NaBH4 - A novel method for the deprotection of Nω-nitro-arginine

Article abstract of DOI:10.1016/j.tetlet.2015.12.081

The selective deprotection of N^ω-nitro-arginine derivatives represents a major preparative challenge. This problem can be circumvented by the use of catalytic hydrogenation, but often high pressure, elevated temperature, and/or long reaction times are needed. In certain cases hydrogenation is not suitable, for example, small-scale reactions, parallel synthesis, or due to selectivity issues. Herein, we demonstrate for the first time, the use of NaBH₄ in the presence of a metal ion catalyst for the removal of the N^ω-nitro moiety under simple, 'open-vessel' conditions. This process using NaBH₄ does not remove the benzyloxycarbonyl-protecting group; thus the method is orthogonal for this protecting scheme.

Full text of DOI:10.1016/j.tetlet.2015.12.081

Tetrahedron Letters 57 (2016) 546–548
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
x
NaBH4 – a novel method for the deprotection of N -nitro-arginine
Mónika Sebestyén a, György Kóczán a, Antal Csámpai b, Ferenc Hudecz a,b,
⇑
a MTA-ELTE Research Group of Peptide Chemistry, Hungaryyà
b Department of Organic Chemistry, Institute of Chemistry, ELTE, Hungaryà
a r t i c l e i n f o
a b s t r a c t
x
Article history:
The selective deprotection of N -nitro-arginine derivatives represents a major preparative challenge. This
problem can be circumvented by the use of catalytic hydrogenation, but often high pressure, elevated
temperature, and/or long reaction times are needed. In certain cases hydrogenation is not suitable, for
example, small-scale reactions, parallel synthesis, or due to selectivity issues. Herein, we demonstrate
Received 3 October 2015
Revised 3 December 2015
Accepted 19 December 2015
Available online 21 December 2015
x
for the first time, the use of NaBH4 in the presence of a metal ion catalyst for the removal of the N -nitro
moiety under simple, ‘open-vessel’ conditions. This process using NaBH4 does not remove the benzyloxy-
carbonyl-protecting group; thus the method is orthogonal for this protecting scheme.
Ó 2015 Elsevier Ltd. All rights reserved.
Keywords:
Sodium borohydride
x
N
-Nitro–Arg deprotection
Selective removal
Catalyst
Introduction
N-nitration is one possible way to achieve guanidino-NH2-pro-
tection. The cleavage of this protecting group is the standard
The benzyloxycarbonyl (Z-) protecting group has been widely
method of deprotection, but the use of NaBH4 for this purpose
has not been documented so far.
Herein, we report our findings on the development of an effec-
tive approach for the selective deprotection of a guanidino-nitro
protecting group in the presence of a benzyloxycarbonyl-group,
using NaBH4 and selected metal ion catalysts (Scheme 1).16
utilized in traditional peptide synthesis in combination with the
N -nitro protected arginine derivative.1,2 This protecting scheme
x
is used in large scale solution-phase peptide synthesis,3,4 and solid
phase chemistry.5 Of the two protecting groups the benzyloxycar-
bonyl group has been well-established to be selectively depro-
tected by strong acids, for example, HBr/acetic acid (Scheme 1).
However, no reports are available regarding the selective removal
x
Results and discussion
of the N -nitro groups of protected arginine in the presence of Z-
protected amino groups. The development of an effective method
for the selective deprotection of amino group(s) in the presence
of benzyloxycarbonyl-protected amino group(s) should broaden
the scope of using these protecting schemes not only in the field
of peptides, but perhaps also in general organic synthesis, for
example, in cases where catalytic hydrogenation is not applicable.6
The reduction of C–NO2-groups is often a key step in organic
synthesis. A large selection of reducing agents are available to con-
vert the NO2 group to NH2 (HF, catalytic hydrogenation), but does
not include NaBH4, which is well known for its selectivity toward
oxo-groups in the presence of nitro-groups. However, with the
aid of catalysts, NaBH4 can be made to reduce NO2-groups as
well.7–10
In order to demonstrate the usefulness of this new approach,
N -Z-N -nitro-L-arginine (4) was deprotected to give N -Z-L-argi-
a
x
a
nine with NaBH4 in the presence of a metal ion catalyst (Scheme 2
and Table 1).17 The conversion rate was determined by RP-HPLC
and it was found that 8 equiv of NaBH4 was not enough to com-
plete the reaction, while 10 equiv resulted in almost full transfor-
mation under the conditions used.12
We then studied the effect of various metal ion catalysts under
the same experimental conditions (Table 1). We found copper(II)
acetylacetonate (Cu(acac)2) and copper(II) N,N,N’,N’-tetram-
ethylethylenediamine (Cu-TMEDA) to have the optimal effect. Cop-
per-phthalocyanine and nickel acetylacetonate (Ni(acac)2) were
less effective as catalysts. Using cobalt(II) acetylacetonate contain-
ing crystal water (Co(acac)2ꢀ2H2O) the target compound was
not detected, and the use of water-free Co(acac)2 resulted in the
formation of several unidentified side-products. A possible expla-
nation for this finding is the known catalytic effect of hydrated
cobalt-salts in reducing carboxylic esters.11
⇑
Correcponding author. Tel.: +36 1 372 2828; fax: +36 1 372 2620.
