Synthesis of N-(hydroxy)amide- and N-(hydroxy)thioamide-containing peptides

Article abstract of DOI:10.1021/jo991589r

Methods developed with N-(benzoyloxy)amines and hydroxamic acids were used in the synthesis of N-(hydroxy)amide-containing pseudopeptides. Acylation of N-(benzoyloxy)phenethylamine with the acid chloride of N(α)- Fmoc-L-leucine provided a N(α)-Fmoc-N-(benzoyloxy)-L-leucinamide in 90% yield. Deprotection of the benzoyl group (using 10 vol % NH₄OH/MeOH) provided the N(α)-Fmoc-N-(hydroxy)-L-leucinamide in 87% yield. In general, the appended Fmoc group allowed for further elaboration of the N-hydroxy-N- (alkyl)amides using classic peptide-coupling methods. A practical synthetic strategy was developed, and racemization issues were addressed using diastereomeric Val-Leu derivatives. In addition, N-(hydroxy)thioamides were generated from the corresponding N-(benzoyloxy)thioamides. N- (Benzoyloxy)thioamides were obtained in moderate yields (53-76%) from the reaction of the corresponding N-(benzoyloxy)amides with Lawesson's reagent (i.e., 2,4-bis(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane-2,4-disulfide). In summary, this new technology allows for the introduction of either N- hydroxyamide or N-(hydroxy)thioamide linkages into pseudopeptide chains without racemization.

Full text of DOI:10.1021/jo991589r

1442
J . Org. Chem. 2000, 65, 1442-1447
Syn th esis of N-(Hyd r oxy)a m id e- a n d
N-(Hyd r oxy)th ioa m id e-Con ta in in g P ep tid es
Lu Wang and Otto Phanstiel IV*
Department of Chemistry, Center for the Discovery of Drugs and Diagnostics,
University of Central Florida, Orlando, Florida 32816-2366
Received October 13, 1999
Methods developed with N-(benzoyloxy)amines and hydroxamic acids were used in the synthesis
of N-(hydroxy)amide-containing pseudopeptides. Acylation of N-(benzoyloxy)phenethylamine with
the acid chloride of NR-Fmoc-L-leucine provided a NR-Fmoc-N-(benzoyloxy)-L-leucinamide in 90%
yield. Deprotection of the benzoyl group (using 10 vol % NH4OH/MeOH) provided the NR-Fmoc-
N-(hydroxy)-L-leucinamide in 87% yield. In general, the appended Fmoc group allowed for further
elaboration of the N-hydroxy-N-(alkyl)amides using classic peptide-coupling methods. A practical
synthetic strategy was developed, and racemization issues were addressed using diastereomeric
Val-Leu derivatives. In addition, N-(hydroxy)thioamides were generated from the corresponding
N-(benzoyloxy)thioamides. N-(Benzoyloxy)thioamides were obtained in moderate yields (53-76%)
from the reaction of the corresponding N-(benzoyloxy)amides with Lawesson’s reagent (i.e., 2,4-
bis(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane-2,4-disulfide). In summary, this new technology
allows for the introduction of either N-hydroxyamide or N-(hydroxy)thioamide linkages into
pseudopeptide chains without racemization.
In tr od u ction
methods, which allow facile access to the internal hy-
droxamic acids, would be of clear value to medicinal
chemists interested in using N-(hydroxy)amides as
pseudopeptides.
The replacement of the amide bond by a thioamide
bond in physiologically active peptides is another back-
bone modification introduced in the search for compounds
that are more potent than their parent structures. The
resistance afforded by the thioamide bond against enzy-
matic cleavage may enhance the potency of the pseudopep-
tide.10 During our development of the synthetic methods
to access N-hydroxyamides,11,12 we recognized that the
Structural modifications of biologically active peptides
are often performed in order to improve the properties
of these peptides for research in medicine and biochem-
istry. An important class of compounds in this field are
the backbone-modified peptides or “pseudopeptides.”
