Surprising behavior of NXO-peptides toward the lithium hydroxide solvolysis

Article abstract of DOI:10.1016/j.tetlet.2013.05.010

An unexpected rearrangement of NXO peptides was observed during solvolysis of the methyl ester using lithium hydroxide as the base. It was found that the NXO-compounds rearranged into semioxamazide derivatives in which the ester is derived from the alcoho

Full text of DOI:10.1016/j.tetlet.2013.05.010

Tetrahedron Letters 54 (2013) 3679–3682
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Surprising behavior of NXO-peptides toward the lithium hydroxide solvolysis
Farhan A. Khan a,b, , Constantin Rabong a, Ulrich Jordis a, Jaywant Phopase c
⇑
a Institute of Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt 9/163, 1060 Vienna, Austria
b Department of Chemistry, COMSATS Institute of Information Technology, 22060 Abbottabad, KPK, Pakistan
c Department of Physics, Chemistry and Biology (IFM), Linköping University, Campus Valla, 58183 Linköping, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
An unexpected rearrangement of NXO peptides was observed during solvolysis of the methyl ester using
lithium hydroxide as the base. It was found that the NXO-compounds rearranged into semioxamazide
derivatives in which the ester is derived from the alcohol used as the reaction solvent.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 19 January 2013
Revised 19 April 2013
Accepted 3 May 2013
Available online 11 May 2013
Keywords:
Peptidomimetics
NXO-building blocks
L
-Phenylalanine
Modified amino acids
Rearrangement
Peptidomimetics have emerged as an important synthetic tool
for drug discovery1 to circumvent some of the problems associated
with natural peptides. Peptide mimetics are designed to mimic the
biological activity of peptides while offering the advantages of
increased bioavailability, biostability, bioefficiency, and bioselec-
tivity against the natural biological target of the parent peptide.2
Amino acids, being the smallest repeating elements of peptides,
are the obvious candidates for structural and chemical exploitation
toward peptidomimetics. Extensive research efforts have been
focused on developing modified amino acid building blocks which
can improve stability profiles and the pharmacokinetic properties
of the parent peptide.3 However, these have not been able to keep
pace with the emergence of new potential binding targets and the
search for new methods continues.
In an endeavor to create new building blocks for peptide mod-
ification, we have developed NXO building blocks (Fig. 1) via mod-
ification of amino acids by introducing an oxalic acid functionality
at the N-terminus and a hydrazine functionality at the C-termi-
nus.4 NXO compounds are easy to prepare and can mimic the
acceptor–donor pattern of a natural tripeptide (Fig. 2). Further-
more, NXO modification can impart conformational constraints
on the peptide sequence to give new pseudo peptides selectively
adopting predictable secondary structures.4 NXO-amino acids have
proved to be valuable as building blocks which can be integrated
efficiently into peptide fragments using both conventional liquid
phase peptide synthesis and solid supported synthesis protocols.4
X
R
N
O
R1
N
O
R2
OR4
N
H
N
R3
O
O
Figure 1. General formula for NXO-compounds. Where X = amino acid, R1–R4 = H,
alkyl, aryl, protecting groups.
R2
H
N
O
H
N
O
H
N
O
H
N
O
R2
N
H
N
H
N
H
1
R3
R
O
O
O
Figure 2. The donor–acceptor pattern of a tripeptide (left) versus an NXO-peptide.
During studies performed to assess the efficiency and compati-
bility of orthogonally protected NXO building blocks for integra-
tion into peptide fragments using different coupling agents and
solvents, methyl ester solvolysis of compound 1 repeatedly pro-
duced 2 as a side product (Scheme 1).
A simple and efficient method for hydrolysis of methyl ester is
typically saponification with aqueous alkali, in the presence of
organic solvents such as methanol, ethanol, dimethylformamide,
1,4-dioxane etc.5 The structure of rearranged product 2 was
⇑
Corresponding author. Tel.: +92 334 7286 986; fax: +92 992 383 441.
E-mail address: farhankhan@ciit.net.pk (F.A. Khan).
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.05.010

