Advances in proline ligation

Article abstract of DOI:10.1021/ja212182q

Application of native chemical ligation logic to the case of an N-terminal proline is described. Two approaches were studied. One involved incorporation of a 3R-substituted thiyl-proline derivative. Improved results were obtained from a 3R-substituted selenol function, incorporated in the context of an oxidized dimer.

Full text of DOI:10.1021/ja212182q

Article
pubs.acs.org/JACS
Advances in Proline Ligation
Steven D. Townsend,† Zhongping Tan,† Suwei Dong,† Shiying Shang,† John A. Brailsford,†
and Samuel J. Danishefsky*,†,‡
†Laboratory for Bioorganic Chemistry, Sloan-Kettering Institute for Cancer Research, 1275 York Avenue, New York, New York
10065, United States
‡Department of Chemistry, Columbia University, Havemeyer Hall, 3000 Broadway, New York, New York 10027, United States
S
* Supporting Information
ABSTRACT: Application of native chemical ligation logic to
the case of an N-terminal proline is described. Two approaches
were studied. One involved incorporation of a 3R-substituted
thiyl-proline derivative. Improved results were obtained from a
3R-substituted selenol function, incorporated in the context of
an oxidized dimer.
yielding, aqueous−compatible, SH specific, metal-free dethiy-
lation (MFD) method served to enhance, in a major way, the
reach of NCL logic.4 Through recourse to these MFD
conditions, NCL was extended, in our laboratory and others,
to include Ala,4 Val,5,6 Thr,7 Leu,8,9 and Lys10,11 (Scheme 1, eq
2). MFD conditions are both mild and highly selective and may
be employed in the presence of a range of peptide
functionalities, including Met residues, Acm-protected Cys
residues, and sensitive glycan domains.
INTRODUCTION
■
The development and application of novel strategies for the
convergent synthesis of homogeneous proteins represents a
prominent challenge in synthetic chemistry. The seminal
discovery, by Kent and co-workers, of native chemical ligation
(NCL) marked a striking advance in the field of peptide
synthesis.1 According to this powerful logic, a peptide bearing a
C-terminal aryl thioester (1) reacts with a second peptidyl
fragment, 2, which is equipped with an N-terminal cysteine
residue (Scheme 1, eq 1). Following rate-determining trans-
RESULTS AND DISCUSSION
■
Thio-Proline Ligation. We wondered about the possibility
of expanding the menu of noncysteine NCL inspired ligations
to include the significant challenge of proline ligation.12 It has
been observed that C-terminal proline thioesters are relatively
poor acyl donors, even in standard cysteine-based NCL
settings.13 The poor reactivity of the proline thioester
functionality has been attributed to deactivation of the thioester
by the proline amide carbonyl, rather than to steric factors. C-
terminal proline p-nitrophenyl esters are somewhat more
reactive, but these types of substrates are quite susceptible to
hydrolysis under ligation conditions. In light of the inherent
difficulties of accomplishing ligation at a C-terminal Pro
residue, we explored an alternative strategy, whereby an
appropriate thio-proline surrogate would reside at the N-
terminus of a peptide, 8 (Scheme 1, eq 3). Following thiol-
mediated ligation, the resultant intermediate, 9, would be
subjected to MFD to deliver the target peptide, 10, bearing a
Pro residue at the site of ligation. The successful development
of this strategy would represent an important expansion of the
NCL paradigm, in that it would involve S→N acyl transfer at a
secondary amine in the context of a ring system and would be
required to pass through a bridged tetrahedral intermediate
(see 8 → 9).
Scheme 1. Native Chemical Ligation (NCL)
thioesterification, a rapid, irreversible S→N acyl transfer (3→4)
generates the native amide bond.