These are peptides or peptide-like molecules that contain
one or more non-peptide linkages between the R-amino
acids or R-amino acid-like motifs. The hydroxamic acid
functionality is a key structural constituent of a wide
spectrum of bioactive agents including various antibacte-
rial, antifungal, and anticancer agents.1,2 Hydroxamic
acids (R-CONHOH) are very effective metal-ion chelators
and have led to the rational design and discovery of
potent inhibitors of metalloenzymes such as thermolysin,3
angiotensin-converting enzyme (ACE),4 and the matrix
metalloprotease (MMP) family of enzymes.5 The terminal
hydroxamic acids (RCONHOH) are readily accessible by
a variety of methods and can be accessed from com-
mercially available hydroxylamine hydrochloride.6 Con-
versely, the internal hydroxamic acid motifs (R1CONR2-
OH) are often found in siderophores7,8 and apoptosis-
inducing agents such as polyoxypeptin A.9 These internal
architectures often require the synthesis of an N-O bond
either by a direct amine or amide oxidation step. New
(5) (a) Gowravaram, M. R.; Tomczuk, B. E.; J ohnson, J . S.; Delecki,
D.; Cook, E. R.; Ghose, A. K.; Mathiowetz, A. M.; Spurlino, J . C.; Rubin,
B.; Smith, D. L.; Pulvino, T.; Wahl, R. C. Inhibition of Matrix
Metalloproteinases by Hydroxamates Containing Heteroatom-Based
Modification of P-1′ Group. J . Med. Chem. 1995, 38, 2570-2581. (b)
Rockwell, A.; Melden, M.; Copeland, R. A.; Hardman, K.; Decicco, C.
P.; Degrado, W. F. Complementarity of combinatorial chemistry and
structure-based ligand design: Application to the discovery of novel
inhibitors of matrix metalloproteinases. J . Am. Chem. Soc. 1996, 118,
10337-10338. (c) Hagmann, W. K.; Lark, M. W.; Becker, J . W.
Inhibition of matrix metalloproteinases. Bristol, J . A., Ed. Ann. Rep.
Med. Chem. 1996, 31, 231-240.
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Organic Synthesis. A Review. Org. Prep. Proced. Int. 1996, 28(3), 249-
288 and references therein.
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T. A versatile synthesis of Deferrioxamine-B. J . Org. Chem. 1995, 60,
109-114.
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nochelin-A, a novel cinnamoyl hydroxamate-containing siderophore.
J . Org. Chem. 1992, 57, 7140-7143.
(1) Miller, M. Hydroxamate Approach to The Synthesis of â-Lactam
Antibiotics. Acc. Chem. Res. 1986, 19, 49-56. (b) Stearn, B.; Losee, K.
A.; Bernstein, J . Hydroxyurea. A New Type of Potential Antitumor
Agent. J . Med. Chem. 1963, 6, 201. (c) Young, C. W.; Schachetman, C.
S.; Hodas, S.; Bolis, M. C. Inhibition of DNA Synthesis by Hydroxy-
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540.
(9) Umezawa, K.; Nakazawa, K.; Ikeda, Y.; Naganawa, H.; Kondo,
S. Polyoxypeptins A and B Produced by Streptomyces: Apopotosis-
Inducing Cyclic Depsipeptides Containing the Novel Amino Acid
(2S,3R)-3-hydroxy-3-methylproline. J . Org. Chem. 1999, 64, 3034-
3038.
(2) Miller, M. J . Chem. Rev. 1989, 89, 1563-1579.
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62, 3808-3809.
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for Thermolysin. Peptides Containing Zinc Coordinating Ligands and
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of O-Benzoyl Protected Hydroxamates. J . Org. Chem. 1997, 62, 8104-
8108.
(4) Petrillo, E. W., J r.; Ondetti, M. A. Angiotensin-converting
Enzyme-Inhibitors Medicinal Chemistry and Biological Actions. Med.
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(12) Wang, Q. X.; Phanstiel, O., IV. Total Synthesis of Acinetoferrin.
J . Org. Chem. 1998, 63, 1491-1495.
