9.
On the basis of these observations, we next sought to
synthesize the bovine insulin C peptide (Ac-EVEGPQVG
ALELAGGPGAGGLEGPPQ-NH2) by establishing a
dual kinetically controlled NCL (with simultaneous satis-
faction of KC1 and KC2), along with the use of an HSPro-
peptide in the SEAlide-mediated NCL (Figure 5). The
three requisite peptide fragments, i.e., Ac-EVEGPQVG-
SCH2CH2CO-A-NH2 (14; 4 mM), H-CLELAGG-NAr-L-
NH2 (15; 4 mM), and H-trans-HSPro-GAGGLEGPPQ-
NH2 (16; 8 mM), were simultaneously mixed in 6 M
Gd HCl/0.2 M sodium 3-[4-(2-hydroxyethyl)piperazin-
3
1-yl]propane-1-sulfonate (HEPPS) buffer (pH 7.8) in the
presence of 100 mM TCEP and 50 mM MPAA at 37 °C.
The first NCL between 14 and 15 proceeded under dual
kinetic control to completion within 3 h to afford the
ligated peptide 14 þ 15. Then the second ligation was
initiated by the addition of 1 M phosphate buffer (pH
7.8)20b,22 at 37 °C (final concentration: peptide 14 þ 15 (ca.
2 mM) and 16 (4 mM) in 3 M Gd HCl/0.1 M HEPPS/0.5
3
Figure 5. HPLC monitoring of ligations for the synthesis of the
insulin C peptide. (A) First ligation (t = 2 h): Peptide fragments
14 (4 mM), 15 (4 mM), and 16 (8 mM) were simultaneously
mixed. (B) Second ligation (t = 24 h): After 2 h of the first
ligation, the second ligation was initiated by the addition of
phosphate buffer. HPLC conditions are described in the Sup-
porting Information.
M phosphate, 50 mM TCEP, and 25 mM MPAA). The
addition of phosphate and continuous reaction for 24 h
allowed the SEAlideunittofunction asathioester, yielding
the correctly ligated C peptide precursor17in50% isolated
yield. Peptide 17 was subsequently subjected to a desulfur-
ization reaction3d identical to that employed for NMU to
afford the bovine insulin C peptide in 78% isolated yield.
In conclusion, we achieved the synthesis of protected
HSPro derivatives and developed an HSPro-mediated
NCL reaction based on an unexpected OꢀN thiocarbonyl
transfer. A combination of the proline ligation and the
SEAlide-peptide-mediated NCL allowed three peptide
fragments to be correctly ligated in a dual kinetically
controlled manner. Further extensions of the kinetic reac-
tion are currently underway.
anticipated reaction should succeed in yielding a correctly
ordered peptide by satisfying all of the following require-
ments (Figure 4). (1) The cysteinyl residue in M-Fr inter-
molecularly reacts with the thioester in N-Fr, not with the
intramolecular SEAlide moiety (k1 . k2: kinetic control
1 (KC1)). (2) Of the two sulfanyl amino acids, the thioester
in N-Fr preferentially reacts with the cysteinyl residue in
M-Fr (k1 . k3: kinetic control 2 (KC2)). (3) The trans-
HSPro moiety in C-Fr reacts with the SEAlide unit under
appropriate conditions.
Requirement (1) has already been confirmed to be
satisfied by our recent investigation.20b With respect to
requirement (2), the aforementioned experimental results
(Figure 3) seem to meet this requirement. In order to
confirm the possible involvement of the trans-HSPro in
the SEAlide-mediated NCL, peptide 9 was subjected to a
model NCL with a SEAlide peptide (H-VQGSG-NAr-L-
Acknowledgment. This research was supported in part
by a Grant-in-Aid for Scientific Research (KAKENHI).
A.O. and A.S. are grateful for research grants from the
Takeda Science Foundation.
Supporting Information Available. Experimental pro-
cedures and NMR spectra for key compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
NH2 13) in 3 M Gd HCl/0.5 M phosphate buffer (pH 7.4)
3
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