Racemization free longer N-terminal peptide hydroxamate synthesis on solid support using ethyl 2-(tert-butoxycarbonyloxyimino)-2-cyanoacetate

Article abstract of DOI:10.1016/j.tetlet.2015.09.084

A facile, efficient, racemization-free, and environment friendly protocol for the synthesis of peptide hydroxamic acids directly from carboxylic/amino acids by ethyl 2-(tert-butoxycarbonyloxyimino)-2-cyanoacetate in the presence of DIPEA/DMAP at room temperature is described. The compatibility of this method with Fmoc based solid phase peptide synthesis (SPPS) is also demonstrated by synthesizing three relatively large N-terminal peptide hydroxamic acids on resin. Also, some biologically important hydroxamates are synthesized using this protocol.

Full text of DOI:10.1016/j.tetlet.2015.09.084

Accepted Manuscript
Racemization Free Longer N-Terminal Peptide Hydroxamate Synthesis on Solid
Support Using Ethyl 2-(tert-butoxycarbonyloxyimino)-2-cyanoacetate
Srinivasa Rao Manne, Kishore Thalluri, Rajat Subhra Giri, Ashim Paul,
Bhubaneswar Mandal
PII:
S0040-4039(15)30126-X
DOI:
http://dx.doi.org/10.1016/j.tetlet.2015.09.084
TETL 46756
Reference:
To appear in:
Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
7 August 2015
15 September 2015
19 September 2015
Please cite this article as: Manne, S.R., Thalluri, K., Giri, R.S., Paul, A., Mandal, B., Racemization Free Longer
N-Terminal Peptide Hydroxamate Synthesis on Solid Support Using Ethyl 2-(tert-butoxycarbonyloxyimino)-2-
cyanoacetate, Tetrahedron Letters (2015), doi: http://dx.doi.org/10.1016/j.tetlet.2015.09.084
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Racemization Free Longer N-Terminal
Peptide Hydroxamate Synthesis on Solid
Support Using Ethyl 2-(tert-
Leave this area blank for abstract info.
butoxycarbonyloxyimino)-2-cyanoacetate
a
a
Srinivasa Rao Manne, Kishore Thalluri, Rajat Subhra Giri, Ashim Paul and Bhubaneswar Mandal*

1
Tetrahedron Letters
journal homepage: www.elsevier.com
Racemization Free Longer N-Terminal Peptide Hydroxamate Synthesis on Solid
Support Using Ethyl 2-(tert-butoxycarbonyloxyimino)-2-cyanoacetate
a
a
Srinivasa Rao Manne, Kishore Thalluri, Rajat Subhra Giri, Ashim Paul and Bhubaneswar Mandal*
Department of Chemistry, Indian Institute of Technology, Guwahati, Guwahati-781039, India.
Email id: bmandal@iitg.ernet.in
a
S. R. Manne and K. Thalluri contributed equally.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A facile, efficient, racemization-free and environment friendly protocol for the synthesis of
peptide hydroxamic acids directly from carboxylic/amino acids by Ethyl 2-(tert-
butoxycarbonyloxyimino)-2-cyanoacetate in presence of DIPEA/DMAP at room temperature is
described. The compatibility of this method with Fmoc based Solid Phase Peptide Synthesis
(SPPS) is also demonstrated by synthesizing three relatively large N-terminal peptide
hydroxamic acids on resin. Also, some biologically important hydroxamates are synthesized
using this protocol.
Available online
Keywords:
Boc-Oxyma
Hydroxamic acid
Peptide hydroxamic acid
Racemization free
SAHA
2009 Elsevier Ltd. All rights reserved.
Introduction
Peptide hydroxamates are found in many active pharmaceutical
1
ingredients (APIs) and natural products. They exhibit wide range
of biological activities including, antibacterial, antifungal, anti-1
inflammatory, anti-asthmatic, anticancer and antiarthritis.
Recently, a series of hydroxamic acid derivatives is discovered to
2
be promising agents for the management of Chagas disease.
Hydroxamic acids are strong bidentate metal-ion-chelating
agents, that interact with zinc (II)-containing proteins and thus
3
act as inhibitors of metalloenzymes.
SAHA (suberoylanilide hydroxamic acid, Figure 1) is an
anticancer agent that works against both hematologic and solid
4
tumors. Another important hydroxamic acid, Deferoxamine (a
5
siderophore), is widely used in medicine to remove excess iron
from body (Figure 1). Many N-terminal peptide hydroxamates
exhibit highly potent tumor necrosis factor converting enzyme
6
inhibitor (TACE, ADAM17) activity. Some of the commercially
available MMP/ADAM (Matrix metalloproteinase/A disintegrin
and metalloproteinase) inhibitors are also shown in Figure 1.
Figure 1. Medicinally important hydroxamic acids
Several methods have been developed for the synthesis of
hydroxamic acids directly from carboxylic acids by reacting with
hydroxylamine hydrochloride in the presence of various
catalysts, including8 cyanuric chloride, 9cyclic phosphonic
anhydride (PPAA), alkylcholoroformate, and TFFH/PTF
Hydroxamic acids have also been synthesized from esters
11
12
using sodium methoxide and potassium cyanide (KCN). The
synthesis of peptide hydroxamic acids on solid supports are also
7
13
reported. However, most of these reported solid support based
methods are limited to compounds with relatively low
molecular mass. Later, Stefanowicz and co-workers developed a
(
Tetramethylfluoroformamidiniumhexafluorophosphate/benzyltri
10
phenyl phosphonium dihydrogen trifluoride).
14
new resin for synthesizing longer peptide hydroxamic acids,
but the method is limited to only C-terminal peptide hydroxamic
acids. Also, many of these methods cause racemization and