10.1021/jo991589r CCC: $19.00 © 2000 American Chemical Society
Published on Web 02/02/2000

Synthesis of N-(Hydroxy)amide-Containing Peptides
J . Org. Chem., Vol. 65, No. 5, 2000 1443
Sch em e 1a
related N-hydroxythioamides (R2CdSNR1OH) could also
be generated.13 These architectures are of interest due
to the vast amount of literature illustrating the success
of related hydroxamic acid containing MMP inhibitors.14
The sulfur derivatives would be of importance for com-
parison to their oxygen analogues and may actually be
more potent. Of the three heteroatoms (nitrogen, oxygen,
and sulfur) typically used to bind zinc (the metal cofactor
in MMPs), replacement of oxygen by sulfur (in the
carbonyl position) may be important for several rea-
sons: (1) the observed stronger coordination of sulfur over
oxygen for zinc15 may enhance the binding affinity of
these substrates over their hydroxamic acid counterparts,
(2) the fact that in a related zinc metalloprotease, i.e.,
angiotensin converting enzyme (ACE), many amine ad-
ducts (including guanidine derivatives) were not as
effective as their sulfur analogues,16 (3) the fact that
sulfur is naturally involved in the “cysteine switch” [i.e.,
proforms of certain MMPs are inhibited by internal
coordination of the active site zinc atom by a cysteine
side chain (e.g., R-SH) in the prosegment, which is
cleaved upon activation].17
In 1997, we published an improved biphasic method,
which allows access to hydroxamic acids in high yield and
purity.11 In particular, this method selectively oxidizes
a primary amine to a N-(benzoyloxy)amine.18 This paper
illustrates the subsequent acylation of a N-(benzoyloxy)-
amine with an N-Fmoc-amino acid chloride to introduce
the hydroxamic acid functional group into peptides. This
synthetic methodology provides R-amino hydroxamic acid
containing peptides in high yield without racemization.
The retention of chirality throughout this synthetic
method was confirmed with a pair of Val-Leu diastereo-
mers. This work also provided several R amino thiohy-
droxamic acid analogues by conversion of the N-(benzoyl-
oxy)amide into the corresponding N-(benzoyloxy)thioamide
using Lawesson’s reagent.19,20 Future work will compare
a
Reagents: (a) SOCl2 in CH2Cl2 reflux, 2 h; (b) BPO (2 equiv)/
CH2Cl2, pH )10.5 aqueous sodium carbonate buffer, rt.
the efficacy of the sulfur-containing ligands with their
oxygen counterparts.
Resu lts a n d Discu ssion
The mild conditions (room temperature and pH 10.5)
associated with the previously described biphasic method
make it ideally suited for converting a primary amine to
the corresponding N-(benzoyloxy)amine.11,18 Initial efforts
to apply this biphasic method to the direct oxidation of
R-amino acids were unsuccessful. For example, attempts
to generate N-(benzoyloxy)-L-phenylalanine ethyl ester
via the oxidation of L-Phe ethyl ester gave a 0% yield of
the desired product. An alternative strategy envisioned
acylation of the N-benzoyloxyamine with an N-protected
amino acid. Unfortunately, the use of the classic peptide
coupling reagents such as DCC,21 or the BOP21 reagent,
gave low yields of the N-(benzoyloxy)amides.22 Carpino
has pointed out that the Fmoc amino-protecting group
(e.g., the fluorenylmethoxycarbonyl group) is stable
enough to survive conversion from the Fmoc-protected
amino acid into an Fmoc-protected acid chloride form.23
Under appropriate conditions, the amine coupling occurs
without loss of chirality adjacent to the carboxylic acid
site.23 As we had previously demonstrated the facile
acylation of N-benzoyloxyamines with acid chlorides,11
Carpino’s Fmoc-protected amino acid chlorides offered a
promising alternative. As shown in Scheme 1, acylation
of N-(benzoyloxy)phenethylamine18 with either the L- or
D-Fmoc-protected leucine acid chloride23 afforded the
respective hydroxamate 1 (90%) or 2 (85%). This suc-
(13) Walter, W.; Schaumann, E. The Chemistry of Thiohydroxamic
Acids. Synthesis 1971, (3), 111-130.
