Insights into the finer issues of native chemical ligation: An approach to cascade ligations

Article abstract of DOI:10.1002/anie.201005513

Maximizing by minimizing: An efficient and broadly useful two-step ligation protocol was developed. Important mechanistic issues of ligation were probed from competition studies on the formation of diastereomeric ligation products. This study provides a valuable approach to facilitate polypeptide synthesis by minimizing protecting group manipulations and intermediate isolations. Copyright

Full text of DOI:10.1002/anie.201005513

Communications
DOI: 10.1002/anie.201005513
Peptide Coupling
Insights into the Finer Issues of Native Chemical Ligation:
An Approach to Cascade Ligations**
Zhongping Tan, Shiying Shang, and Samuel J. Danishefsky*
We have been studying the total synthesis of complex
glycopeptide and glycoprotein targets.[1] Pursuant to this
goal, we hope to discover broadly useful methods to join large
peptide and glycopeptide fragments while minimizing the
need for side-chain protection.[2] A field-changing contribu-
tion to the problem of polypeptide ligation, termed native
chemical ligation (NCL), was provided by Kent and co-
workers in 1994 (Scheme 1).[3] NCL involves the merger of a
We next turned to the possibility of extending NCL logic
to accomplish leucine ligation.[7] We first prepared the
diastereomeric b-thioleucine surrogates 1 [leu(SSMe)] and 2
[which we term leu(SSMe)*] (Scheme 2a). The synthesis of 1
commenced with commercially available (2S,3S)-3-hydroxy-
leucine (3) and passed through 4 and 5 as shown in Scheme 2.
Compound 2 was prepared through an analogous sequence
from (2S,3R)-3-hydroxyleucine (6).[4c,6a]
In addition to studying the feasibility and quality of the
projected leucine ligations, we anticipated that the availability
of two epimeric leucine surrogates (compounds 1 and 2) as
potential probes, could also provide a basis for studying rather
subtle, otherwise hidden issues of native chemical ligation.
Accordingly, we prepared the peptides described below
(Scheme 2b). In all cases, the acyl donor component of the
ligation was presented as a masked thiol ester of a type we had
previously described.[2a] The required peptides (vide infra)
with C-terminal methyl esters or free carboxylic acids and N-
terminal leucine surrogates were prepared using HATU-
Scheme 1. NCL and alanine ligation.
peptide domain possessing a C-terminal thioester fragment
with a second peptide bearing an N-terminal cysteine residue.
The key mechanistic features of NCL—transthioesterification
and S!N acyl transfer—are outlined in Scheme 1. Clearly if
the primary thio group could be desulfurized, the NCL
method can be used to accommodate alanine ligation.[4] Of
course, for this to work well, other sulfur moieties within the
construct must withstand the desulfurization reaction. A
major advance in this regard was accomplished in a metal-free
fashion, using classical mechanistic insights in free radical
mediated desulfurization.[5]
We then set about to apply, more generally, the overall
logic of NCL to other proteogenic amino acids. The thought
was to synthesize non-proteogenic amino acids, bearing
strategically placed thiol groups, to serve as the N-terminal
residues in the ligation event. In this way the logic of NCL
could, in principle, be broadly extended. In each case, the
concluding step would exploit our metal-free desulfurization
method. Indeed, this was accomplished for valine, lysine, and
threonine ligations.[6]
Scheme 2. Synthesis of peptide substrates. Reagents and conditions:
A) a) Boc2O, Na2CO3, THF/H2O, RT, 91%; b) TMSE-OH, DCC, DMAP,
CH2Cl2, 08C!RT, 99%; c) MsCl, Et3N, CH2Cl2, 08C; d) AcSK (excess),
DMF, RT, 408C!608C, 82% over two steps; e) NaOH, MeOH, 08C;
f) MMTS, DIEA, CH2Cl2, RT, 79% over two steps; g) TBAF, THF, RT,
98%. B) a) MeOH, DCC, DMAP, CH2Cl2; b) piperidine, CH2Cl2; c) Boc-
Leu(SSMe)-OH, HATU, DIEA, DMSO; d) TFA/H2O/TIS (95:2.5:2.5);
e) EDCI, HOOBt, CHCl3/TFE. TMSE=trimethylsilylethyl, DCC=dicy-
clohexylborane, DMAP=4-dimethylaminopyridine, AcSK=potassium
thioacetate, DMF=dimethylformamide, MMTS=methane methylthio-
sulfonate, DIEA=ethyldiisopropylamine, TBAF=tetrabutylammonium
fluoride, Boc=tert-butyloxycarbonyl, HATU=O-(7-azabenzotriazol-1-
yl)tetramethyluronium hexafluorophosphate, DMSO=dimethylsulfox-
ide, TFA=trifluoroacetic acid, TIS=triisopropylsilane, EDCI=N’-(3-
dimethylaminopropyl)-N-ethylcarbodiimide, TFE=2,2,2-trifluoroetha-
nol.
