Peptide ligation assisted by an auxiliary attached to amidyl nitrogen

Article abstract of DOI:10.1016/j.tetlet.2010.01.108

New thiol-containing auxiliaries were developed for peptide ligation. They were placed at the amidyl N-atom in the second amino acid residue of a peptide fragment. With the new auxiliaries, peptide ligation could be conducted at non-Cys and non-Gly sites. Compared to other recently developed auxiliaries, an important feature of the present design was that the new auxiliaries were generally applicable and readily removable.

Full text of DOI:10.1016/j.tetlet.2010.01.108

Tetrahedron Letters 51 (2010) 1793–1796
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Peptide ligation assisted by an auxiliary attached to amidyl nitrogen
*
Juan Li, Hong-Kui Cui, Lei Liu
Department of Chemistry, University of Science and Technology of China, Hefei 230026, China
Department of Chemistry, Tsinghua University, Beijing 100084, China
a r t i c l e i n f o
a b s t r a c t
Article history:
New thiol-containing auxiliaries were developed for peptide ligation. They were placed at the amidyl
N-atom in the second amino acid residue of a peptide fragment. With the new auxiliaries, peptide ligation
could be conducted at non-Cys and non-Gly sites. Compared to other recently developed auxiliaries, an
important feature of the present design was that the new auxiliaries were generally applicable and read-
ily removable.
Received 4 November 2009
Revised 25 January 2010
Accepted 29 January 2010
Available online 2 February 2010
Ó 2010 Elsevier Ltd. All rights reserved.
The efficiency of intramolecular catalysis due to the large in-
crease of ‘effective molarity’ is a subject that has attracted escalat-
ing attention.1 An elegant application of this strategy to bioorganic
chemistry is the thiol-capture method of protein native chemical
ligation (NCL) developed by Kent and co-workers.2 NCL harnesses
the high efficiency of intramolecular catalysis for the purpose of
effecting amide ligation between peptide fragments. It employs
Herein, we describe a new design of the auxiliary for the thiol-
capture method of peptide ligation. In the design the thiol-contain-
ing group is attached to the amidyl N-atom at the second amino
acid residue of the peptide (Scheme 3). This design has several
advantages over the previous ones: (1) The N-terminal group re-
mains a primary amine. This may allow for the ligation at non-
Gly sites. (2) Almost every peptide contains an amidyl N–H at its
second amino acid residue. Thus the new design is more generally
applicable as compared to e.g. sugar-assisted auxiliary.7 (3) Many
previous studies have shown that an auxiliary on an amidyl N-
atom can be readily removed after ligation.5
an S-to-N acyl transfer reaction via
a five-membered ring
intermediate.3
Currently NCL and its related methods (e.g., expressed protein
ligation) are extensively used for chemical protein synthesis.4 An
often encountered problem of NCL is that cysteine (Cys) is not al-
ways observed in all the natural proteins. To avoid the demand
of Cys at the ligation site, several methods have been developed
by attaching a thiol-containing auxiliary at the N-terminus of the
peptide (Scheme 1).5 A secondary amino group is generated in this
type of auxiliary, leading to a relatively slow S-to-N acyl transfer.
As a result, the ligation methods assisted by N-terminal auxiliaries
can only be used for very few junctions such as Gly–Gly.
To examine the effectiveness of the new design, we notice that
the chemical structure of the spacer in the thiol-containing auxil-
R
R
peptide
peptide
HN
HN
HS
HS
O
O
Alternatively one can also put the thiol-containing auxiliary at
the other locations instead of the N-terminus. This strategy results
in a larger ring size in the acyl transfer, whose efficiency may be low-
er than the five-membered ring acyl transfer in standard NCL. None-
theless, Kemp et al. have shown that through strategic design it was
possible to achieve an efficient acyl transfer through a medium-size
ring intermediate (Scheme2).6 More recently, Wong and co-workers
extended Kemp’s method by anchoring an auxiliary to the carbohy-
drate moiety of a glycosylated amino acid to facilitate the S-to-N acyl
transfer.7 This method allows for the fragment condensation of gly-
copeptides at junctions containing neither Cys nor Gly. However, the
use of a sugarauxiliaryfor ligationis expensive, andits application to
the synthesis of sugar-free proteins is limited.
