Exploiting the MeDbz Linker To Generate Protected or Unprotected C-Terminally Modified Peptides

Article abstract of DOI:10.1002/chem.201703380

C-terminally modified peptides are important targets for pharmaceutical and biochemical applications. Known methods for C-terminal diversification are limited mainly in terms of the scope of accessible modifications or by epimerization of the C-terminal amino acid. In this work, we present a broadly applicable approach that enables access to a variety of C-terminally functionalized peptides in either protected or unprotected form. This chemistry proceeds without epimerization of C-terminal Ala and tolerates nucleophiles of varying nucleophilicity. Finally, unprotected peptides bearing nucleophilic side chain groups can be selectively functionalized by strong nucleophiles, whereas macrocyclization is observed for weaker nucleophiles. The potential utility of this method is demonstrated through the divergent synthesis of the conotoxin conopressin G and GLP-1(7-36) and analogs.

Full text of DOI:10.1002/chem.201703380

A Journal of
Accepted Article
Title: Exploiting the MeDbz Linker to Generate Protected or
Unprotected C-Terminally Modified Peptides
Authors: Christine A Arbour, Hasina Y Saraha, Timothy F McMillan,
and Jennifer Stockdill
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To be cited as: Chem. Eur. J. 10.1002/chem.201703380
Link to VoR: http://dx.doi.org/10.1002/chem.201703380
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COMMUNICATION
Exploiting the MeDbz Linker to Generate Protected or
Unprotected C-Terminally Modified Peptides
Christine A. Arbour, Hasina Y. Saraha, Timothy F. McMillan, and Jennifer L. Stockdill*
accessible upon activation of the MeDbz linker.18 We envisioned
Abstract: C-terminally modified peptides are important targets for
pharmaceutical and biochemical applications. Known methods for C-
terminal diversification are limited mainly in terms of the scope of
accessible modifications or by epimerization of the C-terminal amino
acid. In this work, we present a broadly applicable approach that
enables access to a variety of C-terminally functionalized peptides in
either protected or unprotected form. This chemistry proceeds
without epimerization of C-terminal Ala and tolerates nucleophiles of
varying nucleophilicity. Finally, unprotected peptides bearing
nucleophilic side chain groups can be selectively functionalized by
strong nucleophiles, while macrocyclization is observed for weaker
nucleophiles. The potential utility of this method is demonstrated
through the divergent synthesis of the conotoxin conopressin G and
GLP-1(7-36) and analogs.
that these peptides could be directly cleaved to afford protected
C-terminally modified peptides 2 or treated with trifluoroacetic
acid (TFA) to afford side-chain-deprotected MeNbz peptides 3.
Upon exposure to various nucleophiles, unprotected peptides 3
would be diversified at the C terminus (4). The susceptibility of
this linker to attack by weaker nucleophiles than thiols4 and
hydrazine 19 remains under-explored. 20 Additionally, the direct
nucleophilic cleavage of this linker from resin has not been
reported.
Resin
Cleavage
(TFA)
Me
N
Me
N
CONH2
-Gly-
Resin
O
O
NH
PG
N
N
O
MeNbz
Peptide
Peptide
PG
H
O
O
O
C-Terminally modified peptides are important for the
development and delivery of peptide based pharmaceuticals
because they improve peptide activity,1 stability,2 hydrophobicity,
and membrane permeability.3 Additionally, the vulnerability of C-
terminal esters to cleavage by endogenous esterases makes
them excellent pro-drugs.3 Meanwhile, C-terminal thioesters4 and
hydrazides5 are critical to the synthesis of larger peptide targets
via native chemical ligation (NCL) 6 and non-cysteine NCL. 7
Despite the demand for C-terminally modified peptides, there
remain significant limitations in the available strategies to access
them by chemical synthesis. 8 , 9 Variations at the C-terminus
traditionally require repetition of the solid-phase peptide
synthesis (SPPS) on a different linker for each desired C-
terminal moiety2a,2c, 10 or the use of a C-terminal glycine. 11
Solution-phase activation of protected C-terminal acids risks
epimerization.12 Side-chain anchoring strategies are limited by
the need for particular amino acids at the C-terminus and by
epimerization during the activation of the C-terminal carboxylic
PG
1
3
Direct
Nucleophilic
Cleavage
Nucleophilic
Attack
PG
Nuc
O
Nuc
O
Peptide
PG
Peptide
H
PG
4
2
Protected peptides
Unprotected peptides
Scheme 1. Proposed strategy for C-terminal peptide modification.