[*] Prof. S. J. Danishefsky
Department of Chemistry, Columbia University
3000 Broadway, New York, NY 10027 (USA)
Fax: (+1)212-772-8691
E-mail: s-danishefsky@ski.mskcc.org
Dr. Z. Tan, Dr. S. Shang, Prof. S. J. Danishefsky
Laboratory for Bioorganic Chemistry
Sloan-Kettering Institute for Cancer Research
1275 York Avenue, New York, NY 10065 (USA)
[**] Support for this research was provided by the National Institutes of
Health (CA28824 to S.J.D.).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201005513.
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Table 1: Substrate scope of leucine ligation and desulfurization.
mediated peptide coupling reactions in the presence of
1 equivalent of synthetic leucine precursor.[1c] The peptides
bearing ortho-disulfide phenolic esters at the C-termini were
synthesized using EDCI mediation under the non-epimeriz-
ing conditions developed by Sakakibara and co-workers.
(Scheme 2b)[8]
Entry
C-Terminal
peptide
Ligation
product
Conv.
[%]
t
[h]
We began by comparing the quality of ligation of the two
leucine epimers by joining both 10 and 11 with peptide 9,
presenting a C-terminal phenylalanine residue (Scheme 3).[6a]
The impact of thioleucine configuration on the ligation was
striking. Thus, under standard conditions, peptide 10 readily
underwent coupling with 9 to afford 12. Within 15 min, the
reaction had achieved ca. 50% conversion (by UPLC-MS),
and a 75% yield was obtained as shown. In the case of peptide
11, less than 7% conversion was observed after 15 min. After
30 h, an 18% yield of 13 could be obtained. The mechanistic
implications of this type of finding will be discussed below.
With the preferred leucine amino acid surrogate estab-
lished (i.e. 1), we next probed the versatility of the leucine
ligation protocol. Not surprisingly, the rate and efficiency of
ligation was found to be qualitatively dependent on the level
of steric hindrance at the C-terminus residue. Thus, under
standard conditions, peptide 10 underwent rapid ligation with
the C-terminal glycine peptide, 14, to furnish adduct 15 in
95% conversion within 2 h (Table 1, entry 1). Although
ligation with the C-terminal alanine peptide, 16, was some-
what less facile, a reasonable conversion of ligation product
was obtained (85%, Table 1, entry 2). As expected, peptides
18 and 20, presenting C-terminal valine and proline residues,
respectively, were significantly less reactive as acyl donors and
gave accordingly lower yields (Table 1, entries 4 and 5). The
ability to efficiently convert the b-thioleucine surrogates to
leucines in the context of their primary ligation products,
upon exposure to our standard metal-free desulfurization
conditions, is shown in entries 3 and 4 in Table 1.[5]
1
2
95
85
2
2.5
3
83
2.5
4
5
50
21
8
9
[a] 6m Gn·HCl, 100 mm NaH2PO4, 50 mm TCEP, pH 7.5; ratio C-
terminal peptide/10 = 1.5:1. [b] TCEP, VA-044, tBuSH, 0.8 h. Peptide 1:
ꢀ
ꢀ
GKHLNSAERVE ; Peptide 2: RKKLQDVHNFVALG-OMe.