Though originally limited to couplings that deliver a Cys
residue at the site of ligation, the scope of the NCL technology
was soon expanded, by Dawson and colleagues, via postligation
metal-based desulfurization. In this way, NCL could, in the end,
lead to ligations at Ala2 and Phe3 residues. While metal induced
dethiylation served to enhance the range of NCL logic, there
remained a significant need to accomplish RSH→RH
conversion under conditions which are compatible with water
as the solvent. The discovery, in our laboratory, of a high
Received: December 30, 2011
Published: February 15, 2012
© 2012 American Chemical Society
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Happily, our efforts along these lines were indeed successful,
and we recently described utilizing reduction in the envisioned
thio-proline ligation protocol.12 Interestingly, but not surpris-
ingly, reaction viability was found to be highly dependent on
the stereochemistry of the thio-Pro surrogate used. As shown in
Scheme 2, only the trans-thioproline surrogate was found to be
supported by the comparatively facile formation of adduct 23
from peptide 12 and an unsubstituted Pro amino acid, under
standard coupling conditions.
Thio-Proline Ligation: Application to a Possible
Synthesis of Homogeneous Erythropoietin. A long-term
effort in our laboratory is devoted to accomplishing the
chemical synthesis of homogeneous erythropoietin (EPO).14
Found in nature as a 166-residue protein bearing four sites of
glycosylation, EPO is the primary regulator of erythropoiesis
and is widely prescribed for the treatment of anemia. EPO
represents a particularly compelling synthetic target, due to its
challenging structure and therapeutic relevance. Moreover, at
least in principle, successful realization of our mission would
enable access, for the first time, to homogeneous erythropoie-
tins containing defined, but variable, glycoforms at the
conserved glycosylation sites. Such access could set the stage
for a combined chemistry/glycobiology program to learn more
about why nature glycosylates so many of its important
proteins.
Scheme 2. Thio-Proline Ligation with Two Pro(SH)
a
Diastereomers
a
In the context of our EPO synthetic effort, we recently
sought to assemble the bis-glycosylated hEPO(79−166)
fragment, 24 (Scheme 4).15 In a retrosynthetic sense, we
Key: (a) 6 M Gn•HCl, 100 mM NaH2PO4, 50 mM TCEP, pH 7.5.
P1: ALLVNSS−; P2: −WEPLN; Ar = 2-(ethyldithio)-phenyl; TCEP
= tris(2-carboxyethyl)phosphine.
a
a viable participant in the ligation step. In the productive trans
series, the large COP2 moiety is situated in a favorable exo
position in the presumed tetrahedral S→N acyl transfer
intermediate, while, in the unproductive cis series, the COP2
group would need to occupy a hindered endo position.
Scheme 4. Synthesis of hEPO(79-166) Glycopeptide, 24
In a substrate scope study, we observed a strong correlation
between the steric bulk of the C-terminal amino acid residue
and the quality of the reaction (Scheme 3). Thus, while ligation
a
Scheme 3. Thio-Proline Ligation: Substrate Scope
a
Key: (a) (1) 6 M Gn•HCl, 100 mM NaH2PO4, 50 mM TCEP, pH
7.5, 67%; (2) piperidine, DMSO, 61%; (3) 0.2 M MeONH2, 60%; (b)
6 M Gn•HCl, 100 mM NaH2PO4, 50 mM TCEP, pH 7.5, 23%; (c) 6
M Gn•HCl, 100 mM NaH2PO4, 50 mM TCEP, 200 mM MPAA, pH
7.8, 40%; (d) TCEP, VA-044, tBuSH. Ar = 2-(ethyldithio)-phenyl; R =
CH2CH2CO2Et; VA-044 = 2,2′-azobis[2-(2-imidazolin-2-yl)propane]
dihydrochloride.
a
Key: (a) 6 M Gn•HCl, 100 mM NaH2PO4, 50 mM TCEP, pH 7.5.