To establish the reactivity of the resin-bound MeNbz
group, we treated MeNbz-linked tripeptides (5a-c) with a
variety of nucleophiles (Table
1). A Gly residue was
installed at the C-terminus (5a) to avoid concerns related to
epimerization at this stage. The generation of thioesters
18b
from MeNbz is well established,
so we began our
acid.13 Recent efforts to diversify the C-terminus from a single
investigation with nitrogen nucleophiles. As the smallest
14
SPPS effort suffer from epimerization,
require extended
nucleophile, we expected that bubbling with ammonia would
reaction times or heating, 15 are incompatible with common
cysteine protecting groups,15,16 or require pre-functionalization of
the peptide.17
21
readily induce cleavage from the resin.
Indeed, we
observed complete removal of the peptide from the resin
after bubbling with a balloon of NH3 for 1.5 h. The
corresponding tripeptide carboxamide was isolated in 41%
yield. Primary amines of varying nucleophilicity were
For broad utility, the ideal functionalization method should
employ a commercially available resin/linker, use convenient
reagents for activation, undergo reaction with a variety of
nucleophiles of varying steric and nucleophilic properties, and
proceed at ambient temperature without epimerization of the C-
terminal residue. Furthermore, the approach should enable the
user to select between the production of protected peptides and
the solution-phase diversification of unprotected peptides. To
date, no report has demonstrated the achievement of this set of
objectives. In this manuscript, we commandeer the commercially
available MeDbz linker18 to realize these goals for the first time.
Our strategy for divergent C-terminal functionalization is
outlined in Scheme 1. MeNbz-Linked resin-bound peptides 1 are
22
23
evaluated.
Butylamine and 3-azidopropylamine
proceeded with complete conversion. Propargyl amine and
benzyl amine are slightly less nucleophilic than primary alkyl
amines, but both are efficient in displacing MeNbz.
Importantly, azide and propargyl containing products can be
further diversified via azide-alkyne cycloaddition.24 The least
nucleophilic amine tested was aniline, which led to minimal
product formation. However, in the presence of 5 equiv
Hünig’s base, aniline displacement proceeded with 53%
conversion, allowing access to peptide N-aryl amides via
this strategy.
Because of the unique biological properties of C-terminal
esters,3 we were interested in the ability of oxygen
nucleophiles to displace MeNbz. To maximize the amount of
RO– in solution, alcohols were combined with KOtBu and
added to the swelled resin.14 Primary alkoxides reacted with
complete conversion to afford the corresponding esters.
Steric hindrance in the nucleophile slows the reaction
[a]
C. A. Arbour, H. Y. Saraha, T. F. McMillan, Prof., Dr. J. L. Stockdill
Department of Chemistry
Wayne State University, Detroit, MI 48202
stockdill@wayne.edu
Electronic Supplementary Information (ESI) available: See
DOI: 10.1039/x0xx00000x
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10.1002/chem.201703380
Chemistry - A European Journal
COMMUNICATION
considerably, with isopropoxide proceeding to only 62% linkage. The resin was divided into 3 vessels and treated with
conversion. Although benzyl oxide was also less efficient ammonia, propargyl-amine, or MeOH/KOtBu, cleaving the
than the alkoxides, phenoxide led to complete conversion. peptide from the resin and generating the fully protected
Finally, treatment with aqueous NaOH resulted in >99% conopressins. The remaining protecting groups were removed,
conversion to the corresponding carboxylic acid. Overall, a affording native conopressin (7-NH2) in 29% isolated yield,
variety of strong, weak, and even branched N- and O- propargyl conopressin (7-NHpropargyl) in 7% yield, and
nucleophiles are effective in cleaving MeNbz from the resin. conopressin methyl ester (7-OMe) in 11% isolated yield.