To approach this question more precisely, we designed a
competition experiment that would allow for rough compar-
ison of the acyl acceptor qualities of the two epimeric N-
termini under identical conditions. In the event, peptides 10
and 24, bearing N-terminal leu(SSMe) and leu(SSMe)*
residues were treated with the C-terminal glycine peptide 14
under standard ligation conditions. The results are shown in
Scheme 4. Since the yield of the ligation of the N-terminal
leu(SSMe)* epimer is poor (Scheme 3, vide supra), it is not
possible to extract hard numbers for the relative rates of acyl
acceptor reactivity of epimers leu(SSMe) and leu(SSMe)*. By
examining the relative amount of ligation products in the
early stages of the experiment, we estimate the ratio to be at
least 20:1 in favor of fast reacting epimer leu(SSMe). In the
limiting case, assuming the inherent acyl acceptor properties
of the thiol groups in leu(SSMe) and leu(SSMe)* were nearly
the same, and each intermediate suffered S!N acyl transfer
with comparable efficiency, the ratio of effective ligation
would be ca. 1:1. The actual comparative yields reported in
Scheme 3 strongly suggest that the acyl transfer step is
substantially slower in the case of leu(SSMe)* relative to
leu(SSMe). This finding can be rationalized, since in the case
of leu(SSMe), S!N acyl transfer requires a trans relationship
of the substituents on the 5-membered ring, while in the case
of the epimeric leu(SSMe)* system, the corresponding
isopropyl and peptidic residues are cis. The attenuated rate
of acyl transfer in the case of leu(SSMe)* presumably renders
its thioester intermediate more vulnerable to competitive
adventitious hydrolysis.
The substantial difference in the performance of peptides
terminating in surrogate 1 and surrogate 2 as acyl acceptor
encouraged us to probe more intensively into some of the
mechanistic issues associated with native chemical ligation.[9]
The widely held notion is that the transthioesterification step
is rate determining, and that the acyl transfer of the C-
terminal coupling component to nitrogen is rapid (see
Scheme 1). This accounts for the inability to clearly identify
any intermediate thioesters en route to ligation.[9]
Scheme 3. Leucine ligation with two leu(SSMe) diastereomers.
Reagents and conditions: a) 6m Gn·HCl, 100 mm NaH2PO4, 50 mm
ꢀ
ꢀ
TCEP, pH 7.5. Peptide 1: GKHLNSAERVE ; Peptide 2:
RKKLQDVHNFVALG-OMe. Gn·HCl=guanidine hydrochloride,
TCEP=tris(2-carboxyethyl)phosphine.
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Scheme 5. The competition reaction between leu(SH) and cys to form
peptide-coupling products. a) 6m Gn·HCl, 100 mm NaH2PO4, 50 mm
ꢀ
TCEP, pH 7.5. Peptide 1: GKHLNSAERVE ; Peptide 3:
ꢀ
RKKLQDVHNFVALG-OH.
Scheme 4. The competition reaction between leu(SH) and leu(SH)* to
form peptide-coupling products. a) 6m Gn·HCl, 100 mm NaH2PO4,
Finally, we were able to demonstrate the complexity-
building capacity of this chemistry. The thought was to
develop a kinetically controlled re-iterative coupling strategy,
which would exploit the reactivity differential between the N-
terminal thioleucine surrogate and cysteine acyl acceptors.[11]
In keeping with a major concern of our laboratory, we
envisioned, as a goal, the rapid assembly of the human
erythropoietin (hEPO) peptide, 34, from three individual
fragments through sequential cysteine and thioleucine liga-
tions.
ꢀ
50 mm TCEP, pH 7.5. Peptide 1: GKHLNSAERVE ; Peptide 2:
ꢀ
RKKLQDVHNFVALG-OMe; Peptide 3: RKKLQDVHNFVALG-OH.
Furthermore, the rates of the transthioesterification steps
may also be quite different for the two epimeric surrogates.
For intramolecular transfer to occur, the proton on the thiol
acyl acceptor group must be transferred to a putative “base”
or to the bulk solvent under weakly basic conditions (pH
ꢁ 7.5). A possibility in this regard is that a proton of the
neighboring NH3+ group is donated to medium, thus allowing
the nitrogen to remove the proton from the sulfur. Once
again, in the case of leu(SSMe), such a transfer would require
a trans relationship of the large substituents, while in the case
of leu(SSMe)*, they are cis (see Scheme 4).
We then posed the question as to how the productive
epimer, leu(SSMe), might compare with cysteine in a com-
petitive experiment. Required substrates were prepared as
described and the experiment crafted as shown in Scheme 5.
Because intermolecular transthioesterification is sup-
posed to be rate determining, one might anticipate that the
primary thiol of the cysteine residue would react more rapidly
than the secondary thiol of the leu(SSMe) surrogate.[9]
Indeed, as shown in Scheme 5, peptide 29, arising from
ligation of the cysteine-bearing peptide, 27, was found to be
the predominant product (29/28 ꢁ 4:1). Thus, the observed
margin of selectivity was less than that expected on the basis
of “A-value” comparisons between the H and isopropyl
substituents. Apropos of the arguments outlined above,
explanations for this apparent discrepancy may be proposed.