P1: ALLVNSS−; P2: −WEPLN; Ar = 2-(ethyldithio)-phenyl.
proceeded readily with relatively unhindered C-terminal Gly
(14), Gln (19), or Phe (20) thioesters, reaction was
significantly lower yielding with Val (21) residues. Moreover,
incorporation of a C-terminal Pro residue led to complete
inhibition of ligation (22). In the Val−Pro ligation en route to
21, we observed that, while the trans-thioesterification step was
rapid, the resulting thioester (cf. 18) was not adequately
reactive to permit efficient S→N acyl transfer. In contrast, the
Pro thioester substrate was not able to undergo the initiating
transthioesterification step. We attribute the failure of the Pro−
Pro ligation to the deactivating nature of the peptide chain
(P1) on the C-terminal Pro residue, a supposition which is
envisioned organizing the target glycopeptide into two long
polypeptide segments (26 and 28) and two shorter
glycopeptide domains (25 and 27). These would be iteratively
merged through a series of thiol-assisted ligations, to generate
the full glycopeptide backbone, 29, bearing three extraneous
sulfur functionalities. Finally, global MFD would deliver the
target EPO glycopeptide, 24. The success of this particular
proposed retrosynthesis would be predicated on our ability to
achieve efficient Pro ligation between the polypeptide, 26,
bearing the trans-thio-Pro surrogate at its N-terminus, and the
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glycopeptide fragment, 25, incorporating a C-terminal ortho-
thiophenolic ester and a Gln residue.
The first objective was to synthesize the previously unknown
trans-selenoproline, 37.20 Beginning with commercially avail-
able pyrrolidine 33, Mitsunobu inversion provided the cis-iodo
proline derivative, 34, in high yield (Scheme 6).21 Nucleophilic
In the event, the projected sequence of ligations did proceed
as expected. Thus, Cys-mediated ligation between 27 and 28,
followed by Fmoc removal and thiazolidine ring opening,
generated the ligation acceptor corresponding to hEPO(125−
166). We were pleased to observe that, under our optimized
conditions, the desired Pro ligation between 25 and 26 could
be achieved in a modest but serviceable isolated yield (23%).
The two glycopeptide fragments were subsequently merged to
generate 29, bearing three erstwhile thiol groups (in red).
Finally, threefold dethiolation was achieved under our standard
MFD conditions, to afford the target system, 24. The successful
realization of this challenging synthetic route serves to further
demonstrate the complexity-building potential of our newly
developed thio-proline ligation protocol, coupled with our mild
and selective MFD conditions, while exploiting our recently
developed O-mercapatouryl rearrangement to reveal, in site, the
required thioester.
Proline Ligation via a Selenol Surrogate. Though the
thio-proline dethiylation sequence had been applied to fairly
complex settings, we were nonetheless mindful of its potential
limitations. Thus, as we have shown (Scheme 3), ligation
efficiency is severely compromised by the presence of a bulky
amino acid on the C-terminal coupling partner. In an effort to
expand the scope of our Pro ligation method, we took note of
some recent methodological advances that have focused on
overcoming issues apparently arising from steric congestion in
ligation chemistry. In particular, Durek and Alewood have
described the conversion of thioesters to selenoesters as a
means to form highly reactive C-terminal ligation partners.16
Similarly, Dawson and colleagues have reported the ligation and
selective deselenization of peptides feature N-terminal
selenocysteine residues.17
a
Scheme 6. Synthesis of trans-Seleno-Pro, 37
a
Key: (a) PPh3, DIAD, CH3I, THF, 0 °C → 23 °C, 88−92% yield; (b)
BzSeH, DIPEA, DMF, 60 °C, 84%; (c) K2CO3, aq. MeOH, 79%; (d)
HCl/CH2Cl2, 95%.
displacement with selenobenzoic acid22 generated 35 in 84%
yield. This intermediate displayed the key diagnostic 13C
resonance of ∼200 ppm for a selenocarboxylate.23 Removal of
the benzoate and saponification of the methyl ester occurred in
concert to provide, upon aqueous workup, N-Boc selenoproline
dimer 36, in 79% yield. Finally, cleavage of the Boc group under
acidic conditions afforded “oxidatively dimerized” 37, in near-
quantitative yield.
