Synthesis and application of N-hydroxylamine derivatives as potential replacements for HOBt

Article abstract of DOI:10.1002/ejoc.200801179

Several heterocycles containing N-hydroxylamine were prepared and tested as coupling additives to replace the use of N-hydroxybenzolriazole (HOBt.) derivatives. On the basis of our results on coupling yield and racemization-suppressing properties, we propose N-hydroxyindolin-2-one and 6- cfiloro-N-hydroxy-2-phenylbenzimidazole as suitable substitutes for HOBt. Wiley-VCH Verlag GmbH & Co. KGaA.

Full text of DOI:10.1002/ejoc.200801179

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
SHORT COMMUNICATION
DOI: 10.1002/ejoc.200801179
Synthesis and Application of N-Hydroxylamine Derivatives as Potential
Replacements for HOBt
Ayman El-Faham*[a,b,c] and Fernando Albericio*[a,d,e]
Keywords: Amino acids / Coupling reagents / N-Hydroxylamine derivatives / Peptides / Racemization
Several heterocycles containing N-hydroxylamine were pre-
pared and tested as coupling additives to replace the use of
N-hydroxybenzotriazole (HOBt) derivatives. On the basis of
our results on coupling yield and racemization-suppressing
properties, we propose N-hydroxyindolin-2-one and 6-
chloro-N-hydroxy-2-phenylbenzimidazole as suitable substi-
tutes for HOBt.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2009)
Introduction
During the last decades, aminium/uronium and phos-
phonium derivatives of HOBt (5, 6) have been proposed for
the synthesis of peptides.[5] In the presence of base, these
stand-alone coupling reagents react with the protected
amino acids to render the corresponding oxybenzotriazole
esters.[6]
One of the main drawbacks associated with the use of
HOBt derivatives is their explosive properties, which are
often not always properly referenced in the literature.[5,7]
This explosive tendency is associated with the triazo system,
For many years, 1-hydroxybenzotriazole (HOBt, 1)[1] is
present in almost all peptide couplings, especially as an ad-
ditive in combination with carbodiimides (2).[1] Later, other
benzotriazole derivatives, such as 6-chloro-1-hydroxybenzo-
triazole (6-Cl-HOBt, 3)[2] and 1-hydroxy-7-azabenzotriazole
(HOAt, 4),[3] were introduced. The main role of HOBt de-
rivatives is to react with O-acylisourea, formed by reaction
of the protected amino acid, to reduce racemization associ-
ated with O-acylisourea and to prevent its rearrangement
to the inactive N-acylurea.[4]
which must be handled with care.[8] This risk is greater dur-
ing the industrial production of peptides, which often in-
Barcelona Science Park, Baldiri Reixac 10, 08028 Barcelona, volves the handling of tons of these derivatives.
[a] Institute for Research in Biomedicine,
Spain
E-mail: albericio@irbbarcelon.org
[b] Department of Chemistry, Faculty of Science, Alexandria Uni-
versity,
Ibrahimia 21321, Alexandria, Egypt
E-mail: aymanel_faham@hotmail.com
[c] Department of Chemistry, College of Science, King Saud Uni-
versity,
P. O. Box 2455, Riyadh, Saudi Arabia
[d] Department of Organic Chemistry, University of Barcelona,
Martí i Franqués 1-11, 08028 Barcelona, Spain
[e] CIBER-BBN, Networking Centre on Bioengineering, Biomate-
rials and Nanomedicine, Barcelona Science Park,
Baldiri Reixac 10, 08028 Barcelona, Spain
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
Eur. J. Org. Chem. 2009, 1499–1501
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1499

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
A. El-Faham, F. Albericio
SHORT COMMUNICATION
with good to excellent yields (45–85%, yields not opti-
mized) and characterized by NMR (1H, 13C) spectroscopy
and elemental analysis (Table 1).
Results and Discussion
Here we describe the preparation of a number of N-hy-
droxylamine-containing heterocycles for tests as coupling
additives. Both coupling yield and racemization-sup-
pressing properties were studied.
In addition to the presence of the hydroxylamine moiety,
none of the derivatives prepared contained two or more ni-
trogen atoms in consecutive positions as occurs in hydroxy-
benzotriazole derivatives. Furthermore, some extra Cl
atoms were introduced in an attempt to mimic the positive
effect shown by Cl in the case of the 6-Cl-HOBt vs.
HOBt.[2,9]
Compound 7 was prepared by reductive cyclization of
(2-nitrophenyl)acetic acid (14) in the presence of Zn/H2SO4.
Compounds 8–11 were prepared by reaction of substituted
isatoic acid derivatives 15–18, respectively, with hydroxyl-
amine or benzyl derivatives, followed by reaction with
(HCO)2O or (AcO)2O. Compounds 12[10] and 13 were pre-
pared by reaction of o-nitroaniline derivatives 19 and 20
with NaH, followed by addition of benzyl bromide in dry
THF, followed by catalytic hydrogenation with Pd/C in
Table 1. Yield and racemization during the formation of Z-Phg-
Pro-NH2 in DMF at room temp. by using distinct additives (solu-
tion-phase synthesis). HPLC system, linear gradient over 30 min
of 0.045% TFA in ACN and 0.036% aqueous TFA from 2:8 to
1:1; flow rate 1.0 min–1, detection 220 nm, Waters Symmetry C18
columns (5 mm, 4.6ϫ150 mm), RtLL
= 26.01 min, RtDL =
27.40 min.
Entry
Coupling reagent
Yield
(%)

