Synthesis of arginine-containing hydroxamate dipeptidomimetics

Article abstract of DOI:10.1016/j.tetlet.2006.03.190

The syntheses of arginine-containing hydroxamates using various peptide coupling methods are described. Fmoc-Arg(NO₂)-Cl prepared at low temperature did not undergo intramolecular δ-lactam formation and effectively provided desired hydroxamates

Full text of DOI:10.1016/j.tetlet.2006.03.190

Tetrahedron Letters 47 (2006) 4069–4073
Synthesis of arginine-containing hydroxamate dipeptidomimetics
Jiwon Seo and Richard B. Silverman*
Department of Chemistry, Department of Biochemistry, Molecular Biology, and Cell Biology,
The Center for Drug Discovery and Chemical Biology, Northwestern University, Evanston, IL 60208-3113, USA
Received 24 January 2006; revised 28 March 2006; accepted 29 March 2006
Available online 2 May 2006
Abstract—The syntheses of arginine-containing hydroxamates using various peptide coupling methods are described.
Fmoc-Arg(NO2)-Cl prepared at low temperature did not undergo intramolecular d-lactam formation and effectively provided
desired hydroxamates (8 and 10) in good yields. Fmoc and N-nitro protecting groups can be easily removed. Therefore, this report
provides a facile synthesis of arginine-containing peptidomimetic compounds.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
L-Arginine is a guanidino-containing basic amino acid,1
which is positively charged at neutral pH, and is
involved in many important physiological processes.2
Many enzymes show a preference for arginine residues
in natural substrates and synthetic inhibitors, for
example, nitric oxide synthases3 and trypsin-like serine
proteases.4 Therefore, peptidomimetics of arginine-
containing biologically active molecules have been of
interest for medicinal chemists and have provided
molecules with better bioavailability, biostability, and
potency.1
HN
NHR
NH
N
NHR
NH
P
N
H
X
P
N
O
H
O
1: P = Boc
2: P = Fmoc
R = H or NO2
P = protecting group
X = activated group
Figure 1. Intramolecular cyclization of arginine.
Despite the importance of arginine mimetics, the scope
of their syntheses has been limited because of the
difficulty of the chemistry associated with arginine.
Firstly, the highly basic nature and nucleophilic charac-
ter of the guanidino moiety in arginine normally
requires appropriate protection of this group before
subsequent chemical manipulation.1 Ideally, protecting
groups have to be removed easily under mild conditions
and should be orthogonal to the other protecting
groups. Secondly, acylation reactions of activated
arginine usually compete with the side reaction of intra-
molecular d-lactam formation (Fig. 1).5 The latter case is
a serious problem, especially when the reaction involves
a weak nucleophile.
In the course of our research program of neuronal
isoform selective nitric oxide synthase inhibitors dis-
covery, we needed to synthesize nitroarginine-contain-
ing dipeptide peptidomimetic compounds (8 and 10,
Scheme 2). The key feature in the inhibitor synthesis
was the hydroxamate-forming coupling reaction. This
reaction involves weakly nucleophilic N-alkyl hydroxyl-
amines (5 and 6, Scheme 1), and the reaction requires
strong activation of the other reactant, namely, pro-
tected arginine (3 or 4, Scheme 2). For this purpose,
we investigated a way to activate nitroarginine, while
avoiding common side reactions, and to provide a read-
ily useful intermediate not only for the hydroxamate
coupling, but also for the other arginine-containing
pseudopeptides syntheses.
Keywords: Arginine; Hydroxamate; Peptidomimetics; Acid chloride.