R'
R'
Scheme 1. Auxiliaries at N-terminal nitrogen producing a secondary amine for acyl
transfer.
O
O
H
N
H2N
Peptide
HO
peptide
Peptide
O
O
O
Peptide
O
S
S
S
S
O
* Corresponding author. Tel.: +86 10 62780027; fax: +86 10 62771149.
E-mail address: lliu@mail.tsinghua.edu.cn (L. Liu).
Scheme 2. Kemp’s ligation via a 12-membered ring intermediate.
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.01.108

1794
J. Li et al. / Tetrahedron Letters 51 (2010) 1793–1796
COOMe
O
COOMe
Br
NBS
Ph3CSH
Peptide
O
R2
SR
K2CO3
71%
(PhCOO)2
98%
H
N
ligation
Peptide
Peptide
+
N
O
R2
O
R1
HS
O
COOMe
SCPh3
CHO
OH
H2N
HS
Peptide
LiAlH4
PCC
46%
N
removable
THF
63%
R1
O
after ligation
SCPh3
SCPh3
S-to-N
acyltransfer
transthio-
esterification
O
R2
N
H
Ph
PhCH(NH2)CH3
N
H
Ph
1) TFA/TIPS
H2N
Peptide
N
2) tBuSH,I2
NaBH3CN
54%
R1
O
S
59%
Peptide
S
SCPh3
O
S
spacer
O
BocHN
Boc-Gly-OH
N
Ph
Scheme 3. The design of a new thiol-containing auxiliary.
TFA/DCM
81%
1e
EDC/DMAP
76%
iary remain to be optimized. Accordingly we have synthesized a
series of model compounds 1a–1e which all possess a thiol-con-
taining side chain at their amidyl N-atoms (Fig. 1). The length
(from two to four carbons) and nature (sp3 or sp2 carbons) of the
spacer are systematically examined to probe their effects on the
intramolecular acyl transfer. Scheme 4 outlines the synthesis of
these model compounds by using 1e as an example. An important
step in the synthesis is the change of the protecting group on the
thiol group from the trityl group to the t-butylthio group.8
The efficiency of the different spacers in the acyl transfer reac-
tion was examined by the model ligation reactions carried out un-
der normal NCL conditions (Scheme 5). It was found that with
compound 1a, 1c, 1d, and 1e, the ligation yields were relatively
low. Fortunately, when compound 1b was used, the ligation yield
reached 71%. Note that for 1b to undergo the S-to-N acyl transfer,
a nine-membered ring intermediate must be formed (Scheme 5).
This observation supports the previous results that an efficient acyl
transfer may proceed via a medium-size ring intermediate.6,7,9
The results from the above model study indicate that the amidyl
auxiliary strategy is experimentally feasible and may constitute a
new method for the bioorthogonal ligation. Our next tasks include:
(1) to modify the spacer so that it can be removed after ligation; (2)
to show that the new method can be applied to ligation at non-Cys
and non-Gly sites; and (3) to further optimize the spacer to achieve
S
S
Scheme 4. Synthesis of compound 1e.
S
S
O
Aux
N
S
+
Ph
N
H
H2N
O
O
Ph
2 (5 mM)
1a-1e (7.5 mM)
6M GnHCl/NMP (1:1)
200 mM Na2HPO4
TCEP, 40 oC, pH = 7.5, Ar
HS
Aux
O
S
N
Ph
N
H
+
H2N
O
O
Ph
transthioesterification
O
H
Aux
Ph
N
S
O
N
H2N
O
Ph
O
intramolecular
O
O
S
H2N
S-to-N acyl transfer
H2N
N
H2N
N
N
Substrate Yield (%)
HS
O
Aux
N
H
N
1a
1b
1c
1d
1e
52
71
55
24
22
Ph
N
H
S
S
S
S
O
O
Ph
S
ligation product
1a
1b
1c
Scheme 5. Model ligation reactions with 1a–1e.
O
O
H2N
H2N
N
higher ligation yields. In the following work we have solved the
first two problems.
