An advance in proline ligation

Article abstract of DOI:10.1021/ja204277b

Native chemical ligation (NCL) is widely applicable for building proteins in the laboratory. Since the discovery of this method, many strategies have been developed to enhance its capability and efficiency. Because of the poor reactivity of proline thioesters, ligation at a C-terminal proline site is not readily accomplished. Here, we demonstrate that ligation at an N-terminal protein is feasible using the combined logic of NCL and metal-free dethiylation (MFD).

Full text of DOI:10.1021/ja204277b

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An Advance in Proline Ligation
Shiying Shang,† Zhongping Tan,† Suwei Dong,† and Samuel J. Danishefsky*,†,‡
†Laboratory for Bioorganic Chemistry, SloanÀKettering Institute for Cancer Research, 1275 York Avenue, New York, New York 10065,
United States
‡Department of Chemistry, Columbia University, 3000 Broadway, New York, New York 10027, United States
S Supporting Information
b
Scheme 1. NCL, Desulfurization, and Extended NCLa
ABSTRACT: Native chemical ligation (NCL) is widely
applicable for building proteins in the laboratory. Since the
discovery of this method, many strategies have been devel-
oped to enhance its capability and efficiency. Because of the
poor reactivity of proline thioesters, ligation at a C-terminal
proline site is not readily accomplished. Here, we demon-
strate that ligation at an N-terminal protein is feasible using
the combined logic of NCL and metal-free dethiylation
(MFD).
he groundbreaking discovery of native chemical ligation
(NCL) by Kent and colleagues was a seminal event in the
a P1 and P2 are peptide fragments. R can be alkyl or aryl. R1 can be
varied according to the amino acid structure.
T
chemical synthesis of protein-based biopolymers with precisely
defined structures.1,2 Typically, NCL involves the reaction be-
tween an unprotected peptidyl C-terminal thioester with another
peptide bearing an N-terminal cysteine. Following rate-deter-
mining trans-thioacylation, a fast SfN acyl transfer presumably
passing through a five-membered tetrahedral intermediate leads
to product (Scheme 1, eq 1, Kent et al.).3
While the original discovery of NCL was based on a peptidyl
N-terminal cysteine which appeared at the ligation site, it was soon
extendedto alanineligation, via desulfurization ofthe thiol function
by Raney Nickel reduction,4 to phenylalanine ligation, using nickel
boride as a reductant,5 and to serine ligation, through cyanobro-
mide-mediated cleavage of the methylcysteine precursor.6 The
finding that high yielding metal-free dethiylation is feasible in
an aqueous medium and specific to SH groups constituted a
major expansion of NCL (Scheme 1, eq 2). Not only did it enable
alanine ligation in the presence of existing ACM protected cys-
teines (as well as methionines), it opened up an entirely new set
of possibilities for ligation. One could now incorporate a thiol con-
taining unnatural amino acid at the N-terminus.7,8 The combina-
tion of thiol and amino groups allows for simulations of the
qualitative mechanism of cysteine based NCL. Metal-free dethiy-
lation (MFD) generates either a proteogenic or unnatural amino
acid at the ligation site (Scheme 1, eq 3).7a
C-terminal proline thioesters are not particularly effective acyl
donors, even inthe contextof cysteine-based NCL.17À19 While the
corresponding C-terminal proline p-nitrophenyl esters are more
effective,10 they too carry a serious liability, i.e. susceptibility to
hydrolysis in a ligation context. We werethus led to ask whether, in
light of the considerations discussed above, a proline-like moiety at
the N-terminus of a peptide might serve as a ligation site. This led us
to consider the possibility of utilizing an N-terminal thioproline at
the ligation site. In so doing, we would be extending the mechan-
istic concepts of NCL in two major respects. First, the SfN acyl
migration would have to occur even at a secondary amine in a ring
context. Furthermore, the transfer would be occurring in the con-
text of a bridged ring tetrahedral intermediate. To probe the fea-
sibility of the idea, we took recourse to the commercially available
diastereomeric 4-thioproline building blocks 1 and 2.