(%)
1
2
3
4
5
6
7
8
9
HOI (7)/DIC
HOI (7)/DIC[a]
HOQ (8)/DIC
80.0
81.4
75.6
74.9
76.3
76.1
76.5
78.9
87.2
88.0
81.4
81.9
1.6
3.3
38.1
32.9
36.1
31.2
39.4
34.0
19.5
16.0
3.3
6-Cl-HOQ (9)/DIC
HOMQ (10)/DIC
6-Cl-HOMQ (11)/DIC
HOMQ (10)/DIC[a]
6-Cl-HOMQ (11)/DIC[a]
HOBI (12)/DIC
10
11
12
6-Cl-HOBI (13)/DIC
HOAt (4)/DIC
HOBt (1)/DIC
9.3
MeOH (Scheme 1). All these compounds were obtained [a] 2 min preactivation.
Scheme 1. Synthesis of the N-hydroxy derivatives.
1500
www.eurjoc.org
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 1499–1501

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
N-Hydroxyamine Derivatives as Potential Replacements for HOBt
temp. for 1 h, and then H2O (20 mL) was added. The precipitate
was collected by filtration, washed with cold water, and dried to
give the pure product [yield 1.69 g (80.3%)]. M.p.183–185 °C. 1H
NMR ([D6]DMSO): δ = 2.11 (s, 3 H, CH3), 7.63 (dd, 1 H), 7.87
(d, 1 H), 8.45 (d, 1 H), 10.93 (s, 1 H, OH) ppm. 13C NMR([D6]-
DMSO): δ = 25.57, 119.17, 122.56, 126.83, 130.80, 134.21, 140.18,
168.83, 169.26 ppm. C9H7ClN2O2 (210.6): calcd. C 51.32, H 3.35,
N 13.30; found C 51.60, H 3.55, N 13.52.
To run a preliminary study of the performance of these
new additives in carbodiimide-based coupling, we chose to
synthesize the dipeptide Z-Phg-Pro-NH2 model in solution
by using DIC.
The phenylbenzimidazole derivatives (12,13) (Entries 9,
10) gave better coupling yields than HOBt and HOAt (En-
tries 11, 12). Furthermore, the coupling yield for N-hy-
droxyindolin-2-one (7) (Entries 1, 2) was similar to that of
HOBt and HOAt (Entries 11, 12). In contrast, the remain-
ing additives showed slightly worse performance than the
parent compounds. Regarding racemization-suppressing
properties, HOI (7) (Entries 1, 2) gave an excellent result,
whereas HOBI (12) and 6-Cl-HOBI (13) (Entries 9, 10)
showed poorer performance, but clearly better than the rest
of the coupling additives tested. Again, as with the HOBt/
Cl-HOBt system, the chloro derivatives gave better results
than the parent compounds (Entries 4, 6, 8, 10 versus En-
tries 3, 5, 7, 9).
6-Chloro-N-hydroxy-2-phenylbenzimidazole (6-Cl-HOBI, 13): Syn-
thesized according to a similar strategy as that described for the
1
parent compound.[14] M.p. 262–264 °C. H NMR ([D6]DMSO): δ
= 7.25 (dd, 1 H), 7.53–7.57 (m, 4 H, Ph), 7.68 (d, 1 H), 8.20–8.25
(m, 2 H), 12.19 (s, 1 H, OH) ppm. 13C NMR ([D6]DMSO): δ =
123.72, 125.90, 130.35, 131.35, 132.36, 133.87, 134.88, 135.58,
136.35, 147.34, 148.17, 159.99, 165.26 ppm. C13H9ClN2O (244.68):
calcd. C 63.81, H 3.71, N 11.45; found C 64.07, H 3.92, N 11.67.
Supporting Information (see footnote on the first page of this arti-
1
cle): H and 13C NMR spectra of the compounds prepared.
Acknowledgments
Conclusions
This work was partially supported by the Ministerio de Ciencia e
Innovación (CTQ2006-03794/BQU), the Instituto de Salud Carlos
III (CB06_01_0074), the Generalitat de Catalunya (2005SGR
00662), the Institute for Research in Biomedicine, and the Barce-
lona Science Park.
Although the model peptide is highly demanding in
terms of racemization, HOI (7) and even 6-Cl-HOBI (13)
are worth considering as potential substitutes for HOBt de-
rivatives.
[1] W. König, R. Geiger, Chem. Ber. 1970, 103, 788–798.
[2] M. Ueki, T. Yanagihira, in Peptides 1998, Proceedings of the
25th European Peptide Symposium (Eds.: S. Bajusz, F. Hudecz),
Akademiai Kiado, Budapest, 1998, pp. 252–253.
[3] L. A. Carpino, J. Am. Chem. Soc. 1993, 115, 4397–4398.