Additionally, hindered C-terminal amino acids and bulky Additionally, we synthesized the active portion of glucagon-like
protecting groups are tolerated. Tripeptide 5b (X=Ile) and 5c peptide-1, GLP-1(7-36). GLP-1 receptor agonists are state-of-
(X=Arg(Pbf)) reacted with excellent conversion when treated the-art pharmaceutical agents, with 16 different GLP-1 agonists
with ammonia, butylamine, or MeOH/KOtBu.25
To demonstrate the convenience of this approach, we treated with ammonia in DMF for 1.5 h to afford native GLP-1(7-
synthesized conopressin (7), C-terminal carboxamide- 36) in >99% conversion and 4% isolated yield (27% crude).
containing vasopressin homolog isolated from the venom of With the viability of the method established, our next focus
in clinical trials as of 2015.27 GLP-1(7-36)–MeNbz-Gly-Resin was
G
a
piscivorous Conus snails, and 2 analogs (Scheme 2).26 This was evaluating the extent of epimerization under these
neuroactive peptide contains a single disulfide bridge and a C- conditions using Fmoc-AW(Boc)A–MeNbz-Gly-Wang (8).25 To
terminal carboxamide. Following SPPS, the disulfide bond was our delight, no epimerization was observed upon displacement of
accessed via iodine-mediated Trt cleavage/oxidation. The MeNbz by butylamine (Table SI-1). Given the extent of
MeDbz linker was activated in the presence of the disulfide epimerization observed by Meldal and co-workers in the
presence of KOt-Bu (50%),14,
we expected to observe
28
Table 1. Access to C-terminally modified protected peptides by direct
cleavage of MeNbz-linked peptides on resin.
epimerization during treatment with KOt-Bu/MeOH. Indeed,
under these conditions, 15% epimerization was observed.
Dawson reported <2% epimerization during the activation of C-
terminal Tyr-Dbz in the presence of Hünig’s base.18 Thus, we
hypothesized that under similar conditions, an epimerization-free
ester modification might be feasible. Indeed, treatment with 5
equiv Hünig’s base in MeOH led to complete conversion with no
observable epimerization. Analogously, employing Hünig’s base
in water led to the carboxylic acid with no epimerization.
Additionally, H-AW(Boc)H(Trt)-MeNbz-Gly-Wang was prepared
and cleaved with ammonia. No epimerization was observed.25
An advantage of the MeDbz linker is that the activated linker
(MeNbz) is stable to typical post-SPPS manipulations including
resin cleavage, purification,18 and storage.29 Thus, we imagined
that solution-phase modifications of unprotected peptides would
be feasible. The ability to diversify the C-terminus of an
unprotected peptide in solution would be ideal for situations
where the SPPS itself led to multiple close-eluting products.
Rather than diversification during resin cleavage followed by
several challenging purifications, a single purification could be
executed, followed by solution-phase diversification of the pure
MeNbz peptide. For simplicity during evaluation of the
nucleophile scope, we generated the tripeptide H-AWA-MeNbz-
Gly-NH2 (9), which does not have any nucleophilic side chains.
The crude peptide was dissolved in MeCN then treated with the
nucleophile. To avoid epimerization, Hünig’s base was employed
when a stoichiometric base was needed. A variety of primary
amines were tolerated, leading to complete conversion in 30 min
(Table 3). In contrast to the resin-bound approach, complete
conversion was also observed for less nucleophilic amines such
as aniline. Hydrazine and hydroxylamine were excellent
30
nucleophiles,
affording the corresponding hydrazide and
hydroxamide31 with >99% conversion. The reaction has some
sensitivity to steric effects, as demonstrated by the low
conversion observed with Weinreb amine. Primary alkyl, benzyl,
and phenyl alcohols were competent nucleophiles in the
presence of Hünig’s base, whereas i-PrOH was more sluggish
(61% conversion, 5 h). In the presence of aqueous NaOH, the
carboxylic acid was observed. Finally, the b-amino alcohol could
be generated by NaBH4 addition.
The solution-phase C-terminal diversification of MeNbz-
linked peptides is notable in the ready accessibility of the
Scheme 2. Divergent synthesis of conopressin G and analogs.
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Table 3. Selectivity for intermolecular attack vs intramolecular attack.
Table 2. Scope of nucleophilic substitution of MeNbz in solution.
activated peptide, the mild conditions, the short reaction times,
and the scope of nucleophiles demonstrated. For maximum
utility, the functionalization of unprotected peptides should be
compatible with residues bearing nucleophilic side chains.17 Thus,
we synthesized H-AKTWA-MeNbz-Gly (11) and subjected it to
various nucleophiles for 30 min (Table 3). The reactions were
quenched by dilution with 1:1 MeCN:H2O, and the product
distribution (12a:12b:12c) was analyzed by HPLC integration.