N
As to the first problem, our solution is to attach the oxyalkyl
substitution on the amide bond. This substitution was previously
examined by Kent and co-workers in their study on the ligation
S
S
S
S
a
of N (oxyethanethiol)-peptide.10 In Kent’s study, the Boc-chemis-
try was utilized so that HF was used to deprotect the thiol in the
preparation of N (oxyethanethiol)-peptide. In our study, we focus
1d
1e
a
on the milder Fmoc-chemistry and therefore, we need to develop
new synthesis to make the N (oxyethanethiol)-amino acid.
Figure 1. Model compounds possessing
amidyl N-atoms.
a thiol-containing side chain at their
a

J. Li et al. / Tetrahedron Letters 51 (2010) 1793–1796
1795
O
Br
O
O
As to the second problem, we examined the ligation yields be-
tween different amino acid residues (Table 1). Gly, Ala, and Val
were selected to represent amino acid residues with very low,
modest, and very high steric hinderance, respectively. It was found
that when both amino acids at the ligation site are glycines, the
yield is as high as 92% (entry 1). When one of the two amino acids
is glycine (either at the thioester side or the other side), the ligation
yield is around 60–70% (entries 2–4). Importantly, when both ami-
no acids are alanine, the ligation yield is 45% (entry 5). Therefore,
the new type of auxiliary can allow for the ligation at a non-Cys
and non-Gly site. This property is of importance because the earlier
auxiliaries attached to the N-terminal nitrogens cannot be used for
ligations at non-Gly sites under normal NCL conditions. Only the
sugar-assisted auxiliaries and the present method can allow for
ligations at non-Gly sites because in both cases the nucleophile
in the S-to-N acyl transfer is a primary amine. Nonetheless, the
ligation between Val and Ala exhibits a low yield in our study (en-
try 6), presumably because the steric hinderance in this particular
case is too high.
At this point we have demonstrated that the new auxiliary can
be attached to the peptide in a removable fashion. More impor-
tantly, the new auxiliary can allow for the ligation between non-
Cys and non-Gly residues. Admittedly the ligation yields at non-
Gly sites are relatively low. However, we believe that this problem
can be solved by the next generation of spacer design in our ensu-
ing study, in which we will incorporate a ring structure (e.g., a
cyclohexanyl group like that in sugar-assisted auxiliary7) into the
spacer.
Br
STrt
TrtSH
Br
N
OH
N
O
N O
NaH
87%
47%
O
O
O
O
TFA/TIPS
tBuSH, I2
70%
O
S
NaBH4
80%
H2N
S
H2N
STrt
3
Scheme 6. Synthesis of compound 3.
Ph
O
S
Ph
HO
H2N
S
Tf2O
3
O
O
TfO
2,6-Lutidine
65%
Ph
O
O
Ph
H
n-BuNH2
14%
S
O
O
N
S
O
N
S
S
N
H
Bu
Bu
H
O
O
4
Ph
H
Ph
O
O
H
N
FmocHN
N
H2N
N
O
Bu
N
O
piperidine
Fmoc-Ala-Cl
54%
O
O
DCM
70%
S
S
S
S
5b
To conclude, in this Letter we report the first generation of new
thiol-containing auxiliaries that are placed at the amidyl N-atom in
the second amino acid residue of a peptide fragment.11–13 The
experimental results provide proof of principle for the utility of
the new auxiliaries that ligation can now be conducted at non-
Cys and non-Gly sites with modest yields. Compared to other re-
cently developed auxiliaries (e.g., sugar-assisted auxiliaries7), an
important feature of the current design is that the new auxiliaries
are generally applicable and readily removable. Further studies are
in progress to evolve a second generation of removable, thiol-con-
taining auxiliaries attached to the amidyl N-atom that allows for
ligation at non-Cys and non-Gly sites with more satisfactory yields.
Scheme 7. Synthesis of the removable auxiliary.
Table 1
Ligation yields between different amino acid residuesa
tBuSS
R
R'
O
N
O
Cbz
S
ligated
+
N
H
H2N
Aa2
NHBu
tri-peptide
O
O
Ph
Aa1
6a-6c
Acknowledgments
5a-5b
Aa2
Entry
Aa1
Yieldb (%)
This work was supported by NSFC (Nos. 20802040 and
20932006), and the Specialized Research Fund for the Doctoral
Program of Higher Education of China (Grant No. 200800030074).