(14) (a) Brown, F. K. Brown, P. J .; Bickett, P. M.; Chambers, C. L.;
Davies, H. G.; Deaton, D. N.; Drewry, D.; Foley, M.; McElroy, A. B.;
Gregson, M.; McGeehan, G. M.; Myers, D. L.; Norton, D.; Salovich, J .
M.; Schoenen, F. J .; Ward, P. Matrix Metalloproteinase inhibitors
containing a (carboxyalkyl) amino zinc ligand modification of the P1
and P2′ residues. J . Med. Chem. 1994, 37, 674-688 and references
therein. (b) Odake, S.; Okayama, T.; Obata, M.; Morikawa, T.; Hattori,
S.; Hori, H.; Nagai, Y. Vertebrate collagenase inhibitor. 1. Tripeptidyl
hydroxamic acids. Chem. Pharm. Bull. 1990, 38, 1007-1011 and
references therein. (c) Toba, S., Damodaran, K. V., Merz, K. M., J r.
Binding Preferences of Hydroxamate Inhibitors of the Matrix Metal-
loproteinase Human Fibroblast Collagenase. J . Med. Chem. 1999, 42,
1225-1234. (d) Yamamoto, M., Tsujishita, H., Hori, N., Ohishi, Y.,
Inoue, S., Ikeda, S., Okada, Y., Inhibition of Membrane-Type 1 Matrix
Metalloproteinase by Hydroxamate Inhibitors: An Examination of the
Subsite Pocket. J . Med. Chem. 1998, 41, 1209-1217.
(15) Cushman, D. W.; Ondetti, M. A. Inhibitors of Angiotensin-
converting Enzyme. Prog. Med. Chem. 1980, 42-93.
(16) Redshaw, S. In Medicinal Chemistry: The Role of Organic
Chemistry in Drug Research; Ganellin, C. R., Roberts, S. M., Eds.;
Academic Press: London, 1993; pp 170-172.
(17) Springman, E. B.; Angleton, E. L.; Birkedalhansen, H.; Van-
wart, H. E. Multiple-Modes of activation of latent human Fibroblast
collagenase-Evidence for the role of a Cys-73 active-site zinc complex
in latency and a cysteine switch mechanism for activation. Proc. Natl.
Acad. Sci. U.S.A. 1990, 87, 356-368.
(18) This procedure was later improved in: Phanstiel, O., IV; Wang,
Q. X.; Powell, D. H.; Ospina, M. P.; Leeson, B. A. Synthesis of
Secondary Amines via N-(Benzoyloxy)amines and Organoboranes. J .
Org. Chem. 1999, 64, 803-806.
(21) Bodanszky, M. Principles of Peptide Synthesis 1994, 118, 124,
155.
(22) For the corresponding condensation of the related N-(methyl)-
O-benzylhydroxylamine with N-Boc alanine and DCC, see ref 6, p 266.
(23) Carpino, L. A.; Cohen, B. J .; Stephens, K. E., J r.; Sadat-Aalaee,
S. Y.; Tien, J .; Langridge, D. C. ((9-Fluorenylmethyl) oxy) carbonyl
(Fmoc) Amino Acid Chlorides. Synthesis, Characterization, and Ap-
plication to the Rapid Synthesis of Short Peptide Segments. J . Org.
Chem. 1986, 51, 3732-3734.
(19) J ensen, D. E.; Lawesson, S. O. Studies on Amino Acids and
Peptides VIII. Tetrahedron 1985, 41, 5595-5606.
(20) Clausen, K.; Spatola, A. F.; Lenieux, C.; Schiller, P. W.;
Lawesson, S. O. Evidence of a Peptide Backbone Contribution Toward
Selective Receptor Recognition for Leucine Enkephalin Thioamide
Analogue. Biochem. Biophys. Res. Commun. 1984, 120, 305-310.