We began by synthesizing peptides 4 and 7 incorporating
N-terminal γ-thioprolines [(2S,4R)-Mpt and (2S,4S)-Mpt],
respectively, by the solid-phase peptide synthesis (SPPS) meth-
od using the Fmoc chemistry. We went on to evaluate the ligation
of both 4 and 7 with peptide 3, presenting a C-terminal glycine
residue. For this purpose, we took recourse to the surrogate acyl
donor system bearing a 2-(ethyldithiophenyl)ester, previously
invented in our laboratory as a more durable thioester equi-
valent.20,21 The required thioester is generated in situ, as previously
described, via unidirectional OfS acyl transfer.21 The reaction
between 4 and 3 was initiated by the addition of the standard phos-
phate ligation buffer andwas monitored by ultraperformance liquid
Using this MFD enabled logic, we and others have demon-
strated valine,9,10 leucine,11,12 threonine,13 and lysine ligations.14,15
Thus, MFD has brought with it a major expansion in the syn-
thesis of reasonably sized proteins, using the powerful underlying
logic of NCL.
The research described herein was directed to a remaining
but important problematic issue, i.e. that of proline ligation.16 It
turns out that, for reasons that have been elegantly interpreted,
Received: May 10, 2011
Published: June 23, 2011
r
2011 American Chemical Society
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Scheme 2. Proline Ligation with Two Pro(SH)
Diastereomersa
Table 1. Substrate Scope of Proline Ligationa
a Key: (a) 6 M Gn•HCl, 100 mM NaH2PO4, 50 mM TCEP, pH 7.5;
(b) TCEP, VA-044, tBuSH, MeCN/H2O, 37 °C, 10 min, 88%. P1:
ALLVNSSÀ; P2: ÀWEPLN; Ar = 2-(ethyldithio)phenyl.
chromatography (UPLC). As shown in Scheme 2, under stan-
dard ligation conditions, peptide 4 readily underwent trans-
thioesterification with 3 to afford 5 (90% conversion),22 within
10 min at room temperature.23 At a considerably slower rate, the
thioester intermediate 5 underwent the SfN acyl transfer reac-
tion to afford the desired peptide product in 85% yield within
2.5 h. By contrast, under identical reaction conditions, peptide 7,
incorporating the diastereomeric γ-thioproline, [(2S,4S)-Mpt],
demonstrated very low proclivity toward ligation with peptide 3.
Although the trans-thioesterification could be achieved as effi-
ciently as in the case of 5, progression to peptide product 9
essentially failed. After 28 h, less than 5% conversion was ob-
served. Instead, about 90% of thioester 8 was hydrolyzed to
peptide 7.
This finding of a large difference is readily interpreted. Thus, in
the case of the productive trans series, in the presumed tetra-
hedral SfN acyl transfer intermediate, the large COP2 sub-
structure is exo. By contrast, in the unproductive cis series, based
on 2, the large COP2 moiety would have to be contained in a
highly hindered endo disposition.
Having established an efficient and practical γ-thioproline
ligation protocol, albeit only with a terminal glycine, we sought to
probe the versatility of this protocol in the context of varying the
nature of the C-terminal amino acid residue. Toward this end, we
prepared several peptide esters, which would allow us to evaluate
the effects of steric hindrance on peptide bond formation. In
order to avoid any potential complications associated with resi-
dues not involved in amide bond formation, these peptide esters
only differ by a side chain at the C-terminus. In accordance with
previous NCL studies, the rate and efficiency of ligation were
found to be dependent on the steric hindrance of the C-terminal
residue.9,10,12,16 Under standard conditions, peptide 10 and 12
underwent rapid trans-thioesterification with the peptide 4 to
generate the corresponding thioester intermediates. Although
more time was required to complete the SfN acyl transfer, good
yields were obtained (entries 1 and 2). As expected, the coupling
reactions at the valine and proline sites were significantly less
efficient (entries 3 and 4). Interestingly, the factors that caused the
low conversion in these two reactions are different. In the case of
valine, the trans-thioesterification step was fast and apparently effi-
cient. However, the resulting thioester intermediate was insufficiently
a Reaction conditions: 6 M Gn•HCl, 100 mM NaH2PO4, 50 mM TCEP,
pH 7.5. P1: ALLVNSSÀ; P2: ÀWEPLN; P3: ÀPPWEPLN; Ar =
2-(ethyldithio)phenyl.
reactive to allow for reasonable rates of acyl transfer. By contrast,
the proline peptide ester 16 was found to exhibit remarkably
diminished levels of reactivity in the trans-thioacylation step.
Only about 5% of 16 was converted to the thioester intermediate,
which was rapidly hydrolyzed under attempted ligation condi-
tions to peptide 4.