E-mail address: fhudecz@elte.hu (F. Hudecz).
MTA: Hungarian Academy of Sciences, Széchenyi István sqr. 9., H-1051 Budapest,
y
Hungary.
à
ELTE: Eötvös L. University, Pázmány Péter st. 1/A, H-1117 Budapest, Hungary.
http://dx.doi.org/10.1016/j.tetlet.2015.12.081
0040-4039/Ó 2015 Elsevier Ltd. All rights reserved.

M. Sebestyén et al. / Tetrahedron Letters 57 (2016) 546–548
547
NO2
NH
BnOOC
HN
NO2
NH
BnOOC
HN
(a)
(b)
H2N
NH2
HBr/acetic acid
NaBH4/metal catalyst
known
this work
1
2
3
x
Scheme 1. Selective deprotection of Z- and N -nitro protecting groups.
O
OH
O
OH
O
NaBH4
O
H
N
H
N
H
N
NH2
NH
O
N
H
O
N
H
NO2
cat.
EtOH
NH
4
5
x
Scheme 2. Deprotection of N -nitro-arginine.
Table 1
Catalysts studied. ethanol was used as solvent
Catalyst (10 mol %)
Conversion (%)
After 30 min
After 120 min
Cu(acac)2
Cu-TMEDA
Cu-phthalocyanine
Ni(acac)2
69
74
33
43
—
>99
>99
70
62
Co(acac)2ꢀ2H2O
70*
>99**
a
Co(acac)2
92
a
The water-free complex (violet color vs. the pink color of the hydrate) was
produced by azeotropically distilling off the solvent from an acetonitrile-solution of
the hydrated complex.
*
Approx. 100% of various side products.
Approx. 30% of various side products.
**
Figure 1. RP-HPLC chromatograms of (Z)-Phe-Arg(NO2)-Ala-NH2 model tripeptide
deprotection. Reaction mixtures recorded after separation on Phenomenex
Synergi C12, 4
, 120 A, 4.6 ꢁ 300 mm column, using TFA/H2O (V/V), 0.1% TFA/
By far the most suitable solvent for the reaction was dry etha-
nol. In methanol the formation of several side-products could be
observed and additionally it is well-known that the borohydride
reagent is rapidly consumed by the solvent. Longer chain alcohols
(propyl alcohol, butyl alcohol) or ethers (tetrahydrofuran, dioxane,
diisopropyl ether) were also tested as solvents, but the reaction
was typically very slow, or no reaction could be detected, perhaps
due to the poor solubility of the reactants. Even traces of water
were found to jeopardize the reaction causing the formation of
side-products, for example, by transesterification or by the forma-
tion of metal-boride reactive intermediates.12
a
l
80% acetonitrile-H2O (V/V), gradient elution 20–80% B (5-20 min), and detection:
UV k = 220 nm. (a) without NaBH4, (b) 5 equiv NaBH4, injected after 30 min, (c) an
additional 5 equiv NaBH4, injected after 30 min. Peaks: [1] (Z)-Phe-Arg(NO2)-Ala-
NH2, [2] (Z)-Phe-Arg(NO)-Ala-NH2, [3] (Z)-Phe-Arg-Ala-NH2 were identified using
electrospray ionization mass spectrometry.
nitro-L-arginyl-L-alanine-amide ((Z)-Phe-Arg(NO2)-Ala-NH2) (6,
ESI, Scheme 4) by conventional solid-phase methods.18 The peptide
was cleaved from the solid support, and used without further
purification.13 This compound was deprotected using NaBH4 in
the presence of copper(II) acetylacetonate (Scheme 3).14 Both etha-
nol and methanol was used as solvents with similar results. The
reaction process was monitored by RP-HPLC (Fig. 1). The NaBH4
(10 equiv) was added in two equal portions, and 30 min later each
sample was analyzed. Neither the formation of a de-benzyloxycar-
bonyl peptide derivative, nor amide hydrolysis or epimerisation
was detected. The identity of the product was verified by IR and
NMR. The yield was acceptable using this method and the 10 mg
reaction-scale used.19
It is known, that methyl-, ethyl-13, and benzotriazole-esters14
can be cleaved using NaBH4 without catalysts, or benzyl esters in
the presence of excess nickel-salt.15 We studied the stability of var-
ious ester type protecting groups under the optimal experimental
conditions for the deprotection of the guanidino-nitro group, thus
a
demonstrating the orthogonality. It was found that N -Boc, benzyl-
esters, and ethers, typical protecting groups in Boc/Bzl peptide syn-
thesis protocols, remained intact (ESI, Table 2).