1444 J . Org. Chem., Vol. 65, No. 5, 2000
Wang and Phanstiel
Sch em e 2
Sch em e 3a
a
Reagents: (a) 10% NH4OH/MeOH; (b) 10% DEA, DMF; (c)
hydrocinnamoyl chloride; (d) acetyl chloride.
cessful strategy was then used to generate several
hydroxamic acids and N-hydroxythioamide-containing
peptides. Significantly, this modular synthetic approach
allows access to a diverse number of structures from a
common N-(benzoyloxy)amine intermediate.
Having incorporated the protected N-hydroxy-N-alkyl-
hydroxamic acid motif within an amino acid scaffold (see
1 in Scheme 2), we wanted to further elaborate the
peptide chain via the R-amine. Upon addition of a 10%
(by volume) solution of diethylamine (DEA) in DMF to
compound 1, the NR-Fmoc group was removed. The newly
generated amine rapidly deprotected the benzoyl group
from the adjacent hydroxamate to give the corresponding
benzamide hydroxamic acid 3 in 90% yield through a
putative intramolecular transformation (Scheme 2).8
To generate the free amine without rearrangement, the
benzoyl group was removed prior to the Fmoc deprotec-
tion step. The benzoyl groups of 1 and 2 were removed
with a solution of 10% (by volume) concentrated NH4-
OH in MeOH at -23 °C to give the “free” hydroxamic
acids 4 and 5 in 87% and 90% yield, respectively (Scheme
3). Subsequent deprotection of the Fmoc group with 10%
DEA in DMF and acylation with either hydrocinnamic
acid chloride or acetyl chloride gave the hydroxamic acids
6 and 7 (each in 75% yield).
F igu r e 1. (A) Ko’s reagent. (B) Lawesson’s reagent.
Sch em e 4
agent (B in Figure 1) has been shown to convert amides
to thioamides and does not usually react with esters.19,20
Since the N-(benzoyloxy)amide system (e.g., RCONRO-
COPh) contained both functional groups, it seemed
reasonable that the reagent might selectively convert the
amide portion to its thioamide derivative, while leaving
the benzoyloxy group intact. Reaction of model compound
C11 with Lawesson’s reagent gave the desired thioamide
This method was also extended to the synthesis of
N-hydroxythioamide-containing peptides.24 A recent pa-
per by Ko et al.10 illustrated the use of thioacylating
agents in synthesizing thiopeptides. Even though Ko’s
reagent (A in Figure 1) was shown to selectively convert
amines to thioamides, it was not successful in thioacy-
lating N-(benzoyloxy)phenethylamine. Lawesson’s re-
1
8 (76%) (Scheme 4). H NMR chemical shift and mass
spectrum (see the Experimental Section) differences
allowed for the confirmation of sulfur incorporation into
C [δ CH3: 8 (2.5 ppm) vs C (2.0 ppm)]. Indeed, reaction
of 1 with Lawesson’s reagent gave the N-(benzoyloxy)-
thioamide derivative 9 in 53% yield (Scheme 5). The mass
spectrum of 9 further corroborated the incorporation of
sulfur into 1.
(24) Shalaby, M. A.; Grote, C. W.; Rapoport, H. Thiopeptide Syn-
thesis. Amino Thionoacid Derivatives of Nitrobenzotriazole as Thio-
acylating Agents. J . Org. Chem. 1996, 61, 9045-9048 and references
therein.

Synthesis of N-(Hydroxy)amide-Containing Peptides
J . Org. Chem., Vol. 65, No. 5, 2000 1445
Sch em e 5a
Sch em e 6a
a
Reagents: (a) Lawesson’s reagent, THF, rt; (b) 10% DEA,
a
Reagents: (a) 10% NH4OH/MeOH; (b) 10% DEA, DMF, 4 h;
DMF, 4 h; (c) 10% NH4OH/MeOH.
(c) BOC-L-valine.
Treatment of 9 with 10% NH4OH/CH3OH afforded the
rearranged benzamide adduct 10, albeit in 39% yield.