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F. A. Khan et al. / Tetrahedron Letters 54 (2013) 3679–3682
MeOH
O
O
LiOH.H2O
H
N
S
O
RT; 30 min
O
N
H
N
H
O
O
O
2
57%
EtOH
LiOH.H2O
O
O
O
O
H
N
H
N
S
S
O
RT; 30 min
O
O
N
N
O
N
H
N
H
H
H
O
O
O
O
O
3
51%
1
74%
i PrOH
LiOH.H2O
RT; 30 min
O
O
H
N
S
O
N
H
N
H
O
4
42%
Scheme 1. Solvolytic rearrangement of NXO-compounds using different solvents.
O
O2N
O
O
O
H
N
DMF
NH2
+
O
N
H
O
O
N
H
O
O
RT; 45 min
O
O
25%
MeOH/LiOH
RT; 30 min.
S
O
HCl.NH2
O
O
O
O
O
H
O
H
N
N
S
O
N
H
OH
O
N
N
H
DCC; HOBt
H
O
CH2Cl2; NMM
RT; Overnight
O
O
25%
26%
2
Scheme 2. Alternative route for the synthesis of the rearranged product 3.6
H
N
O
NH
O
O
N
H
s
Figure 3. Proposed cyclized structure of the NXO-compound.7
O2N
1) DMF
1.5 h; 60 °C
O
O
O
H
N
S
O
S
O
R
OH
O
+
2) RNHNH2
HOBt; DCC
RT; 12 h
N
H
N
H
O
H2N
O
O
O
O
1
5
R = Boc
R = Cbz
74%
69%
Scheme 3. One pot synthesis of NXO-compounds.4
confirmed by NMR analysis and also by an alternative synthetic
route (Scheme 2).6
Initially, it was thought that this rearrangement was the result
of an internal cyclization, but attempts to synthesize the proposed
cyclized structure were unsuccessful (Fig. 3).
To investigate this unusual behavior, a series of experiments
were designed and performed. Firstly, the influence of the alcohol
used as the solvent was examined because it was not clear whether
the alkoxy function in the newly formed ester in 2 originated from
the solvent alcohol or from the alcohol liberated from the starting