*
Corresponding author. Tel.: +1 847 491 5653; fax: +1 847 491
7713; e-mail: Agman@chem.northwestern.edu
0040-4039/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.03.190

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J. Seo, R. B. Silverman / Tetrahedron Letters 47 (2006) 4069–4073
O
O
O
O
O
(i)
HO
HO
HO
a
O
d
b
c
OCH3
OH
OH
OCH3
O2N
NHFmoc
NHFmoc
NH2
NHFmoc
5c
5a
5b
HO
HN
O
O
Boc
Boc
O
N
O
HO
OCH3
OCH3
OCH3
f
e
NHFmoc
NHFmoc
NHFmoc
5d
5e
5
(ii)
O
O
O
Boc
Boc
O
N
O
HO
HN
O
O
g
HO
HO
h
i
j
OCH3
OH
OCH3
OCH3
N
Fmoc
6a
N
N
N
N
Fmoc
Fmoc
Fmoc
Fmoc
6b
6c
6
Scheme 1. Synthesis of N-alkylhydroxylamines 5 and 6. Reagents and conditions: (a) Fmoc-Cl, 10% Na2CO3, dioxane, 86%; (b) MeI, NaHCO3,
DMF, 95%; (c) p-nitrobenzoic acid, DEAD, PPh3, THF, 94%; (d) MeOH, NaN3, 40 °C, 98%; (e) Boc-NH-O-Boc, DEAD, PPh3, THF, 60%; (f) 50%
TFA, CH2Cl2, 96%; (g) DIAD, PPh3, THF, 85%; (h) MeOH, NaN3, 40 °C, 96%; (i) Boc-NH-O-Boc, DEAD, PPh3, THF, 45%; (j) 50% TFA,
CH2Cl2, 98%.
Mitsunobu reaction with N,O-diBoc protected hydr-
HN
HN
NHNO2
oxylamine, and a Boc deprotection.6
Attempted reactions to obtain 7 or 9 using uronium- or
phosphonium-based peptide coupling reagents7 resulted
in no isolable product formation.8 In these reactions,
most of the starting material (5 or 6) remained
unchanged, indicating that 5 or 6 are weak nucleophiles;
consequently, side product 1 (Fig. 1)9 was observed.
Because it has been reported that one of the halophos-
phonium salts, PyBroP,10 is highly efficient for difficult
coupling reactions with essentially no epimerization,11
we employed this reagent for the coupling of 4 with 5.
PyBroP/DIEA reagent provided an 8% isolated yield
of 8. The yield was slightly improved by using the
in situ acid fluoride forming reagent TFFH/Na2CO3.
Acid fluoride is reported to be an effective reagent for
sterically demanding peptide coupling reactions.12 How-
ever, the yield of 8 did not exceed 20% with TFFH, and
2 was recovered as a major product.
OH
N
O
P
N
OCH3
H
HN
HN
NHNO2
O
5
NHFmoc
7 (P = Boc)
8 (P = Fmoc)
OH
O
P
N
H
P = Boc (3) or Fmoc (4)
6
HN
HN
NHNO2
OH
N
O
P
N
OCH3
H
O
N
Fmoc
9 (P = Boc)
10 (P = Fmoc)
To compare the reactivity of hydroxylamine 5 with
an O-protected hydroxylamine analogue, a coupling
reaction using O-benzyl protected hydroxylamine 11
(Scheme 3) was attempted. Under peptide coupling
conditions such as EDC/HOAt, HATU/DIEA, and
PyBroP/DIEA, compound 11 did not couple with 4.
Therefore, we concluded that free hydroxylamine 5
was more reactive than protected hydroxylamine 11 in
the coupling reaction with 4, and investigated further
for a better coupling system with 5.
Scheme 2. Nitroarginine-containing dipeptidomimetic intermediate
syntheses.
2. Results and discussion
TFA salts of N-alkylhydroxylamines 5 and 6 were
prepared according to Scheme 1. For the synthesis of
5, commercially available (S)-(ꢀ)-4-amino-2-hydroxy-
butyric acid was protected followed by a series of two
Mitsunobu reactions to obtain the hydroxylamine
functionality with the desired chirality. Fmoc-trans-4-
Hydroxy-L-proline was converted to 6 by the following
successive reactions: an intramolecular Mitsunobu
reaction, a sodium azide catalyzed methanolysis, a
To overcome the low reactivity problem of hydroxyl-
amines 5 or 6, a highly activated protected nitroarginine,
such as an acid chloride, seemed desirable, if not neces-
sary, for the coupling reaction. Previously, the use of
sulfonamide (i.e., tosyl) protected arginines for the

J. Seo, R. B. Silverman / Tetrahedron Letters 47 (2006) 4069–4073
4071
HN
HN
NHNO2
O
BnO
HN
O
OBn O
i) Tf2O, 2,6-lutidine, dry CH2Cl2, -78 o
ii) BnONH2, dry CH2Cl2, r.t., 48%
C
HO
4
OCH3
OCH3
N
Fmoc
N
OCH3
H
O
NHFmoc
NHFmoc
NHFmoc
11
12
Scheme 3. Hydroxamate coupling reaction with benzyl protected hydroxylamine 11.
