Solid-Phase Synthesis of Oxetane Modified Peptides

Article abstract of DOI:10.1021/acs.orglett.7b01466

Solid-phase peptide synthesis (SPPS) is used to create peptidomimetics in which one of the backbone amide C=O bonds is replaced by a four-membered oxetane ring. The oxetane containing dipeptide building blocks are made in three steps in solution, then integrated into peptide chains by conventional Fmoc SPPS. This methodology is used to make a range of peptides in high purity including backbone modified derivatives of the nonapeptide bradykinin and Met- and Leu-enkephalin.

Full text of DOI:10.1021/acs.orglett.7b01466

Letter
pubs.acs.org/OrgLett
Solid-Phase Synthesis of Oxetane Modified Peptides
Jonathan D. Beadle,† Astrid Knuhtsen,‡ Alex Hoose,‡ Piotr Raubo,§ Andrew G. Jamieson,*,‡
and Michael Shipman*,†
†Department of Chemistry, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, U.K.
‡School of Chemistry, University of Glasgow, Joseph Black Building, University Avenue, Glasgow G12 8QQ, U.K.
§IMED Oncology, AstraZeneca, 310 Cambridge Science Park, Milton Road, Cambridge CB4 0WG, U.K.
S
* Supporting Information
ABSTRACT: Solid-phase peptide synthesis (SPPS) is used to
create peptidomimetics in which one of the backbone amide
CO bonds is replaced by a four-membered oxetane ring. The
oxetane containing dipeptide building blocks are made in three
steps in solution, then integrated into peptide chains by
conventional Fmoc SPPS. This methodology is used to make a
range of peptides in high purity including backbone modified
derivatives of the nonapeptide bradykinin and Met- and Leu-
enkephalin.
espite resurgent interest in the use of peptides as drugs,1
Scheme 1. SPPS Route to Oxetane Modified Peptides
Dtheir development is often hampered by their poor oral
bioavailability and short plasma half-lives. As a consequence,
there is intense interest in the design, synthesis, and study of
peptidomimetics that mimic the structure and biological
function of native peptides yet possess better drug-like
properties.2−4 Independently, Shipman5 and Carreira6 intro-
duced a new type of peptide bond isostere in which the
heterocyclic 3-aminooxetane unit was used as a replacement for
one of the amide bonds (Figure 1).7 Several features of these
oxetane modified peptides (OMPs) make them of particular
interest as peptidomimetics. First, deletion of one of the amide
bonds can improve peptide half-lives through increased stability
to proteases while retaining bioactivity.6b Second, oxetanes are
known to make excellent bioisosteric replacements for CO
bonds, and are commonly used in conventional, small molecule
drug discovery.8 Third, the 3-aminooxetane unit can act as both
a H-bond donor and acceptor, supporting the types of
noncovalent interactions available to peptides. Finally, con-
To date, access to OMPs has been by way of solution-based
formational changes arising from removal of the double bond
methods.5,6 However, to study the impact of oxetane
character of the peptide bond change the conformational bias
modification on the structure and properties of larger,
of OMPs,5a opening up new areas of peptide structural space to
biologically important peptides, we turned to solid-phase
explore.
peptide synthesis (SPPS).9 Our plan was to synthesize Fmoc-
protected dipeptide building blocks such as 1, then integrate
them into a growing peptide chain using automated SPPS
techniques (Scheme 1). Here, we describe the successful
development of this methodology and use it to make a range of
OMPs including novel enkephalin and bradykinin analogues.
Received: May 15, 2017
Published: June 6, 2017
Figure 1. Oxetane based peptidomimetics.
© 2017 American Chemical Society
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Table 1. Synthesis of Oxetane Containing Dipeptide Building Blocks
a
b
c
Prepared in situ from oxetan-3-one and nitromethane (see Supporting Information). After column chromatography. Made directly from α-amino
d
e
f
g
ester without Fmoc deprotection. Reference 5b. Reference 5a. Using 1.2 equiv of FmocOSu. 60% yield on higher 3 mmol scale (see Supporting
Information).
Initially, we sought a practical route to Fmoc-protected
dipeptide building blocks in which the oxetane residue is based
on glycine. Conjugate addition of the appropriate amino ester,
made from 2a−i by initial Fmoc cleavage, to 3-(nitromethyl-
ene)oxetane readily gave the addition products 3a−i (Table
1).5 Raney Ni reduction of 3a−i in the presence of FmocOSu
provided 4a−i in good yields.6 A range of protected side chain
types were introduced namely: acidic, basic, polar, and
nonpolar.