2
Tetrahedron
a
require resin-bound esters, which are not commercially
Table 1. Synthesis of various hydroxamic acids using I
available. In this article, we wish to report an environment
friendly method for synthesizing racemization free hydroxamic
Product
acids
using
ethyl
2-(tert-butoxycarbonyloxyimino)-2-
Entry
Acid
O
a
Structure
O
Id
Yield
cyanoacetate (Boc-Oxyma, I, Figure 2) at room temperature
without the use of acid chlorides. Further, we have
demonstrated synthesis of larger N-terminal peptide hydroxamic
acids on solid support using this method.
1
2
OH
1a
75
Ph
OH
O
Ph
N
H
O
OH
OH
N
2a
72
1
0
H
1
0
O
O
OH
OH
N
3
H
3a
70
Br
Br
O
O
H
N
4
Ph
O
N
H
COOH
Ph
O
N
OH
72
4a
H
O
H
N
Figure 2. Ethyl 2-(tert-Butoxycarbonyloxyimino)-2-cyanoacetate (Boc-
Fmoc
Fmoc
5
N
H
COOH
N
H
OH
5a
71
Oxyma)
O
H
Fmoc
Fmoc
N
6
7
8
N
H
COOH
N
OH
6
a
69
H
O
Ph
Ph
H
Fmoc
N
Fmoc
N
OH
7a
70
N
H
COOH
H
O
Scheme 1. Synthesis of hydroxamic acids directly from carboxylic acids
Ph
Ph
H
DL-Fmoc
N
8a
69
DL- Fmoc
N
OH
First, we have optimized the reaction conditions using benzoic
acid as a model substrate. Pre-activation of benzoic acid have
been achieved using I in THF in the presence of DIPEA (N, N-
diisopropylethylamine) for 30 min at 0-5 °C. Next, the addition
of hydroxylamine hydrochloride dissolved in DMF to the
reaction mixture has generated moderate yield of the desired
product in 52% yield (entry 1, table S1 in supplementary data)
after stirring at room temperature for 2 h. Use of 0.1 equiv of
DMAP along with DIPEA has increased the yield to 75% (entry
N
H
COOH
H
O
O
O
H
Fmoc
N
N
9a
75
9
Fmoc
OH
N
H
COOH
H
O
Ph Ph
Ph
Ph Ph
Ph
N
N
N
N
10a
71
Fmoc
NHOH
10
N
Fmoc
2
, Table S1, supplementary data). Therefore, we have used of 0.1
N
H
COOH
H
O
equiv of DMAP as a catalyst and 2.5 equiv of DIPEA (in total) as
a base for further exploration. Next, various solvents and solvent
combinations have been examined and THF/DMF solvent
mixture produced better yield.
Ph
Ph
H
N
11
Boc
Boc
N
COOH
N
OH
11a
68
H
H
O
Ph
Ph
A wide variety of peptide hydroxamic acids with various N-
protected amino acids and carboxylic acids have been
synthesized in good to excellent yields (Table 1) under the
optimized reaction condition. Reactions with aromatic carboxylic
acids (entry 1 and 2, Table 1), aliphatic carboxylic acids (entry 3)
and amino acids (entries 4-12) have proceeded well. This method
shows compatibility with common N-terminal protecting groups
such as Cbz (entry 4), Fmoc (entries 5-10) and Boc (entries 11
and 12). The method is also compatible with various side chain
protecting groups including, tert-butyl (entry 9), trityl (entry 10),
and benzyl (entry 12).
O
O
1
2
H
N
Boc
Boc
N
COOH
N
OH
12a
70
H
H
O
a
Reaction conditions: Carboxylic acid (1 mmol), Boc-Oxyma (242 mg, 1
mmol.), DMAP (12.2 mg, 0.1 mmol), DIPEA (322.5 mg, 2.5 mmol),
hydroxylamine hydrochloride (103.5 mg, 1.5 mmol), room temperature, 2 h.
THF/DMF ratio was 4:1; all amino acids are L-amino acids other than that of
entry 8; no evidence of racemization has been observed by HPLC
b
(Chiralpack-ASH column) for the products 4a-7a and 9a-11a. Isolated yield.
O
O
H
N
NH2
I (1 equiv.)
OH
OH
+
HO
DIPEA, DCM
O
Further, we have applied this method for the synthesis of
SAHA (suberoylanilide hydroxamic acid, Figure 1) and its
derivatives (Scheme 2). These compounds have emerged as
important anticancer agents and recently gained FDA approval
R
O
R
I (1 equiv.)
NH2.OH.HCl (1.5 equiv.)
13a R = H
3b R = 3-Cl
13c R = 4-NO2
13d R = 4-F
DIPEA (2.5 equiv.)
1
O
15
H
N
for the treatment of advanced cutaneous T-cell lymphoma. First
step of the synthesis have been carried out as described
OH
N
H
O
R
16
elsewhere and the second step have been performed using the
current protocol. The added advantage of our method over the
Scheme 2. Synthesis of SAHA and its derivatives
17
other published methods is of two fold. Both the steps can be
achieved using the same reagent I, also prior selective protection
of the starting di-carboxylic acid is not necessary. Thus, one step
has been reduced and the reaction can be performed in one pot
manner.
We also have investigated the racemization probability of this
method using HPLC. All the hydroxamic acids that have been
synthesized from L-amino acids (entries 4a-7a and 9a-11a) have
exhibited single peak corresponding to single enantiomeric
product when passed through a chiral HPLC column (Diacel,