preparation of the corresponding acid chlorides was
reported.13 However, sulfonamide is one of the most
stable nitrogen protecting groups, so methods for depro-
tection usually suffer from poor yields and/or harsh
reaction conditions.14 In peptide syntheses, harsh
conditions can lead to unwanted side reactions such as
a loss of stereogenic integrity or the deprotection of
other protecting groups.15 Additionally, a nitro group
attached to arginine at the x-nitrogen was necessary
for our nitric oxide synthase inhibitor synthesis, so we
used the conditions13c to prepare Fmoc-Arg(NO2)-Cl.
Using this activated amino acid, we investigated proper
conditions for the hydroxamate coupling reaction.
Fmoc and N-nitro protecting groups can be deprotected
by secondary amine treatment and hydrogenation,16
respectively. Furthermore, as Carpino et al.17 reported,
Fmoc-amino acid chloride coupling occurs without loss
of chirality at the a-carbon under appropriate condi-
tions. Wang et al. proved minimal loss of stereointegrity
during an acid chloride mediated hydroxamate-forming
reaction.18
et al.,13c it was found that the side reaction could be
suppressed significantly when the acid chloride was used
in situ. In our hydroxamate coupling reaction, we found
that 2,4,6-collidine performed superior to pyridine or
triethylamine as the base, and THF provided excellent
solubility. Using these optimized conditions, Fmoc-
Arg(NO2)-Cl and hydroxylamines 5 and 6 coupled to
form hydroxamates 8 and 10 in 85% and 74% isolated
yields, respectively (Scheme 4).20 The extent of epimer-
ization during the coupling reaction was determined
by HPLC (Supplementary data). Results obtained from
this experiment indicate that the loss of stereointegrity
was about 5%. We have found that anhydrous condi-
tions are a prerequisite for the success of this reaction,
including the suppression of racemization.
In summary, the conditions for arginine-containing
hydroxamate synthesis were investigated. We found con-
ditions that allowed the coupling of Fmoc-Arg(NO2)-Cl
and free hydroxylamine analogs 5 and 6 to be carried out
effectively without significant side reactions. This method
should broaden the scope of arginine containing pepti-
domimetic compound preparation.
Along with histidine, arginine is one of the amino acids
that is not stable in an acid halide form at room temper-
ature.19 At room temperature the d-amino group of
Nx-nitroarginine possesses sufficient reactivity for
intramolecular lactam formation to occur. In fact, the
attempted hydroxamate coupling reaction between 5
and Fmoc-Arg(NO2)-Cl was unsuccessful at ambient
temperature. However, by running the reaction at a
lower temperature (ꢀ10 °C), as reported by Inouye
3. Typical procedure for Fmoc-Arg(NO2)-Cl preparation
and hydroxamate coupling
A completely dissolved solution of Fmoc-Arg(NO2)-
OH (486 mg, 1.10 mmol) in freshly distilled anhydrous
THF (3 mL) was chilled in an ice-acetone bath
HN
HN
NHNO2
HN
HN
NHNO2
HN
HN
NHNO2
5, THF
2,4,6-collidine
SOCl2, THF
-10 oC to 0 o
O
OH
N
C
0 oC to r.t., 85%
OCH3
OH
O
Fmoc
N
H
Cl
O
Fmoc
N
Fmoc
N
O
H
H
NHFmoc
4
8
HN
NHNO2
6, THF
2,4,6-collidine
HN
0 oC to r.t., 74%
OH
N
O
Fmoc
N
OCH3
H
O
N
Fmoc
10
Scheme 4. Fmoc-Arg(NO2)-Cl and hydroxamates 5 and 6 coupling reactions.