Therefore, both the sulfur analogue 9 and the oxygen
analogue 1 were observed to undergo the intramolecular
rearrangement to the benzamide adducts 10 and 3,
respectively. The benzoyl group was removed from 9
using the same procedure described previously with 1 to
give the Fmoc-protected N-(hydroxy)thioamide 11 in 54%
yield (Scheme 5). Although the reactions that involve
sulfur afforded moderate yields, the N-(hydroxy)thio-
amide-containing peptides were still accessible.
Since most biologically active compounds are chiral,
racemization is a very important issue in peptide or
“pseudopeptide” synthesis. To monitor the chiral integrity
throughout the synthesis, a pair of diastereomers (L-L
and L-D of Val-Leu-hydroxamic acid derivatives) were
synthesized (Scheme 6). The respective Fmoc groups in
compounds 4 and 5 were removed in the presence of 10%
DEA in DMF. Addition of BOC-L-valine and dicyclohexyl-
carbodiimide (DCC) to either the free amine of 4 or 5 gave
the respective diastereomer 12 (L-L) or 13 (L-D) in 45%
and 63% overall yield, respectively. The free amine of 4
was also coupled to Boc-L-Valine using the BOP21 reagent
to provide 14 (L-L) isomer in 70% yield. It should be
noted that BOP method gave higher yields and less
byproduct formation by TLC.
dressed by 13C NMR experiments in CDCl3. The L-L and
L-D Val-Leu diastereomers gave distinct CH3 signals at
δ 17.2 and 16.6 ppm, respectively.26 As a control, a
“spiked” sample containing both diastereomers also gave
the two signals at 17.2 and 16.6 ppm. Inspection of the
13C NMR spectrum of 14 (L-L, δ 17.2 ppm) gave no
detectable signal for the other diastereomer (L-D) at δ
16.6 ppm. These results suggest that little (<3%) if any
racemization occurred during the coupling step involving
either the leucine amine or acid functional groups.23
In summary, enantiomeric amino acids containing the
N-hydroxyamide motif can be introduced into peptides
by coupling N-(benzoyloxy)amines with Fmoc-protected
amino acid derivatives. This technology provides promis-
ing reagents to be used in the generation of natural
products such as polyoxypeptin A9 or combinatorial
libraries which contain internal N-hydroxyamide sub-
units. These pseudopeptides, which contain metal bind-
ing ligands within their architecture, may have unique
biological properties. Inhibition studies evaluating these
ligands (4, 5, 13, and 14) with a variety of metalloproteins
are in progress.
Exp er im en ta l Section
Gen er a l Meth od s. Nuclei were observed at 200 and 50
MHz for 1H NMR and 13C NMR, respectively. The only
After isolation the optical rotation of each compound
(4, 5, 12, 13, and 14) was measured.25 The overall
racemization at the leucine R carbon center was ad-
1
exceptions were the H and 13C comparison experiments (see
below).
(25) Under the same conditions (c ) 1, CHCl3), the BOP-generated
(26) Other 13C spectral differences were clearly apparent such as
the BOC carbonyl resonances at δ 154.35 and 154.40 for 13 and 14,
respectively. The 1H NMR (CDCl3) for 14 gave a deceptively simple
doublet centered at 0.82 ppm, whereas derivative 13 gave a more
complex pattern in this range (see the Supporting Information).
peptide 14 gave a higher absolute value for its optical rotation (R25
)
D
-34°) than the corresponding DCC derived 12 (R25 ) -22°). This
D
observation is consistent with partial racemization during the DCC-
mediated coupling step.

1446 J . Org. Chem., Vol. 65, No. 5, 2000
Wang and Phanstiel
Com p a r ison of P ep tid es of 13 a n d 14 by 13C NMR. The
13C NMR comparison studies of 13 and 14 were performed at
75.4 MHz in CDCl3. The concentrations of 13 and 14 were 24.5
and 23.2 mg/ mL, respectively. A spectrum was obtained on
each sample, and then the samples were combined in an
approximately 2:3 volume ratio and the “spiked” sample
spectrum was obtained. Note: Although the chemical shifts
are slightly off (∼1 ppm) from those recorded at 50 MHz, the
trends are identical. The 1H NMR (300 MHz) and 13C APT
(attached proton test) experimental results with 13 and 14 are
available as Supporting Information.