3
2
0
Chiralpack-ASH, supporting information Figure S13, S17, S21,
S25, S30, S34 and S38), whereas, that of a DL-amino acid
derived hydroxamic acid (entry 8, acted as a reference) has
exhibited two distinct peaks corresponding to two enantiomers,
one is for L and the other one is for D enantiomer (see Figure 3
below and supporting information, Figure S25 and S26) in same
experimental condition and same gradient. These results indicate
that the current method of hydroxamic acid synthesis does not
cause detectable racemization.
modified resin is described recently. But, we focused on the
formation of N-terminal hydroxamates of such large peptides on
resin. Succinylation at the N-terminal was preferred, as many
metalloprotein inhibitors bear succinylated hydroxamic acid
21
derivatives at the N-terminal. However, when we have tried to
perform hydroxamidation on the succinylated N-terminal amino
acid of a peptide (sequence: FGLIAG, Figure 5) on solid support,
almost quantitative conversion to the succinimide derivative of
the peptide has been observed. Such succinimide formation is
22
highly probable as five membered ring formations is preferable.
To avoid such five membered ring formation, two other peptides
have been synthesized by SPPS, where N-methylated glycine has
been used at the N-terminal (A and B, Figure 4). On resin
hydroxamidation has been successful for both of these peptides.
However, to demonstrate that N-methylation, which is often not
easy to perform and also N-methylated amino acid derivatives are
costly, is not always necessary, another peptide hydroxamate has
been synthesized using adipic acid at the N-terminal (C, Figure
4
). We have successfully synthesised three peptide hydroxamic
acids A, B and C (Figure 4) with overall yield of 25%, 22% and
9%, respectively. Yields have been calculated with respect to
1
the reported resin loading by the supplier after purification by
semi-preparative HPLC.
Figure 3. HPLC profiles of products 7a (left) & 8a (right).
Further, we have extended this method for the synthesis of
peptide hydroxamic acids using solid phase peptide synthesis
18
(
SPPS) protocol. Amino acids have been coupled on Rink
amide MBHA resin following Fmoc/tBu orthogonal protection
strategy using BOP ((Benzotriazol-1-
yloxy)tris(dimethylamino)phosphonium hexafluorophosphate) as
a coupling reagent. Then, succinylation have been performed
using succinic anhydride on the N-terminal amino acid of the
peptide on solid resin. After that, hydroxamidation has been
performed by current protocol on solid support using
hydroxylamine hydrochloride (3 equiv), Boc-Oxyma (3 equiv),
and DIPEA (5 equiv). DMF has been used as solvent for all the
reactions on solid support. Hydroxamidated peptides have been
finally cleaved using TFA/DCM (85:15) solvent mixture,
precipitated with cold ether, and purified using semi-preparative
HPLC.
Figure 5. Undesired succinimide formation while hydroxamidation on solid
support
A plausible reaction mechanism of the hydroxamic acid
formation starting from carboxylic acid is depicted based on
existing literature (see Scheme S1 in supplementary data). At the
first step, the carboxylic acid group reacts with Boc-Oxyma to
16
form the corresponding ester of Oxyma, III via anhydride II. In
the second step, III reacts with hydroxylamine hydrochloride in
23
the presence of DMAP to form the desired product. However,
hydroxylamine can also react directly with either intermediate II
or the intermediate III and may generate the desired product.
In summary, we have developed a racemization free and
environment friendly method for conversion of carboxylic acids
to the corresponding hydroxamic acids with hydroxylamine
hydrochloride in presence of DIPEA/DMAP under mild reaction
24
conditions using Boc-Oxyma. This method is compatible with
Fmoc based SPPS as well as various acid or base labile
protecting groups. Further, we have demonstrated the utility of
the current protocol for synthesizing biologically important
SAHA derivatives. Thus, this method may be useful for the
synthesis of various long chain MMP/ADAM inhibitors and
other biologically important hydroxamates.
Figure 4. Long chain N-terminal peptide hydroxamic acids synthesized on
solid support; peptide sequences: (A) GVFILG-NH
2
and (B)
GLAKLAELAKG-NH
in A & B.
, (C) VLGFA-NH ; N-terminal Gly is N-methylated
2 2
Acknowledgments
We have selected hydrophobic peptide sequences for our
exploratory studies, as these are known as “difficult sequences”
for synthesis (A and C, Figure 4). The amino acid sequence of
peptide B (Figure 4) is based on the amphiphilic peptides,
applied by Mutter in designing TASP molecules. Solid phase
synthesis of C-terminal hydroxamates of such peptides using
Authors are grateful to Central Instruments Facility (CIF) and
Department of Chemistry of Indian Institute of Technology
Guwahati for NMR and HRMS facilities and to the Department
of Biotechnology, Govt. of India (twinning programme for the
19