4072
J. Seo, R. B. Silverman / Tetrahedron Letters 47 (2006) 4069–4073
5. (a) Semple, J. E.; Levy, O. E.; Minami, N. K.; Owens,
T. D.; Siev, D. V. Bioorg. Med. Chem. Lett. 2000, 10,
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(ꢀ10 °C), and to this was added SOCl2 (262 mg,
2.20 mmol) dropwise. The mixture was stirred under
nitrogen for 1 h. Chilled anhydrous ether (20 mL) was
then introduced to yield a syrupy precipitate. Maintain-
ing low temperature, the solvent was removed under
reduced pressure, and the residue was triturated with
chilled anhydrous ether (20 mL). A white syrupy solid
was obtained after evaporation under reduced pressure.
Immediately after evaporation, an ice-cold solution
of 6 (275 mg, 0.55 mmol) and 2,4,6-collidine (67 mg,
0.55 mmol) in THF (5 mL) was added to the acid
chloride via a cannula. Stirring continued for 20 min
at 0 °C and 20 min more at room temperature. The
solvent was removed under reduced pressure, and the
residue was treated with EtOAc (10 mL). The organic
mixture was washed twice with 5% NaHCO3, water,
0.5 N HCl, and brine, and dried over Na2SO4. The solu-
tion was concentrated in vacuo, and the residue was
purified by flash column chromatography (EtOAc/
MeOH = 19:1, Rf = 0.45) to afford 10 (330 mg,
´
6. (a) Gomez-Vidal, J. A.; Silverman, R. B. Org. Lett. 2001,
´
3, 2481–2484; (b) Gomez-Vidal, J. A.; Forrester, M. T.;
Silverman, R. B. Org. Lett. 2001, 3, 2477–2479.
7. HBTU/HOBt/DIEA, HATU/DIEA, BOP/DIEA, PyBOP/
DIEA.
8. Examples of BOP/DIEA system for hydroxamate coup-
ling: (a) Dong, L.; Miller, M. J. J. Org. Chem. 2002, 67,
4759–4770; (b) Bergeron, R. J.; Liu, C. Z.; McManis, J. S.;
Xia, M. X. B.; Algee, S. E.; Wiegand, J. J. Med. Chem.
1994, 37, 1411–1417.
9. TLC (hexane/EtOAc, 1:1) Rf = 0.50; 1H NMR (500 MHz,
CD3OD) d 4.36 (t, J = 7.5 Hz, 1H), 4.27 (t, J = 6.5 Hz,
1H), 3.69 (s, 1H), 2.21 (q, J = 6.5 Hz, 1H), 1.94 (t,
J = 7.0 Hz, 2H), 1.77 (t, J = 7.0 Hz, 1H), 1.46 (s, 9H); MS
(APCI, CH2Cl2) [M+H+] = 301.7.
10. Reagent abbreviations used: PyBroP = bromo-tris-pyrr-
olidinophosphonium hexafluorophosphate; DIEA = N,N-
diisopropylethylamine; TFFH = fluoro-N,N,N0,N0-tetra-
methylformamidinium hexafluorophosphate; EDC = N-
(3-dimethylaminopropyl)-N0-ethylcarbodiimide; HATU =
O-(7-azabenzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium
hexafluorophosphate; HBTU = O-benzotriazole-N,N,N0,
N0-tetramethyluronium hexafluorophosphate; HOBt =
1-hydroxybenzotriazole; BOP = benzotriazole-1-yl-oxy-
tris-(dimethylamino)-phosphonium hexafluorophosphate;
PyBOP = benzotriazole-1-yl-oxy-tris-(pyrrolidino)-phos-
phonium.