Putative ring character is clearly involved in the intra-
molecular S!N acyl transfer step in NCL. As argued above,
it could also be involved in the required de-protonation of the
thiol group en route to transthioacylation. In either case such
ring formation, in the case of the fast reacting pre-leucine
compound, is favored by an argument similar to the classical
Thorpe–Ingold effect, since the acyclic array is more sub-
stituted.[10] The corresponding pertinent acylic ensemble in
the case of the N-terminal cysteine is substantially less
substituted. Therefore it might benefit far less from cycliza-
tion.
The experiment was crafted as shown in Scheme 6. We
first undertook to connect 32 and 31. The resulting gross
product was capped by a second ligation to 30. In the event, a
61% yield of the desired sequential double ligation product
33 was isolated. This product indeed corresponds to the
sequence 32 + 31 + 30. We were unable to find the single
ligation product corresponding to the self-coupling of 31,
though it could well have been missed (perhaps due to further
oligomerization). Interestingly, we also did not detect a
double ligation product arising from a sequence 31 + 31 + 30.
One product, implicated in ca. 5% yield,[12] presumably
corresponds to initial cyclization (i.e. thiolactonization) of 31.
We also identified a monoligation product arising from the
coupling of 32 with 30. In principle, it is the thiolactonization
side reaction, which is responsible for “leftover” 32 and 30.
It is well to note that every self-coupling of 31 + 31 serves
to deplete two equivalents of the “competing substrates”.
Hence, in principle, even a 4:1 acyl acceptor reactivity ratio of
32/31 would be leveraged to provide an ca. 8:1 factor
“against” the hypothetical (unobserved) product of a single
ligation of 31
+
30.[13] Obviously, any further level of
oligomerization of 31 would provide greater leveraging of
the selectivity for forming 33 rather than the unobserved
products (31 + 31 + 30 or 31 + 30).
In summary, the chemistry described above started with
the central concept of native chemical ligation. To extend this
core idea, we have devised an effective route to generating
the required C-terminal thioester acyl donor through an
intramolecular O!S transfer in the same step where the
cysteine (or surrogate) thiol is exposed. Sterochemically
defined leucine ligation surrogates were synthesized and used
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b) J. Chen, G. Chen, B. Wu, Q. Wan, Z. Tan, Z. Hua, S. J.
Danishefsky, Tetrahedron Lett. 2006, 47, 8013; c) Z. Tan, S.
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2009, 131, 5424; d) Y. Yuan, J. Chen, Q. Wan, Z. P. Tan, G. Chen,
C. Kan, S. J. Danishefsky, J. Am. Chem. Soc. 2009, 131, 5432;
e) C. Kan, J. D. Trzupek, B. Wu, G. Chen, Z. P. Tan, Y. Yuan, S. J.
Danishefsky, J. Am. Chem. Soc. 2009, 131, 5438; f) P. Nagorny, B.
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[2] a) J. D. Warren, J. S. Miller, S. J. Keding, S. J. Danishefsky, J. Am.
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1994, 266, 776; b) J. P. Tam, Y. A. Lu, C. F. Liu, J. Shao, Proc.
Natl. Acad. Sci. USA 1995, 92, 12485.
Scheme 6. Synthesis of EPO(95-120) peptide (34). Reagents and con-
ditions: a) 6m Gn·HCl, 100 mm NaH2PO4, 50 mm TCEP, pH 7.5, 0.5 h;
b) 6m Gn·HCl, 100 mm NaH2PO4, 50 mm TCEP, pH 7.5, 0.5 h;
MESNa, H2O/MeCN (1:1), 1 min, 61% over two steps. c) TCEP, VA-
044, tBuSH, 1 h, 82%. MESNa=sodium-2-mercaptoethane sulfonate,
VA-044=2,2’-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride.
[4] a) L. Z. Yan, P. E. Dawson, J. Am. Chem. Soc. 2001, 123, 526;
b) B. L. Pentelute, S. B. Kent, Org. Lett. 2007, 9, 687; c) D. Crich,
A. Banerjee, J. Am. Chem. Soc. 2007, 129, 10064.
[5] a) Q. Wan, S. J. Danishefsky, Angew. Chem. 2007, 119, 9408;
Angew. Chem. Int. Ed. 2007, 46, 9248; for the seleno analog, see:
b) N. Metanis, E. Keinan, P. E. Dawson, Angew. Chem. 2010,
122, 7203; Angew. Chem. Int. Ed. 2010, 49, 7049.