With trans-seleno-Pro (37) in hand, we first examined the
ligation in the context of a single amino acid elongation
(Scheme 7). The ligation between 38 and 37 was conducted at
Scheme 7. Seleno-Proline Ligation between 38 and 37
Thus, in an effort to enhance the rate of intramolecular acyl
transfer in the pseudoproline ligation, we wondered about the
feasibility of ligating peptides via N-terminal prolines
containing C4 selenol functionality. We hypothesized that the
increased nucleophilicity of the selenol would lend itself to
more rapid trans-esterification. The intermediate selenium
ester, 31, might well be expected to display increased reactivity
relative to the analogous thioester and could conceivably
accommodate productive Se→N transfer even in the presence
of bulky C-terminal residues (Scheme 5).18,19 In the course of
pH 7.4 to 7.6, in degassed buffer in the presence of an aromatic
thiol (4-mercaptophenylacetic acid, MPAA). As previously
described by Dawson,17 MPAA serves to desymmetrize the
selenium dimer, liberating small amounts of selenol to
participate in the opening ligation. Moreover, MPAA further
activates the thioester through trans-thioesterification. Under
these conditions, peptide 38 was consumed within 3 h,
generating adduct 40 in 92% yield. We note that, due to the
oxidative sensitivity of the presumed selenol, standard
degassing by passing a stream of nitrogen through the reaction
was ineffective. Instead, the buffered solution was degassed
according to the freeze−pump−thaw protocol.
Scheme 5. Seleno-Proline Ligation between 1 and 30
In order to gain access to appropriate quantities of material
for further studies, we directed our attention to the
incorporation of a “selenoproline dimer” into a peptide through
SPPS techniques. For optimal reaction efficiency, standard
SPPS protocols require the use of excess amounts of each
amino acid. However, in order to synthesize a dimeric peptide,
we would require submolar quantities of the selenoproline. In
the event, we were pleased to find that our target dimeric
peptides could be smoothly prepared through the use of 0.55
equiv of the dimer, by employing extended reaction times in
the coupling step. Through recourse to this approach, we were
able to readily gain access to our target peptide, 43, in high
yield (see Supporting Information for details).
these investigations, we would also explore the relative rate and
efficiency of deselenization of a secondary selenol (32→10),
compared to desulfurization of a secondary thiol. It was
anticipated that, owing to the ease with which divalent selenium
compounds are oxidized, the selenoproline moiety might
require presentation as a dimer, which would be reductively
activated to reveal its selenol function. Moreover, for
widespread convenience, the capacity to integrate such a
residue into a peptide sequence through solid phase peptide
synthesis (SPPS) techniques would be most helpful.
We next sought to evaluate the relative efficiency of
deselenation compared to dethiolation in model peptidyl
systems, bearing N-terminal seleno-Pro and thio-Pro residues,
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a
respectively. As outlined in Table 1, peptide 41, bearing an N-
terminal thio-Pro surrogate, underwent dethiolation at 37 °C to
Table 2. Selenoproline Ligation: Substrate Scope
Table 1. Comparison of Proline Desulfurization and
a
Deselenation
a
Key: (A) VA-044, TCEP, t-BuSH, aq. CH3CN, 37 °C; (B) VA-044,
TCEP, t-BuSH, aq. CH3CN, 5 °C; (C) TCEP, 6 M Gn•HCl, 200 mM
NaH2PO4, pH 5−6, 23 °C; (D) TCEP, 6 M Gn•HCl, 200 mM
NaH2PO4, pH 5−6, 5 °C; (E) DTT (Dithiothreitol), 6 M Gn•HCl,
200 mM NaH2PO4, then TCEP, pH 5−6.