1
2
3
4
5
6
Gly
Ala
Val
Gly
Ala
Val
Gly
Gly
Gly
Ala
Ala
Ala
92
68
58
66
45
<10
References and notes
1. (a) Cacciapaglia, R.; Stefano, S. D.; Mandolini, L. Acc. Chem. Res. 2004, 37, 113;
(b) Coltart, D. M. Tetrahedron 2000, 56, 3449.
a
Conditions: 6 (5.0 mM), 5 (7.5 mM), TCEP (100 mM), 4:1 v/v NMP/buffer/GnHCl
(6 M)/Na2HPO4 (0.2 M), pH 7.3–7.5, 40 °C.
2. (a) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science 1994, 266,
776; (b) Dawson, P. E.; Kent, S. B. H. Annu. Rev. Biochem. 2000, 69, 923; (c)
Haase, C.; Seitz, O. Angew. Chem., Int. Ed. 2008, 47, 1553; (d) Hackenberger, C. P.
R.; Schwarzer, D. Angew. Chem., Int. Ed. 2008, 47, 10030; (e) Kent, S. B. H. Chem.
Soc. Rev. 2009, 38, 338.
b
HPLC yield.
3. Johnson, E. C. B.; Kent, S. B. H. J. Am. Chem. Soc. 2006, 128, 6640.
4. (a) Warren, J. D.; Miller, J. S.; Keding, S. J.; Danishefsky, S. J. J. Am. Chem. Soc.
2004, 126, 6576; (b) Merkx, R.; Brouwer, A. J.; Rijkers, D. T. S.; Liskamp, R. M. J.
Org. Lett. 2005, 7, 1125; (c) Muir, T. W.; Sondhi, D.; Cole, P. A. Proc. Natl. Acad.
Sci. U.S.A. 1998, 95, 6705; (d) Saleh, L.; Perler, F. B. Chem. Recl. 2006, 6, 183; (e)
Muir, T. W. Annu. Rev. Biochem. 2003, 72, 249.
To this end, we have synthesized compound 3 as outlined in
Scheme 6. From readily available a-hydroxycarboxylic acid (which
can be made from the corresponding amino acid), we then obtain
a
the desired N (oxyethanethiol)-amino acid 4 through a displace-
5. (a) Offer, J.; Boddy, C. N. C.; Dawson, P. E. J. Am. Chem. Soc. 2002, 124, 4642; (b)
Botti, P.; Carrasco, M. R.; Kent, S. B. H. Tetrahedron Lett. 2001, 42, 1831; (c)
Marinzi, C.; Bark, S. J.; Offer, J.; Dawson, P. E. Bioorg. Med. Chem. 2001, 9, 2323;
(d) Clive, D. L. J.; Hisaindee, S.; Coltart, D. M. J. Org. Chem. 2003, 68, 9247; (e)
Tchertchian, S.; Hartley, O.; Botti, P. J. Org. Chem. 2004, 69, 9208; (f) Kawakami,
T.; Aimoto, S. Tetrahedron Lett. 2003, 44, 6059; (g) McGinty, R. K.; Kim, J.;
Chatterjee, C.; Roeder, R. G.; Muir, T. W. Nature 2008, 453, 812; (h) Offer, J.;
Dawson, P. E. Org. Lett. 2000, 2, 23; (i) Macmillan, D.; Anderson, D. W. Org. Lett.
2004, 6, 4659; (j) Low, D. W.; Hill, M. G.; Carrasco, M. R.; Kent, S. B. H.; Botti, P.
ment reaction with inversion of stereochemistry (Scheme 7). With
the amino acid residue 4 in hand, we can easily make the peptide.
Noteworthily, Kent and co-workers have demonstrated that the
oxylalkyl group on the amide bond can be selectively removed
by facile treatment with Zn in acidic medium to give a native pep-
a
tide after the ligation. Therefore, by using N (oxyethanethiol)-ami-
no acid we can solve the first problem.