[4] a) F Albericio, L. A. Carpino, In Methods in Enzymology, So-
lid-Phase Peptide Synthesis (Ed.: G. B. Fields), Academic Press,
Orlando, 1997, vol. 289, pp. 104–126; b) F. Albericio, R. Chin-
chilla, D. Dodsworth, C. Nájera, Org. Prep. Proc. Int. 2001, 33,
203–303.
[5] O. Marder, F. Albericio, Chem. Today 2003, 21, 6–11.
[6] a) L. A. Carpino, A. El-Faham, C. A. Minor, F. Albericio, J.
Chem. Soc., Chem. Commun. 1994, 201–203; b) F. Albericio,
M. Cases, J. Alsina, S. A. Triolo, L. A. Carpino, S. A. Kates,
Tetrahedron Lett. 1997, 38, 4853–4856.
[7] K. D. Wehrstedt, P. A. Wandrey, D. Heitkamp, J. Hazard. Ma-
ter. 2005, 126, 1–7.
Experimental Section
General: TLC was performed on silica plates (8ϫ4 cm) from Albet
by using suitable solvent systems and visualization by a Spectroline
UV Lamp Model CM-10 (254 nm). Melting points were obtained
in open capillary tubes with a Gallenkamp Sanyo melting point
apparatus and are uncorrected. NMR spectra were recorded with
a Varian Mercury 400 MHz spectrometer at room temperature
(r.t.). All solvents used for recrystallization, extraction, column
chromatography and TLC were commercial grade, distilled before
use and stored under dry conditions. Model peptide Z-Phg-Pro-
NH2 was analyzed according to previously described methods.[3]
N-Hydroxyindolin-2-one (HOI, 7),[11] 3-hydroxyquinazolin-4-one
(HOQ, 8),[12] 3-hydroxy-2-methylquinazolin-4-one (HOMQ, 10),[13]
and N-hydroxy-2-phenylbenzimidazole (HOBI, 12)[14] were synthe-
sized as described in the literature.
[8] L. Jiang, A. Davison, G. Tennant, R. Ramage, Tetrahedron
1998, 54, 14233–14254.
6-Chloro-3-hydroxyquinazolin-4-one (6-Cl-HOQ, 9): Synthesized
according to a similar strategy described for the parent com-
pound.[12] We obtained 0.71 g (36.6%) (from a 10 mmol reaction)
[9] O. Marder, Y. Shvo, F. Albericio, Chem. Today 2002, 20, 37–
41.
[10] The organophosphorus and the sulfonyloxy derivatives of
HOBI (12) have been used as stand-alone coupling reagents.
a) N. D. Kokare, R. R. Nagawade, V. P. Rane, D. B. Shinde,
Synthesis 2007, 766–772; b) N. D. Kokare, R. R. Nagawade,
V. P. Rane, D. B. Shinde, Protein Peptide Lett. 2007, 14, 259–
263; and c) N. D. Kokare, R. R. Nagawade, V. P. Rane, D. B.
Shinde, Tetrahedron Lett. 2007, 48, 4437–4440.
[11] K. H. Collins, J. Am. Chem. Soc. 1956, 78, 221–224.
[12] R. H. Clark, E. C. Wagner, J. Org. Chem. 1944, 9, 55–67.
[13] F. Gutierrez, C. Tedeschi, L. Maron, J. P. Daudey, R. Poteau,
J. Azema, P. Tisnes, C. Picard, Dalton Trans. 2004, 1334–1347.
[14] J. M. Gardiner, C. R. Loyns, C. H. Schwallabe, G. C. Barrett,
P. R. Lowe, Tetrahedron 1995, 51, 4101–4110.
1
of the product as a light brown solid. M.p. 268–269 °C. H NMR
([D6]DMSO): δ = 7.74 (d, 1 H), 7.85 (dd, 1 H), 8.09 (d, 1 H), 8.56
(s, 1 H), 12.02 (s, 1 H, OH, exchangeable with D2O) ppm. 13C
NMR ([D6]DMSO): δ = 124.44, 125.44, 130.38, 132.06, 134.78,
146.27, 147.03, 157.01 ppm. C8H5ClN2O2 (196.5): calcd. C 48.88,
H 2.56, N 14.25; found C 49.10, H 2.38, N 14.50.
6-Chloro-3-hydroxy-2-methylquinazolin-4-one (6-Cl-HOMQ, 11):
Synthesized according to a similar strategy as that described for
the parent compound.[13] 2-Amino-5-chloro-N-hydroxybenzamide
(1.86 g, 10 mmol) was suspended in triethylamine (1.3 mL,
10 mmol), and then HOAc (10 mL) was added, followed by AcCl
(1 mL) at room temp. The reaction mixture was stirred at room
Received: November 27, 2008
Published Online: February 18, 2009
Eur. J. Org. Chem. 2009, 1499–1501
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1501