N-(2-P h en ylet h yl)-N-(b en zoyloxy)-Nr-F m oc-L-leu cin -
a m id e (1). A solution of benzoyl peroxide (2.35 g, 9.8 mmol)
in CH2Cl2 was added to a vigorously stirred solution of
phenethyamine (1.20 g, 9.9 mmol) in 25 mL of a carbonate
buffer solution (pH ) 10.5). The reaction was stirred under
argon at room temperature overnight. A new spot was ob-
served on TLC (Rf ) 0.47, 20% EtOAc/hexane).
(broad m, 5H), 2.90 (t, 2H), 1.95 (s, 1H), 1.60 (broad m, 2H),
0.90 (m, 6H); 13C NMR δ 171.6, 157.5, 143.6, 141.6, 138.2,
129.0, 128.8, 128.5, 127.8, 127.0, 126.2, 125.0, 120.0, 67.8, 49.0,
48.5, 47.0, 40.0, 32.5, 24.5, 23.0, 22.0; [R]25 -14.9 (c ) 1,
D
CHCl3). Anal. Calcd for C29H32N2O4: C, 73.70; H, 6.82; N, 5.93.
Found: C, 72.50; H, 6.72; N, 5.83.
N -(2-P h e n yle t h yl)-N -(h yd r oxy)-N r-F m oc-D -le u cin -
a m id e (5). 2 (1.2 g, 2.1 mmol) was dissolved in 10% NH4OH/
MeOH (2.4 mL). The hydroxamic acid 5 (1.08 g, 90%) was
isolated using the same procedure used with 4. Anal. Calcd
for C29H32N2O4‚0.3H2O: C, 72.87; H, 6.87; N, 5.87. Found: C,
1
72.62; H, 6.87; N, 6.27. 29: Rf ) 0.19, 20% EtOAc/hexane; H
NMR (CDCl3) δ 8.90 (s, 1H), 7.70 (d, 2H), 7.50(d, 2H), 7.20
(m, 9H), 5.65 (d, 1H), 4.90 (q, 1H), 4.10 (broad m, 5H), 2.90 (t,
2H), 1.95 (s, 1H), 1.60 (broad m, 2H), 0.90 (m, 6H); 13C NMR
δ 171.6, 157.5, 143.6, 141.6, 138.2, 129.0, 128.8, 128.5, 127.8,
127.0, 126.2, 125.0, 120.0, 67.8, 49.0, 48.5, 47.0, 40.0, 32.5, 24.5,
23.0, 22.0; [R]25 +12.2 (c ) 1, CHCl3).
D
Fmoc-L-leucine (3.9 g, 11 mmol) was dissolved in CH2Cl2
(80 mL) followed by addition of SOCl2 (13.06 g, 110 mmol).
The reaction mixture was refluxed for 2 h under argon. The
solvent and excess of SOCl2 were removed under vacuum. The
remaining residue was added to the biphasic reaction mixture
above at room temperature under argon. The reaction was
monitored by TLC (Rf ) 0.47, 20% EtOAc/hexane) for the
disappearance of starting material. After the coupling was
completed, the organic phase was separated, dried over
anhydrous Na2SO4, filtered, and concentrated. The crude
residue was subjected to flash chromatography and eluted with
20% EtOAc/hexane to furnish the desired O-benzoyl hydrox-
N-(2-P h en yleth yl)-N-(h yd r oxy)-Nr-h yd r ocin n a m oyl-L-
leu cin a m id e (6). 4 (0.20 g, 0.35 mmol) was dissolved in DMF
(7 mL) and DEA (0.78 mL) and stirred at room temperature
until all of the starting material (Rf ) 0.19, 20% EtOAc/
hexane) was consumed. The solvent was removed under
vacuum, and the residue was redissolved in CH2Cl2 (10 mL)
followed by the addition of an aqueous Na2CO3 solution (10
mL).