The last step prior to SPPS was selective removal of the ester
group (R1); a transformation that required considerable
optimization to safeguard the Fmoc and side chain protecting
groups. Initially, phenylalanine derivatives containing methyl
(4a), benzyl (4b), 2,4-dimethoxybenzyl (4c), and 2-phenyl-
isopropyl (cumyl, 4d) esters were studied (Table 1, entries 1−
4). Cleavage of the methyl ester from 4a using LiOH-THF/
H2O led to unwanted N-Fmoc cleavage. Other conditions
reported to leave Fmoc groups unscathed such as Me3SnOH10
11
and NaOH/CaCl2 also proved unsuccessful. Hydrogenolysis
of benzyl ester 4b (H2, 10% Pd/C, MeOH) did provide the
corresponding carboxylic acid in an encouraging 50% yield.
However, optimization of this transformation and its extension
to derivatives containing other side chains proved impractical.12
Poor results were also observed in the attempted removal of the
2,4-dimethoxybenzyl group from 4c using mild acid (1% TFA-
CH2Cl2, anisole).13 Much better outcomes were achieved with
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from the corresponding Fmoc-protected amino acids by
reaction with 2-phenylisopropyl-trichloroacetimidate (see
Supporting Information).14
Scheme 2. Selective Cumyl Ester Deprotection and Solution
Phase Couplings
Initially, the use of the building blocks in solution-phase
couplings was established (Scheme 2). Cleavage of the cumyl
ester from 4e followed by coupling with L-phenylalanine tert-
butyl ester with O-(1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HCTU) and diiso-
propylethylamine (DIPEA) gave (S,S)-5a in excellent yield.
Using D-phenylalanine tert-butyl ester in the same sequence
1
provided (S,R)-5b. Analysis by H NMR confirmed that no
detectable epimerization arose during these couplings (see
Supporting Information). Subjecting 4h bearing a Boc-
protected lysine to the same cleavage/coupling conditions
cleanly gave 6 without evidence of side chain deprotection.
Additionally, 4d was converted to 7 by chain extension at the
N-terminus. In this transformation, no products derived from
acylation of the secondary amine of the 3-aminooxetane unit
were isolated despite the use of 4 equiv of the coupling
partners. This observation encouraged us to explore SPPS
without recourse to protection of the oxetane nitrogen atom.
Next, we examined the use of 4d−i in SPPS. These
experiments were conducted on a 0.1 mmol scale in a Biotage
Alstra microwave synthesizer using preloaded chlorotrityl resin
and HCTU activation (for full details, see Supporting
Information). Initially, six tetrapeptides 8−13 were synthesized
using each of the building blocks, 4d−i (Table 2, entries 1−6).
Two equivalents of the building blocks were used, with a
pretreatment with 2% TFA in CH2Cl2 under anhydrous
conditions to cleave the cumyl ester, immediately prior to
coupling. Standard conditions for Fmoc removal (20%
piperidine, 0.1 M Oxyma pure, DMF),15 coupling [Fmoc-
Trp-OH, HCTU, DIPEA, DMF], and resin cleavage/
deprotection [TFA/triisopropylsilane(TIS)/CH2Cl2
cumyl ester 4d, which was quantitatively deprotected upon
treatment with 2% TFA in CH2Cl2 for 1.5 h.14a On this basis,
we focused our subsequent efforts on cumyl esters 4d−i. From
a practical standpoint, these derivatives proved attractive as they
were bench stable, crystalline solids. Indeed, it proved most
convenient to store the building blocks as the cumyl esters,
then reveal the carboxylic acid using 2% TFA in CH2Cl2
immediately prior to SPPS. These materials were produced
on up to a 3 mmol scale (see Supporting Information).
Moreover, the starting cumyl esters 2d−i were easily made
Table 2. Solid-Phase Synthesis of Oxetane Modified Peptides
HRMS
building
a
purity
e
d
f
entry
block
peptide sequence
W-GOx-F-A, 8
(%)
mass, mg (yield, %) peptide content (%)
calculated
observed
1
2
3
4
5
6
7
8
9
4d
4e
4f
89
10.7 (21)
8.4 (18)
75
55
73
75
48
62
81
79
63
508.2554 [M + H]+
460.2554 [M + H]+
448.2191 [M + H]+
476.2140 [M + H]+
489.2820 [M + H]+
480.2217 [M + Na]+
602.2643 [M + H]+
584.3079 [M + H]+
508.2531 [M + H]+
460.2544 [M + H]+
448.2190 [M + H]+
476.2129 [M + H]+
489.2818 [M + H]+
480.2213 [M + Na]+
602.2659 [M + H]+
584.3056 [M + H]+
W-GOx-V-A, 9
93
g
g
W-GOx-S-A, 10
W-GOx-D-A, 11
W-GOx-K-A, 12
W-GOx-P-A, 13
Y-G-GOx-F-M, 14
Y-G-GOx-F-L, 15
81 (90)
81 (91)
93
12.0 (27)
13.7 (29)
5.0 (10)
4g
4h
4i
94
14.6 (32)
17.0 (28)
16.4 (28)
25.3 (46)
4d
4d
4d
91
91
R-P-P-GOx-F-S-P-F-R, 16
94
544.8036 [M + 2H]2+ 544.8014 [M + 2H]2+
a
b
Treated with 2% TFA in CH2Cl2 for 2 h to reveal the free carboxylic acid from 4d−i prior to coupling with the resin-bound peptide. 30 s, then 3
c
min with fresh reagents. All couplings performed at 75 °C for 10 min except arginine (60 min at rt, then 5 min at 75 °C, repeated with fresh
d
e
reagents). Italicized residues derived from the oxetane containing dipeptide building block. By reverse-phase HPLC (see Supporting Information).