To further explore the suitability of NaBH4-deprotection
a
x
we synthesized
a
model tripeptide N -Z-
L
-phenylalanyl-N -
O
O
CH3
O
O
O
CH3
O
H
H
N
NaBH4
N
NH2
NH2
O
N
H
N
H
O
N
H
N
H
Cu(acac)2
EtOH/MeOH
O
O
6
7
HN
HN
HN
NH
NO2
HN
NH2
Scheme 3. Deprotection of the (Z)-Phe-Arg(NO2)-Ala-NH2 model tripeptide.

548
M. Sebestyén et al. / Tetrahedron Letters 57 (2016) 546–548
12. Ganem, B.; Osby, J. O. Chem. Rev. 1986, 86, 763.
Conclusions
13. Mazzini, F.; Salvadori, P. Synthesis 2005, 2479.
14. Morales-Serna, J. A.; Garcia-Rios, E.; Bernal, J.; Paleo, E.; Gavino, R.; Cardenas, J.
Synthesis 2011, 1375.
15. Khurana, J. M.; Arora, R. Synthesis 2009, 1127.
16. General: Electrospray ionization mass spectrometry (ESI-MS) spectra were
recorded on Bruker Daltonics Esquire 3000+ ion trap mass spectrometer.
Reactions were monitored by TLC on silica gel 60 F254 plates. Melting points
were determined on a Büchi 530 apparatus and are uncorrected. The reactions
were all followed by Reversed Phase High-performance Liquid
In summary, a simple and widely applicable method has been
devised for the guanidino N -NO2 deprotection of the arginine side
x
chain with high yield and chemoselectivity. NaBH4 in ethanol in
the presence of a copper-complex catalyst was an effective reagent.
This method was also applied to an oligopeptide containing an Arg
(NO2) residue. It has been demonstrated that the conditions devel-
Chromatography (RP-HPLC, using Phenomenex Synergi C12,
4 l, 120 Å,
x
oped for the removal of the N -NO2 group were compatible with
4.6 ꢁ 300 mm column, the eluents were: 0.1% TFA/H2O (V/V), 0.1% TFA/80%
acetonitrile-H2O (V/V), gradient elution 20–80% B (5–20 min), detection: UV
k = 220 nm) and ESI-MS. The reaction mixture was purified by RP-HPLC (using
conventional peptide synthesis techniques. To the best of our
knowledge this is the only general method for selective deprotec-
Phenomenex Jupiter C18, 5
l, 10 ꢁ 300 mm column, the eluents were: 0.1%
a
tion of the N-NO2 group leaving the N -Boc, and the benzylic-type
TFA/H2O (V/V), 0.1% TFA/80% acetonitrile-H2O (V/V), gradient elution 5–100% B
(5-60 min), detection: UV k = 220 nm). FT-IR spetra were recorded on Bruker
IFS 28, ATR technics FT-IR Spectrophotometer. The 1H and 13C NMR spectra
were recorded in DMSO-d6 solution in 5 mm tubes at RT, on a Bruker DRX-500
spectrometer at 500 (1H) and 125 (13C) MHz, with the deuterium signal of the
solvent as the lock and TMS as internal standard. The HSQC, HMBC, and COSY
spectra, which support exact assignment of 1H and 13C NMR signals were
obtained by using the standard Bruker pulse programs.
Z-, Bzl-, OBzl- groups intact, thus this reagent could open a ‘new
dimension’ in the orthogonality of traditional peptide protecting
groups.
Acknowledgments
a
a
x
17. N -(Z)-L-arginine (5): To a mixture of N -(Z)-N -nitro-L-arginine (4, 50 mg,
The authors are grateful for the valuable help of Assoc. Prof. E.
Vass for the IR measurements. This work was supported by grant
from the Hungarian National Research Fund (OTKA K104385).