4
Tetrahedron
North Eastern Region, sanction no. BT/347/NE/TBP/2012) for
followed by the addition of hydroxylamine hydrochloride in DMF
0.5 mL), DIPEA (1.5 mmol). The progress of the reaction was
monitored by TLC at room temperature. After completion of the
reaction, the reaction mixture was concentrated using rotary
evaporator and then diluted with 15 mL of ethyl acetate and
(
financial support.
Supplementary Material
washed with 5% HCl (2×10 mL), 5% NaHCO
saturated NaCl solution (2×10 mL) and dried over anhydrous
Na SO and the evaporation of the solvent gave a residue that was
purified on silica gel column chromatography using hexane and
ethyl acetate.
3
(2×10 mL),
The online version of this article contains supplementary
material, which is available to authorized users. The
2
4
supplementary
material
contains,
optimization
table,
1
13
experimental procedures, characterization data, ( H, C) NMR,
HRMS and HPLC spectra for all compounds.
Characterization data of N-Hydroxybenzamide 1a. White solid;
1
(102 mg, 75 %), mp 125-127 °C; H NMR (400 MHz, CDCl
3
) δ
7
7
.65-7.63 (d, J = 6.8 Hz, 2H), 7.42-7.38 (t, J = 7.6 Hz, 1H), 7.33-
13
3
.30 (t, J = 7.6 Hz, 2H); C NMR (100 MHz, CDCl , few drops of
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4. Typical procedure of synthesis of hydroxamic acids:
Ethyl 2-(tert-butoxycarbonyloxyimino)-2-cyanoacetate (Boc-
Oxyma, I) (1 mmol) was added to a stirred solution of carboxylic
acid (1 mmol), DIPEA (1 mmol) and DMAP (0.1 mmol) in THF
2
2
5
(2 mL) at 0-5 °C. Then the reaction mixture was stirred for 30 min