1796
J. Li et al. / Tetrahedron Letters 51 (2010) 1793–1796
Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 6554; (k) Wu, B.; Chen, J. H.; Warren, J. D.;
8. De Koning, M. C.; Petersen, L.; Weterings, J. J.; Overhand, M.; Filippov, D. V.
Tetrahedron 2006, 62, 3248.
9. Haase, C.; Seitz, O. Eur. J. Org. Chem. 2009, 2096.
Chen, G.; Hua, Z. H.; Danishefsky, S. J. Angew. Chem., Int. Ed. 2006, 45, 4116.
6. (a) Kemp, D. S.; Leung, S. L.; Kerkman, D. J. Tetrahedron Lett. 1981, 22, 181; (b)
Kemp, D. S.; Kerkman, D. J. Tetrahedron Lett. 1981, 22, 185; (c) Kemp, D. S.;
Galakatos, N. G.; Bowen, B.; Tan, K. J. Org. Chem. 1986, 51, 1829; (d) Kemp, D. S.;
Galakatos, N. G.; Dranginis, S.; Ashton, C.; Fotouhi, N.; Curran, T. P. J. Org. Chem.
1986, 51, 3320; (e) Fotouhi, N.; Galakatos, N. G.; Kemp, D. S. J. Org. Chem. 1989,
54, 2803.
7. (a) Brik, A.; Wong, C.-H. Chem. Eur. J. 2007, 13, 5670; (b) Brik, A.; Yang,
Y.-Y.; Ficht, S.; Wong, C.-H. J. Am. Chem. Soc. 2006, 128, 5626; (c) Yang, Y.-
Y.; Ficht, S.; Brik, A.; Wong, C.-H. J. Am. Chem. Soc. 2007, 129, 7690; (d)
Brik, A.; Ficht, S.; Yang, Y.-Y.; Bennett, C. S.; Wong, C.-H. J. Am. Chem. Soc.
2006, 128, 15026; (e) Payne, R. J.; Ficht, S.; Tang, S.; Brik, A.; Yang, Y.-Y.;
Case, D. A.; Wong, C.-H. J. Am. Chem. Soc. 2007, 129, 13527; (f) Ficht, S.;
Payne, R. J.; Brik, A.; Wong, C.-H. Angew. Chem., Int. Ed. 2007, 46, 5975; (g)
Payne, R. J.; Ficht, S.; Greenberg, W. A.; Wong, C.-H. Angew. Chem., Int. Ed.
2008, 47, 4411; (h) Lutsky, M.-Y.; Nepomniaschiy, N.; Brick, A. Chem.
Commun. 2008, 1229.
10. (a) Canne, L. E.; Bark, S. J.; Kent, S. B. H. J. Am. Chem. Soc. 1996, 118, 5891; (b)
Shao, Y.; Lu, W.; Kent, S. B. H. Tetrahedron Lett. 1998, 39, 3911.
11. Compound 1a: HR-ESI m/z: 327.15584 [M+H]+ (calcd for C16H26N2OS2
326.52044); compound 1b: HR-ESI m/z: 341.17154 [M+H]+ (calcd for
C17H28N2OS2 340.54702); compound 1c: HR-ESI m/z: 355.18703 [M+H]+
(calcd for C18H30N2OS2 354.57360); compound 1d: HR-ESI m/z: 389.17174
[M+H]+ (calcd for C21H28N2OS2 388.58982); compound 1e: HR-ESI m/z:
403.18720 [M+H]+ (calcd for C16H26N2OS2 402.61640).
12. Ligation product from compound 1b of Scheme 5: ESI-MS m/z: 450.4 [M+Na]+
(calcd for C23H29N3O3S 427.2).
13. Products of Table 1. Entry 1: ESI-MS m/z: 545.2 [M+H]+ (calcd for C27H36N4O6S
544.2); entry 2: ESI-MS m/z: 559.2 [M+H]+ (calcd for C28H38N4O6S 558.3); entry
3: ESI-MS m/z: 587.1 [M+H]+ (calcd for C30H42N4O6S 586.3); entry 4: ESI-MS m/
z: 559.3 [M+H]+ (calcd for C28H38N4O6S 558.3); entry 5: ESI-MS m/z: 573.3
[M+H]+ (calcd for C29H40N4O6S 572.3).