1
74%) as a white foamy solid. H NMR (400 MHz, ace-
tone-d6) d 7.84 (d, J = 5.6 Hz, 4H), 7.64 (m, 4H), 7.40
(d, J = 7.2 Hz, 4H), 7.32 (d, J = 5.2 Hz, 4H), 6.73 (m,
1H), 5.26 (m, 1H), 4.88 (s, 1H), 4.51 (d, J = 5.6 Hz,
1H), 4.29 (m, 6H), 3.70 (m, 4H), 3.39 (s, 2H), 2.62
(m, 1H), 2.19 (d, J = 6.4 Hz, 1H), 2.05 (q, J = 2.5 Hz,
2H), 1.80 (m, 4H); 13C NMR (125 MHz, acetone-d6)
172.73, 170.40, 160.19, 156.60, 154.65, 144.41, 144.24,
141.45, 127.95, 127.43, 127.36, 125.54, 125.32, 120.22,
67.58, 66.71, 59.98, 58.51, 57.94, 53.43, 52.10, 51.87,
49.93, 47.32, 47.22, 40.89, 40.72, 33.88, 32.94, 20.28;
HRMS (ES) (m/z): M+H+ calcd for C42H44N7O10
806.3150, found 806.3156. Anal. Calcd for
C42H43N7O10: C, 62.60, H, 5.38, N, 12.17. Found: C,
62.40, H, 5.38, N, 11.75.
11. Coste, J.; Frerot, E.; Jouin, P. J. Org. Chem. 1994, 59,
2437–2466.
12. Carpino, L. A.; El-Faham, A. J. Am. Chem. Soc. 1995,
117, 5401–5402.
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Garman, S. M.; Hayler, J. F.; Janus, D.; Kane, P. D.;
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5, 1311–1320; (c) Inouye, K.; Sasaki, A.; Yoshida, N. Bull.
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Acknowledgment
The authors are grateful to the National Institutes of
Health (GM 49725) for financial support of this research.
14. Greene, T. W.; Wuts, P. G. M. Protective Groups in
Organic Synthesis, 3rd ed.; John Wiley & Sons: New York,
1999; p 603.
Supplementary data
15. Milburn, R. R.; Snieckus, V. Angew. Chem., Int. Ed. 2004,
43, 892–894.
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Synthetic procedures for all intermediates and HPLC
experiment data. Supplementary data associated with
this article can be found, in the online version, at
doi:10.1016/j.tetlet.2006.03.190.
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20. Compound 8: TLC (EtOAc/MeOH, 19:1) Rf = 0.45;
1H NMR (500 MHz, acetone-d6) d 7.83 (d, J = 7.5 Hz,
4H), 7.66 (m, 4H), 7.37 (d, J = 6.0 Hz, 4H), 7.29
(s, 4H), 6.76 (d, J = 8.0 Hz, 1H), 6.54 (s, 1H), 5.22 (dd,
J = 4.5 Hz, 1H), 4.94 (d, J = 4.5 Hz, 1H), 4.29 (m,
6H), 4.17 (d, J = 6.5 Hz, 1H), 3.70 (s, 3H), 3.41 (s, 2H),
3. (a) Kerwin, J. F., Jr.; Lancaster, J. R., Jr.; Feldman, P. L.
J. Med. Chem. 1995, 38, 4342–4362; (b) Erdal, E. P.;
Litzinger, E. A.; Seo, J.; Zhu, Y.; Ji, H.; Silverman, R. B.
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2000, 43, 305–341.

J. Seo, R. B. Silverman / Tetrahedron Letters 47 (2006) 4069–4073
4073
3.31 (d, J = 5.0 Hz, 1H), 3.19 (d, J = 6.0 Hz, 1H),
2.26 (d, J = 6.5 Hz, 1H), 2.09 (s, 1H), 1.83 (m, 4H);
13C NMR (125 MHz, acetone-d6) 173.84, 170.29,
160.19, 156.84, 144.53, 144.49, 144.35, 144.20, 141.44,
141.41, 127.90, 127.85, 127.30, 125.61, 125.53, 125.46,
120.15, 66.78, 66.41, 56.71, 54.39, 52.08, 51.18, 47.36,
47.31, 40.99, 37.56, 27.88, 25.11; ESI (MeOH)
[M+H+] = 794.4.