f
g
Determined by UV spectroscopy (at 280 nm) except 16 (at 214 nm). Improved to >90% by second purification.
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(70:20:10, v/v/v)] were adopted. After reverse-phase HPLC,
these tetrapeptides were isolated in high purity and acceptable
yields. Pleasingly, the oxetane did not undergo ring opening
under the harsh acidic conditions required for release from the
resin and concomitant side chain deprotection. Encouraged by
these results, we prepared oxetane modified analogues of
biologically relevant peptides. First, we made 14 and 15,
analogues of opioid-binding Met- and Leu-enkephalin,
respectively, in which the central glycine was replaced (Table
2, entries 7 and 8). Similarly, 16 was produced in good yield
and excellent purity as an analogue of the vasodilator
bradykinin. No erosion in peptide yield or purity was seen in
the preparation of this nonapeptide, indicating that the 3-
aminooxetane residue is well tolerated in repetitive rounds of
coupling/Fmoc deprotection. The synthesis of Leu-enkephalin
analogue 15 highlights the benefits of the SPPS approach;
although this material has previously been made by a solution-
phase strategy, it required multiple steps and chromatographic
purifications.6b
In summary, we have developed a practical route to oxetane
modified peptides using solid-phase peptide synthesis techni-
ques. Key findings include: (i) cumyl-protected dipeptide
building blocks 4d−i are easily made in three simple steps and
the ester selectively cleaved with 2% TFA in CH2Cl2; (ii)
efficient amide couplings using these building blocks can be
achieved in solution or on solid-phase without protecting the
secondary amine of the 3-aminooxetane residue; (iii) oxetane
containing peptides (up to 9 amino acids) can be produced in
high purity using conventional SPPS methods for coupling,
resin cleavage, deprotection, and purification, suggesting the
broad applicability of this modification in peptide science.
Current work is focused on using this new methodology to
produce a variety of OMPs so that the impact of this backbone
modification on the secondary structure and physicochemical
and biological properties of the peptides can be systematically
explored.
REFERENCES
■
(1) (a) Fosgerau, K.; Hoffmann, T. Drug Discovery Today 2015, 20,
122. (b) Nevola, L.; Giralt, E. Chem. Commun. 2015, 51, 3302.
(c) Kaspar, A. A.; Reichert, J. M. Drug Discovery Today 2013, 18, 807.
(2) Trabocchi, A.; Guarna, A. Peptidomimetics in Organic and
Medicinal Chemistry: The Art of Transforming Peptides in Drugs; John
Wiley & Sons Ltd: Chichester, 2014.
(3) For reviews, see (a) Qvit, N.; Rubin, S. J. S.; Urban, T. J.;
Mochly-Rosen, D.; Gross, E. R. Drug Discovery Today 2017, 22, 454.
(b) Wang, Z. A.; Ding, X. Z.; Tian, C.-L.; Zheng, J.-S. RSC Adv. 2016,
6, 61599. (c) Klinker, K.; Barz, M. Macromol. Rapid Commun. 2015,
36, 1943. (d) Jayatunga, M. K. P.; Thompson, S.; Hamilton, A. D.
Bioorg. Med. Chem. Lett. 2014, 24, 717. (e) Avan, I.; Hall, C. D.;
Katritzky, A. R. Chem. Soc. Rev. 2014, 43, 3575. (f) Liskamp, R. M. J.;
Rijkers, D. T. S.; Kruijtzer, J. A. W.; Kemmink, J. ChemBioChem 2011,
12, 1626. (g) Grauer, A.; Konig, B. Eur. J. Org. Chem. 2009, 5099.
(h) Vagner, J.; Qu, H.; Hruby, V. J. Curr. Opin. Chem. Biol. 2008, 12,
292.
(4) For illustrative examples, see (a) Nadon, J.-F.; Rochon, K.;
Grastilleur, S.; Langlois, G.; Dao, T. T. H.; Blais, V.; Guerin, B.;
́
Gendron, L.; Dory, Y. L. ACS Chem. Neurosci. 2017, 8, 40.