The functionalized acyclic peptide (12a) was the desired target.
Unreacted peptide 11 was hydrolyzed to the corresponding acid
(12b) during the quench and apart from entry 2, this product
represents unreacted starting material. Attack of either the N-
terminus or the side chain of Lys or Thr would afford a cyclic
peptide. The sole macrocyclic product was assigned as 12c
based on the reactivity of the primary amine relative to an a-
branched amine or alcohol and by independent synthesis of the
head-to-tail cyclized lactam.25
Table 4. Selectivity for cyclization-prone substrates and evaluation of other
nucleophilic side chains.
In 1:1 MeCN:BuNH2, only the intermolecular amide product
was observed. Repeating this reaction with 1:1:1 MeCN:H2O:
BuNH2 still led primarily to the amide with 7% hydrolysis
occurring during the reaction. Thus, the modification has
reasonable tolerance to aqueous conditions when an excess of
nucleophile is employed. Propargylamine was similarly effective,
while the reduced nucleophilicity of aniline resulted in an 8:92
ratio of amide to macrolactam. Peptide hydrazides19, 32 and
hydroxamides31 were generated with no macrocycle formation. In
contrast, treatment with Weinreb amine led to 89% conversion to
the macrolactam. Functionalization with MeOH proceeded with
complete conversion to a 42:58 ratio of methyl ester to lactam. In
the presence of a non-nucleophilic base, the lactam was formed
with 91% conversion. Finally, sodium borohydride reduction was
slower, likely because of the MeCN co-solvent, but no
macrocycle was observed.
Peptides containing Pro and Gly are generally more prone to
macrocyclization. 33 Peptides 13 and 14 were evaluated to
determine whether more cyclization-prone substrates could be
efficiently functionalized (Table 4). The proclivity of these
peptides towards macrocyclization was validated by control
reactions with MeOH/Hünig’s base and Hünig’s base alone
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(entry 2,3,5,6). Peptides 13 and 14 were more prone to
cyclization than AKTWA. Yet, in the presence of a 50:47.5:2.5 [6] (a) P. E. Dawson, T. W. Muir, I. Clark-Lewis, S. B. Kent, Science, 1994, 266,
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[7] S. Shang, Z. Tan, S. J. Danishefsky, Proc. Natl. Acad. Sci. U.S.A. 2011, 108,
amide (13a, 14a) was observed for both peptides (entry 1,4). All
remaining nucleophilic side chains were evaluated by replacing
Lys with Ser (15), Cys (16), and Tyr (17). In all cases, the butyl
amide and the methyl ester could be accessed with no hydrolysis
or macrocyclization. Omission of the exogenous nucleophile
confirmed that these peptides form macrocycles during the short
reaction (entry 9, 12, 15). Overall, intermolecular C-terminal
functionalization with strong, unhindered nucleophiles occurs
with excellent selectivity. Finally, to probe the utility of the in-
solution chemistry, Fmoc-GLP-1(7-36)–MeDbz-Gly-Rink was
activated and cleaved from the resin to afford unprotected GLP-
1(7-36)–MeNbz-Gly-NH2 in 45% crude yield. Subsequent
displacement with butylamine in MeCN/H2O led to GLP-1(7-36)-
NHBu with >99% conversion and 22% isolated yield.
5986–5989.
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nucleophilic side chains are tolerated. We have demonstrated
the utility of this approach via the divergent synthesis of 3
conopressin G derivatives and 2 GLP-1(7-36) derivatives. This
convenient method will facilitate the synthesis of important
bioactive peptides with diverse C-terminal functionalities,
enabling investigation of their potential as pharmaceutical agents.
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Acknowledgements
The authors thank the National Institutes of Health (R00-
GM097095) and Wayne State University (WSU) for generous
financial support (startup funds to JLS, Rumble-Schaap
Fellowship to CAA, Knoller Fellowship to HYS). We acknowledge
Dr. Johnna A. Birbeck and Dr. Bashar Ksebati of the WSU
Lumigen Instrument Center for spectroscopic support and
Shimadzu for a grant supporting the MS.
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Keywords: peptide • C-terminal functionalization • MeDbz linker
• N-acyl urea • epimerization
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