a
Key: (a) MPAA, Ligation Buffer (6 M Gn•HCl, 200 mM NaH2PO4),
pH 7.4−7.6, 23 °C; (b) DTT, Ligation Buffer; then TCEP, pH 5.0−
6.0, 23 °C. P1: ALLVNSS−; P2: −WEPLQ.
generate 42 afer 90 min, in 83% yield (entry 1). No
dethiolation was observed at lower temperatures. By contrast,
as shown in entry 2, peptide 43 rapidly underwent TCEP-
induced deselenation at both room temperature (15 min, 92%
yield) and 5 °C (30 min, 88% yield). We next examined the
deselenation of a selenosulfide-Pro, which represents the
penultimate product of selenoproline ligation. We were pleased
to find that the reduction proceeded within 90 min to deliver
42 in excellent yield (entry 3). Finally, peptide 45,
incorporating both seleno- and thio-Pro residues, was subjected
to reducing conditions. As expected on the basis of the Dawson
precedent, the seleno- functionality was smoothly and
selectively removed, to deliver adduct 46 in 90% yield (entry
4). This finding serves to establish the potential to selectively
remove secondary selenide in the presence of an unprotected
thiol moiety in the proline series.
We now sought to probe the scope of this protocol by
examining the coupling of 43 with a range of peptides with
significant complexity at the C-terminus. The results of this
study are presented in Table 2. As shown, peptides 38 and 48,
bearing C-terminal Gly and Ala residues, respectively, under-
went rapid ligation to generate, following deselenation, 47 and
48 in high yields (entries 1 and 2). The more sterically
demanding Phe-containing peptide, 50, required a longer
ligation time, but after 10 h, the resultant peptide was reduced
to deliver 51 in good yield (80%). A significant improvement in
reaction efficiency was observed with peptide 52, presenting a
challenging Val residue at its C-terminus. As described above
(Scheme 3), this peptide was quite resistant to ligation with the
analogous thio-Pro bearing peptide, proceeding to only ∼15%
conversion within 23 h. However, 52 underwent ligation with
the more reactive seleno-Pro peptide, 43, to generate, following
reduction, a 66% yield of adduct 53 within 12 h. This finding
confirms our hypothesis that the enhanced nucleophilicity of
the selenoester intermediate does, indeed, enable a more facile
Se→N acyl transfer. Unfortunately, the selenol derivative was
not sufficiently reactive to overcome the deactivating effects of
the proline amide carbonyl in peptide 54. Thus, only ca. 5% of
product 55 is currently obtainable, even at elevated temper-
atures.
CONCLUSION
■
In summary we have described herein recent advances from our
laboratory in proline ligation. We have developed a two-step
ligation protocol involving a thio-assisted proline coupling,
followed by a mild and high-yielding MFD. This transformation
is particularly well suited to ligations wherein the C-terminal
residue is relatively unhindered. We have further demonstrated
the utility of this method in synthesizing complex systems,
through application to the synthesis of a doubly glycosylated
fragment of hEPO. Most importantly, we have developed a
second-generation ligation protocol, featuring a seleno-proline
amino acid surrogate. The selenol functionality has been found
to be readily removed following ligation under mild conditions.
Moreover, our seleno-proline mediated ligation appears to be
well suited to more sterically demanding systems and is thus
likely to be of value in building complex glycans.
ASSOCIATED CONTENT
■
S
* Supporting Information
General experimental procedures, including spectroscopic and
analytical data for new compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
s-danishefsky@ski.mskcc.org
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
This work was supported by NIH Grant CA28824 (S.J.D.).
S.D.T. is grateful to Weill Cornell for an NIH postdoctoral
fellowship (CA062948). Special thanks to Dr. George
Sukenick, Hui Fang, and Sylvi Rusli of SKI’s NMR core facility
for spectroscopic assistance and to Dr. Karthik Iyer and Dr.
Jennifer Stockdill for helpful discussions.
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