(b) Busschaert, N.; Thompson, S.; Hamilton, A. D. Chem. Commun.
2017, 53, 313. (c) Krishnan, B. P.; Rai, R.; Asokan, A.; Sureshan, K. M.
J. Am. Chem. Soc. 2016, 138, 14824. (d) Lee, K. J.; Lee, W. S.; Yun, H.;
Hyun, Y.-J.; Seo, C. D.; Lee, C. W.; Lim, H.-S. Org. Lett. 2016, 18,
3678. (e) Wu, H.; Qiao, Q.; Teng, P.; Hu, Y.; Antoniadis, D.; Zuo, X.;
Cai, J. Org. Lett. 2015, 17, 3524. (f) Barnard, A.; Long, K.; Martin, H.
L.; Miles, J. A.; Edwards, T. A.; Tomlinson, D. C.; Macdonald, A.;
Wilson, A. J. Angew. Chem., Int. Ed. 2015, 54, 2960. (g) Newberry, R.
W.; VanVeller, B.; Raines, R. T. Chem. Commun. 2015, 51, 9624.
(h) Doan, N.-D.; Hopewell, R.; Lubell, W. D. Org. Lett. 2014, 16,
̈
2232. (i) Traore,
3588.
́
M.; Doan, N.-D.; Lubell, W. D. Org. Lett. 2014, 16,
(5) (a) Powell, N. H.; Clarkson, G. J.; Notman, R.; Raubo, P.; Martin,
N. G.; Shipman, M. Chem. Commun. 2014, 50, 8797. (b) Beadle, J. D.;
Powell, N. H.; Raubo, P.; Clarkson, G. J.; Shipman, M. Synlett 2016,
27, 169.
(6) (a) McLaughlin, M.; Yazaki, R.; Fessard, T. C.; Carreira, E. M.
Org. Lett. 2014, 16, 4070. (b) Moller, G. P.; Muller, S.; Wolfstadter, B.
T.; Wolfrum, S.; Schepmann, D.; Wunsch, B.; Carreira, E. M. Org. Lett.
2017, 19, 2510.
(7) Oxetane modification of proteins through side chain function-
alization has also recently been described, see Boutureira, O.;
̈
̈
̈
̈
ASSOCIATED CONTENT
■
S
* Supporting Information
́
Martínez-Saez, N.; Brindle, K. M.; Neves, A. A.; Corzana, F.;
Bernardes, G. J. L. Chem. - Eur. J. 2017, 23, 6483.
(8) (a) Bull, J. A.; Croft, R. A.; Davis, O. A.; Doran, R.; Morgan, K. F.
Chem. Rev. 2016, 116, 12150. (b) Burkhard, J. A.; Wuitschik, G.;
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.7b01466.
Rogers-Evans, M.; Muller, K.; Carreira, E. M. Angew. Chem., Int. Ed.
2010, 49, 9052.
(9) Bodanszky, M.; Bodanszky, A. The Practice of Peptide Synthesis;
Springer-Verlag: Berlin, 1994.
(10) Nicolaou, K. C.; Estrada, A. A.; Zak, M.; Lee, S. H.; Safina, B. S.
Angew. Chem., Int. Ed. 2005, 44, 1378.
̈
Experimental procedures and characterization data for all
new compounds, copies of 1H and 13C NMR spectra for
building blocks, and analytical HPLC traces for peptides
(PDF)
(11) Pascal, R.; Sola, R. Tetrahedron Lett. 1998, 39, 5031.
AUTHOR INFORMATION
■
(12) When the side chain of 4b (R = Bn) was replaced with other
i
Corresponding Authors
substituents (R = H or Pr), cleavage of the Fmoc group was seen
during hydrogenolysis. We speculate that the success witnessed with
4b (R = Bn) arises from its insolubility in MeOH. During
hydrogenolysis, the carboxylic acid rapidly precipitates preventing
further Fmoc cleavage.
*E-mail: andrew.jamieson.2@glasgow.ac.uk.
*E-mail: m.shipman@warwick.ac.uk.
ORCID
(13) McMurray, J. S. Tetrahedron Lett. 1991, 32, 7679.
(14) (a) Yue, C.; Thierry, J.; Potier, P. Tetrahedron Lett. 1993, 34,
323. (b) Respondek, T.; Cueny, E.; Kodanko, J. J. Org. Lett. 2012, 14,
150.
Andrew G. Jamieson: 0000-0003-1726-7353
Michael Shipman: 0000-0002-3349-5594
Notes
(15) Subiros
2012, 98, 89.
́
-Funosas, R.; El-Faham, A.; Albericio, F. Biopolymers
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank AstraZeneca for generous financial support.
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