[6] a) J. Chen, Q. Wan, Y. Yuan, J. Zhu, S. J. Danishefsky, Angew.
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Angew. Chem. Int. Ed. 2008, 47, 6807; c) J. Chen, P. Wang, J. L.
Zhu, Q. Wan, S. J. Danishefsky, Tetrahedron 2010, 66, 2277; d) R.
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Soc. 2009, 131, 13592.
[7] During the preparation of this manuscript, Brik and co-workers
reported a native chemical ligation at leucine. However, these
authors did not investigate the relative rates of the two
diastereomeric leucine surrogates as the key selectivity versus
reported herein. Z. Harpaz, P. Siman, K. S. Kumar, A. Brik,
ChemBioChem 2010, 11, 1232.
to probe important mechanistic issues involved in native
chemical ligation. Selectivities in acyl acceptor efficacies in
NCL were explored from several considerations. First,
evidence has been brought to bear that the rate of the S!N
intramolecular acyl transfer (Scheme 1, step b) can be
undermined by imposing a steric hindrance restraint at the
stage where a formation of mechanistically required ring is
required. Similar issues may also impact on the first step of
the process, that is, transthioesterification (Scheme 1, step a).
We postulate that, perhaps, even in this opening step, quasi
ring formation may be a central element in directing a
deprotonation of the acyl acceptor thiol group (see Scheme 4,
structure 24). Finally, we further suggest that increased
substitution in the acyclic system can perhaps be a factor in
the transthioesterification step by favoring ring formation in
the transfer of the thiol proton by intramolecular means (see
Scheme 5).[10]
A combination of these considerations served to establish
a selectivity margin between N-terminal cysteine, itself, and
the N-terminal thiol containing precursor of leucine. Com-
bining the principles, we realized a rather promising sequen-
tial ligation, wherein peptide A (cf. 31) couples with peptide B
(cf. 32) to give peptide C (not purified), which in turn couples
with peptide D (cf. 30) to give rise to peptide F (cf. 33). The
implications of these findings for the synthesis of critical,
biologically relevant peptides and glycopeptides are being
pursued.[14]
[8] S. Sakakibara, Biopolymers 1995, 37, 17.
[9] a) S. B. Kent, Chem. Soc. Rev. 2009, 38, 338; b) E. C. Johnson,
S. B. Kent, J. Am. Chem. Soc. 2006, 128, 6640; c) T. W. Muir,
Annu. Rev. Biochem. 2003, 72, 249; d) T. M. Hackeng, J. H.
Griffin, P. E. Dawson, Proc. Natl. Acad. Sci. USA 1999, 96,
10068; e) L. E. Canne, S. J. Bark, S. B. H. Kent, J. Am. Chem.
Soc. 1996, 118, 5891.
[10] R. M. Beesley, C. K. Ingold, J. F. Thorpe, J. Chem. Soc. Trans.
1915, 107, 1080. Of course, in its pure form, the classical Thorpe–
Ingold effect refers to the inclusion of a fully substituted
(quaternary) carbon in the cyclizing chain. However, as a general
matter, heavier substitution (in the absence of steric hindrance in
the resulting ring) tends to favor cyclization for reasons of
entropy.
[11] D. Bang, B. L. Pentelute, S. B. Kent, Angew. Chem. 2006, 118,
4089; Angew. Chem. Int. Ed. 2006, 45, 3985.
[12] This product was not isolated, per se, but inferred as shown in the
Supporting Information.
Received: September 2, 2010
Published online: November 4, 2010
[13] Of course, the 4:1 ratio established for the benchmark case in
Scheme 5 may not be applicable to the competition of 32 and 31.
[14] a) R. M. Wilson, S. J. Danishefsky, Pure Appl. Chem. 2007, 79,
2189; b) D. P. Gamblin, E. M. Scanlan, B. G. Davis, Chem. Rev.
2009, 109, 131.
Keywords: desulfurization · kinetic control · leucine ·
native chemical ligation · peptides
.
[1] a) B. Wu, Z. Tan, G. Chen, J. Chen, Z. Hua, Q. Wan, K.
Ranganathan, S. J. Danishefsky, Tetrahedron Lett. 2006, 47, 8009;
Angew. Chem. Int. Ed. 2010, 49, 9500 –9503
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
9503