0.142 mmol) and catalyst (10 mol %) in dry ethanol (1 mL) NaBH4 (1.42 mmol)
was added in a single portion. The mixture was stirred at room temperature for
2 h. Glacial acetic acid (10 ll) was added to quench the excess NaBH4. The
solvent was evaporated, and the crude product dissolved in the RP-HPLC
eluents (0.1% TFA/H2O (V/V), 0.1% TFA/80% acetonitrile-H2O (V/V)), and filtered
through a disc filter (hydrophilic PTFE, pore size: 0.45
lm). The conversion was
Supplementary data
followed by RP-HPLC. The collected fractions were lyophilized.
a
x
18. N -(Z)-
L
-phenylalanyl-N -nitro-
L
-arginyl-
L
-alanine-amide
(6):
The
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2015.12.
081.
compound 6 was synthesized by solid phase peptide synthetic methodology
a
on Rink-Amide MBHA resin (0.56 mmol/g), with the Fmoc/Bu strategy. N -
a
x
a
Fmoc-alanine, N -Fmoc-N -nitro-L-arginine and N -Z-phenylalanine were
coupled using DIC/HOBt (4 equiv). Fmoc-deprotection was achieved by
repeated (4 times: 2 min, 2 min, 5 min, 10 min) use of 2% piperidine and 2%
DBU in DMF (V/V). The cleavage of the peptide from the resin was performed
using TFA/H2O/TIS (V/V = 95/2.5/2.5) for 1 h. The product was precipitated
with Et2O, isolated by freeze-drying from 50% acetonitrile/water (V/V) (yield:
88%). The crude product (97% purity by HPLC) was used without further
purification for the NaBH4 reduction step.
References and notes
1. Bergmann, M.; Zervas, L.; Rinke, H. Z. Physiol. Chem. 1934, 224, 40.
2. Gowda, D. C. Tetrahedron Lett. 2002, 43, 311.
3. Tamura, S. Y.; Semple, J. E.; Ardecky, R. J.; Leon, P.; Carpenter, S. H.; Ge, Y.;
Shamblin, B. M.; Weinhouse, M. I.; Ripka, W. C.; Nutt, R. F. Tetrahedron Lett.
1996, 37, 4109.
4. Semple, J. E.; Rowley, D. C.; Brunck, T. K.; Ha-Uong, T.; Minami, N. K.; Owens, T.
D.; Tamura, S. Y.; Goldman, E. A.; Siev, D. V.; Ardecky, R. J.; Carpenter, S. H.; Ge,
Y.; Richard, B. M.; Nolan, T. G.; Håkanson, K.; Tulinsky, A.; Nutt, R. F.; Ripka, W.
C. J. Med. Chem. 1996, 39, 4531.
5. Krishnamoorthy, R.; Vazquez-Serrano, L. D.; Turk, J. A.; Kowalski, J. A.; Benson,
A. G.; Breaux, N. T.; Lipton, M. A. J. Am. Chem. Soc. 2006, 128, 15392.
6. Young, G. T.; Schafer, D. J.; Elliott, D. F.; Wade, R. J. Chem. Soc. C 1971, 46.
7. Richroch, M. N.; Gaudemer, A. J. Organomet. Chem. 1974, 67, 119.
8. Hanaya, K.; Muramatsu, T.; Kudo, H. J. Chem. Soc. Perkin Trans. I 1979, I, 2409.
9. Yoon, N. M.; Choi, J. Synlett 1993, 135.
a
a
19. N -Z-
L
-phenylalanyl-
L
-arginyl-
L
-alanine-amide (7): N -(Z)-
L
-phenylalanyl-
mol) was
x
N
-nitro-
L
-arginyl- -alanine-amide tripeptide (20.0 mg, 6, 35.2 l
L
dissolved in dry methanol or ethanol (4 mL), and stirred with copper(II)
acetylacetonate (2 mg, 7.6 mol) catalyst, then NaBH4 (352 mol) was added.
The mixture was stirred at room temperature for 1 h. Glacial acetic acid (40 l)
was added to quench the excess NaBH4. The reaction mixture was filtered
through a disc filter (hydrophilic PTFE, pore size: 0.45 m). The product was
l
l
l
l
purified using RP-HPLC and the collected fraction lyophilized One unidentified
side product’s peak overlapped on the chromatogram with the product thus
lowering the yield of purification (isolated yield: 76% (1 mg scale) or 27%
(20 mg scale)). The formation of the partially reduced nitroso compound was
demonstrated by MS (ESI, Fig. 2). The product was characterized by ESI-MS,
NMR, and FT-IR.
10. Basu, K.; Chakraborty, S.; Saha, C.; Sarkar, K. A. IOSR-JAC 2014, 7, 30.
11. Jagdale, A. R.; Paraskar, A. S.; Sudalai, A. Synthesis 2009, 660–664.