Next, in order to probe the origin of the observed low reactivity
of the proline peptide ester 16, an additional control ligation was
performed between peptide 4 and proline ester 18. Under the
standard conditions, 4 and 18 underwent rapid transthioesterifica-
tion and acyl transfer to furnish adduct 19 in 73% yield within 3 h
(entry 5). This result agreed well with the observations of Kent
et al., suggesting that the presence of a peptidyl group on the
R-amine of the C-terminal proline is the dominant factor affecting
the reactivity of the proline peptide ester in NCL.17
Furthermore, the versatility of the proline ligation protocol was
demonstrated in the synthesis of the proline-rich peptide 21. In
the event, peptide 20, containing 50% proline, was coupled with
the glutamine peptide ester 10. The result is shown in Table 1,
entry 6. Within 10 h, a 66% isolated yield of 21 was obtained.
Finally, we demonstrated the ability to efficiently convert the
γ-thioproline to the natural amino acid proline in the context of a
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COMMUNICATION
complex peptide. Upon exposure to our standard metal-free de-
sulfurization conditions, the γ-thioprolines containing peptides
11, 13, 19, and 21 were readily converted to the target peptide
products in less than 10 min.7a,10,12,24
(3) Johnson, E. C.; Kent, S. B. J. Am. Chem. Soc. 2006, 128, 6640.
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Since both native chemical ligation and direct aminolysis are
possible options for the synthesis of large peptides and small
proteins, we also directly compared these two methods for
synthesizing desulfurized 11.25,26 The direct aminolysis reaction
was conducted in DMSO using modified BlakeÀAimoto condi-
tions (Supporting Information, Figure S4).27 At room tempera-
ture, it proceeded well to give the coupling product in a slightly
higher yield. However, because of the difficulties related to the
preparation and purification of partially protected peptide frag-
ments28 and the risk of epimerization of the residue on the
C-terminal side of the condensation site,27 in many cases, proline
ligation would be a better choice for coupling two synthetic
fragments at the proline site.
In summary, an efficient and broadly useful two-stage proline
ligation protocol has been developed. The likely basis for the
different ability of the peptide esters to undergo proline ligation
has been rationalized. The proline ligation approach provides a
significant advance in the field of protein chemical synthesis and
will likely find broad application in the study of proline-rich
proteins and glycoproteins. It is of note that proline-rich proteins
frequently participate in diverse signaling pathways and serve
many crucial biological functions.29,30 The unique conforma-
tional properties of proline inserts provide the molecular basis for
highly discriminatory recognition during the formation of multi-
protein signaling complexes. Understanding how these interac-
tions contribute to the stability of the complex should prove
valuable in the rational design of peptide mimics that could
potentially be used to disrupt the interfaces between proline-rich
proteins and their binding partners.31 The methodology devel-
oped herein is expected to facilitate the preparation of proline-
rich polypeptide probe structures, thus enhancing opportunities
for detailed analysis of proteinÀprotein interactions.
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(17) Pollock, S. B.; Kent, S. B. Chem. Commun. 2011, 47, 2342.
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(20) Warren, J. D.; Miller, J. S.; Keding, S. J.; Danishefsky, S. J. J. Am.
Chem. Soc. 2004, 126, 6576.
(21) Chen, G.; Warren, J. D.; Chen, J.; Wu, B.; Wan, Q.; Danishefsky,
S. J. J. Am. Chem. Soc. 2006, 128, 7460.
(22) Thioester intermediate 5 could be observed by UPLC analysis
and was differentiated from the N-linked product 6a by the addition of
MESNa, where the disappearance of 5 would be observed.
(23) Canne, L. E.; Bark, S. J.; Kent, S. B. H. J. Am. Chem. Soc. 1996,
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’ ASSOCIATED CONTENT
S
Supporting Information. Experimental procedures and
b
characterization data. This material is available free of charge via
the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
s-danishefsky@ski.mskcc.org
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1997, 26, 259.
’ ACKNOWLEDGMENT
Support for this research was provided by the National Institute
of Health (CA28824 to S.J.D.). We thank Rebecca Lambert for
valuable discussions. We also thank Dr. George Sukenick, Hui
Fang, and Sylvi Rusli of SKI’s NMR core facility for mass spectral
and NMR assistance and Laura Wilson and Jason Chan for
assistance with the preparation of the manuscript.
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