F. A. Khan et al. / Tetrahedron Letters 54 (2013) 3679–3682
3681
O
O
H
N
S
O
N
H
N
H
O
O
O
1
iPrOH
O
O
LiOH.H2O
H
N
S
R
O
O
N
H
N
H
RT; 30 min
O
O
O
O
H
N
42%
42%
4
6
R = Boc
R = Cbz
S
O
N
N
H
O
H
O
O
5
Scheme 4. LiOH-triggered solvolytic rearrangement of NXO-compounds using isopropanol as solvent.
OH
O
O
O
O
H
N
ROH
O
H
N
S
S
S
O
O
O
O
N
H
N
H
N
H
R
O
N
H
HN
O
O
O
O
O
N
O
N
H
O
(a)
(b)
(c)
Scheme 5. Proposed mechanism for the rearrangement.
material. The second objective was to determine if there was any
effect due to the protecting group used to protect the hydrazide
part of the NXO-compound on the outcome of the reaction. NXO-
an aprotic solvent. The nature of the protecting group did not affect
the yield of the final products.8
compounds were prepared via a reported procedure4 using
L
-phen-
Acknowledgments
ylalanine (Scheme 3) with different protecting groups such as Boc
(tert-butyloxycarbonyl) and Cbz (benzyloxycarbonyl) to protect
the hydrazide end.
Farhan A. Khan is grateful to the Higher Education Commission
(HEC) of Pakistan for financial support.
These NXO-compounds were subjected to methyl ester cleav-
age using lithium hydroxide as a mild base, but with different alco-
hols as the solvent. Synthesis of compound 2 was previously
reported,6 when ethanol and isopropanol were used as the sol-
vents, these reactions resulted in compounds 3 and 4. The results
confirmed that the ester in the rearranged product was derived
from the alcohol used as the solvent (Scheme 1).
References and notes
1. (a) Fry, D. C. Biopolymers 2006, 84, 535–552; (b) Clynen, E.; Baggerman, G.;
Husson, S. J.; Landuyt, B.; Schoofs, L. Expert Opin. Drug Disc. 2008, 3, 425–440; (c)
Daneshtalab, M. J. Pharm. Pharm. Sci. 2008, 11, 44–55; (d) Vagner, J.; Qu, H. C.;
Hruby, V. J. Curr. Opin. Chem. Biol. 2008, 12, 292–296; (e) Lesner, A.; Legowska,
A.; Wysocka, M.; Rolka, K. Curr. Pharm. Des. 2011, 17, 4308–4317; (f) Trabocchi,
A.; Cavalieri, D.; Guarna, A. Pure Appl. Chem. 2011, 83, 687–698; (g) Stamford, A.
W.; Scott, J. D.; Li, S. W.; Babu, S.; Tadesse, D.; Hunter, R.; Wu, Y. S.; Misiaszek, J.;
Cumming, J. N.; Gilbert, E. J.; Huang, C. L.; McKittrick, B. A.; Hong, L. W.; Guo, T.;
Zhu, Z. N.; Strickland, C.; Orth, P.; Voigt, J. H.; Kennedy, M. E.; Chen, X.; Kuvelkar,
R.; Hodgson, R.; Hyde, L. A.; Cox, K.; Favreau, L.; Parker, E. M.; Greenlee, W. J. ACS
Med. Chem. Lett. 2012, 3, 897–902; (h) ‘t Hart, P.; Thomas, D.; van Ommeren, R.;
Lakowski, T. M.; Frankel, A.; Martin, N. I. MedChemComm 2012, 3, 1235–1244.
2. (a) Sallach, R. E.; Cui, W.; Balderrama, F.; Martinez, A. W.; Wen, J.; Haller, C. A.;
Taylor, J. V.; Wright, E. R.; Long, R. C., Jr.; Chaikof, E. L. Biomaterials 2010, 31, 779–
791; (b) Fletcher, J. M.; Hughes, R. A. Bioorg. Med. Chem. 2009, 17, 2695–2702; (c)
Thompson, C.; Cheng, W. P. In Peptide and Protein Delivery; Chris Van Der, W.,
Ed.; Academic Press: Boston, 2011; pp 123–164; (d) Mattos, A.; de Jager-
Krikken, A.; de Haan, M.; Beljaars, L.; Poelstra, K. J. Control. Release 2012, 162, 84–
91.
NXO-compounds containing
L-phenylalanine with different
protecting groups were subjected to solvolysis. It was observed
that both the Boc and Cbz compounds showed similar behavior to-
ward lithium hydroxide, generating rearranged products 4 and 6
(Scheme 4) in comparable yields.
During hydrolysis of an NXO-compound using DMSO as an
aprotic solvent, no rearrangement was observed, and methyl ester
hydrolysis occurred exclusively.
A proposed mechanism for the rearrangement is depicted in
Scheme 5. Initial deprotonation leading to the intramolecular
ring-closure gives a reactive intermediate cyclic piperazine-trione
(b) which subsequently ring-opens to give the rearranged product.
In conclusion, a new rearrangement process was observed in
3. (a) Nucci, M. L.; Shorr, R.; Abuchowski, A. Adv. Drug Delivery Rev. 1991, 6, 133–
151; (b) Modi, N. B. J. Control. Release 1994, 29, 269–281; (c) Arduini, R. M.; Li, Z.;
Rapoza, A.; Gronke, R.; Hess, D. M.; Wen, D.; Miatkowski, K.; Coots, C.; Kaffashan,
A.; Viseux, N.; Delaney, J.; Domon, B.; Young, C. N.; Boynton, R.; Chen, L. L.; Chen,
L.; Betzenhauser, M.; Miller, S.; Gill, A.; Pepinsky, R. B.; Hochman, P. S.; Baker, D.
P. Protein Expr. Purif. 2004, 34, 229–242; (d) Chen, X.; Park, R.; Shahinian, A. H.;
Bading, J. R.; Conti, P. S. Nucl. Med. Biol. 2004, 31, 11–19; (e) Wang, Q.; Graham,
NXO-modified building blocks of L-phenylalanine, with the ester
part in the final products from derived alcohol used as the solvent.
Moreover, such rearrangement was not observed in the presence of

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F. A. Khan et al. / Tetrahedron Letters 54 (2013) 3679–3682
K.; Schauer, T.; Fietz, T.; Mohammed, A.; Liu, X.; Hoffend, J.; Haberkorn, U.;
129.3, 135.8, 155.9, 157.9, 160.4, 170.1. ESI-HRMS: m/z calcd for C18H25N3O6Na
[M+Na]+ 402.1641. Found: 402.1642.
Eisenhut, M.; Mier, W. Nucl. Med. Biol. 2004, 31, 21–30; (f) Jo, Y. W.; Youn, Y. S.;
Lee, S. H.; Kim, B. M.; Kang, S. H.; Yoo, M.; Choi, E. C.; Lee, K. C. Int. J. Pharm. 2006,
309, 87–93.
ESI-HRMS: m/z calcd for C18H25N3O6Na [M+Na]+ 402.1641; found: 402.1642.
Isopropyl N-{[2-(tert butoxycarbonyl)hydrazino] (oxo)acetyl}phenylalaninate
(4):
4. Rabong, C.; Jordis, U.; Phopase, J. B. J. Org. Chem. 2010, 75, 2492–2500.
5. Dayal, B.; Salen, G.; Toome, B.; Tint, G. S.; Shefer, S.; Padia, J. Steroids 1990, 55,
233–237.
White solid 0.40 g (42%); HPLC-purity 99%.
Mp 62–64 °C.
6. Khan, F. A.; Phopase, J.; Jordis, U. ‘Surprise in the Lithium Hydroxide Hydrolysis of a
NXO-Compound, 13th Int. Electron. Conf. Synth. Org. Chem., 2009.
7. Jordis, U.; Phopase, J. B. PCT Int. Appl. WO2007095980, 2007.
8. To a solution of 1 or 5 (1.00 g; 2.7 mmol) in solvent (MeOH/EtOH/iPrOH) (20 mL)
was added LiOHÁH2O (0.115 g; 2.7 mmol) along with two drops of H2O. The
solution was stirred at room temperature for 15 min. The solvent was removed
under reduced pressure and the residue was dissolved in H2O and extracted
with EtOAc (3 Â 50 mL). The organic phase was dried over anhydrous Na2SO4
and the solvent was removed under reduced pressure to give the rearranged
product. This was recrystallized from CH2Cl2.
1H NMR (200 MHz, CDCl3): d = 1.24 (d, J = 6.5 Hz, 6H), 1.36 (s, 9H), 2.96–3.26 (m,
2H), 4.66–4.81 (m, 1H), 4.94–5.09 (m, 1H), 6.58 (br s, 1H), 7.10–7.27 (m, 5H),
7.74 (br s, 1H), 8.37 (br s, 1H).
13C NMR (50 MHz, CDCl3): d = 21.4, 28.1, 37.5, 53.2, 71.8, 82.0, 127.1, 128.7,
129.3, 135.8, 155.2, 156.9, 159.2, 169.6.
ESI-HRMS: m/z calcd for C19H27N3O6Na [M+Na]+ 416.1798; found: 416.1797.
Isopropyl N-[{2-[(benzyloxy)carbonyl]hydrazino} (oxo)acetyl]phenylalaninate
(6):
White solid 0.45 g (42%); HPLC-purity 96%.
Mp 53–54 °C.
Ethyl N-{[2-(tert-butoxycarbonyl)hydrazino](oxo)acetyl}phenylalaninate (3):
White solid 0.48 g (51%); HPLC-purity 99%.
1H NMR (200 MHz, CDCl3): d = 1.19 (dd, J1,2 = 1.8 Hz and 6.3 Hz, 6H), 2.89–3.20
(m, 2H), 4.90–5.05 (m, 3H), 6.93 (br s, 1H), 7.11–7.31 (m, 10H), 7.75 (br s, 1H),
8.53 (br s, 1H).
Mp 60–63 °C.
1H NMR (200 MHz, CDCl3): d = 1.11–1.42 (m, 12 H), 1.92–2.01 (m, 2H), 2.87–
3.28 (m, 2H), 4.66–4.84 (m, 1H), 7.00–7.22 (m, 5H), 8.25 (br s, 1H), 8.76 (br s,
4H), 9.14 (br s, 1H).
13C NMR (50 MHz, CDCl3): d = 16.2, 32.3, 48.0, 62.6, 66.6, 121.9, 122.9, 123.1,
123.3, 123.5, 124.1, 130.2, 130.4, 150.8, 151.8, 153.8, 164.7.
ESI-HRMS: m/z calcd for C22H25N3O6Na [M+Na]+ 450.1641; found: 450.1641.
13C NMR (50 MHz, CDCl3): d = 14.1, 28.0, 37.5, 53.8, 60.5, 82.5, 127.1, 128.7,