In a separate step, hydrocinnamic acid (0.066 g, 0.44 mmol)
and SOCl2 (1.00 mL) were mixed and refluxed for 2 h under
an inert atmosphere. The excess SOCl2 was removed under
vacuum. The residue was added to the biphasic solution
mentioned above and stirred overnight under argon at room
temperature. The organic phase was separated, dried over
anhydrous Na2SO4, filtered, concentrated, and purified by flash
chromatography eluting with 40% EtOAc/hexane to give amide
1
amate 1 (5.08 g, 90%). 1: Rf ) 0.30 in 20% EtOAc/hexane; H
NMR (CDCl3) δ 8.00 (d, 2H), 7.30 (broad m, 16H), 5.40 (d, 1H),
4.60 (m, 1H), 4.30 (d, 1H), 4.15 (m, 2H), 3.00 (t, 2H), 1.50 (broad
m, 3H), 0.65 (broad m, 6H); 13C NMR δ 173.0, 164.0, 156.0,
144.0, 142.0, 141.0, 138.0, 134.2, 130.0, 128.4, 128.2, 127.5,
127.2, 127.0, 125.0, 120.0, 67.0, 50.0, 47.0, 42.0, 33.0, 24.0, 23.0,
1
6 (0.10 g, 75%). 6: Rf ) 0.30 in 40% EtOAc/hexane; H NMR
(CDCl3) δ 10.10 (broad s, 1H), 7.15 (broad m, 10H), 5.05 (m,
1H), 4.00 (m, 1H), 3.70 (m, 1H), 2.80 (m, 4H), 2.45 (m, 2H),
1.60 (m, 1H), 1.40 (m, 2H), 0.85 (m, 6H); 13C NMR δ 174.0,
171.5, 140.6, 138.8, 129.2, 129.0, 128.9, 128.8, 126.5, 49.8, 48.0,
40.0, 38.0, 33.5, 32.0, 25.5, 23.5, 22.5. Anal. Calcd for
21.0; [R]25 +23.2 (c ) 1, CHCl3). Anal. Calcd for C36H36N2O5:
D
C, 74.98; H, 6.29; N, 4.86. Found: C, 74.70; H, 6.31; N, 4.84.
N-(2-P h en ylet h yl)-N-(b en zoyloxy)-Nr-F m oc-D-leu cin -
a m id e (2). Benzoyl peroxide (0.6 g, 2.5 mmol), phenethylamine
(0.3 g, 2.5 mmol), Fmoc-D-leucine (1.0 g, 2.8 mmol), and SOCl2
(3.32 g, 28 mmol) in CH2Cl2 were reacted using the same
procedure described for 1. Column chromatography (20%
EtOAc/hexane) furnished 1.2 g (85%) of 2. 2: Rf ) 0.30 in 20%
EtOAc/hexane; 1H NMR (CDCl3) δ 8.00 (d, 2H), 7.30 (broad
m, 16H), 5.40 (d, 1H), 4.60 (m, 1H), 4.30 (d, 1H), 4.15 (m, 2H),
3.00 (t, 2H), 1.50 (broad m, 3H), 0.65 (broad m, 6H); 13C NMR
δ 173.0, 164.0, 156.0, 144.0, 142.0, 141.0, 138.0, 134.2, 130.0,
128.4, 128.2, 127.5, 127.2, 127.0, 125.0, 120.0, 67.0, 50.0, 47.0,
C
23H30N2O3: C, 72.22; H, 7.90; N, 7.32. Found: C, 72.30; H,
7.91; N, 7.42.
N -(2-P h e n yle t h yl)-N -(h yd r oxy)-N r-a ce t yl-L -le u cin -
a m id e (7). 4 (0.15 g, 0.26 mmol) was dissolved in DMF (5 mL)
and DEA (0.58 mL) and stirred at room remperature until the
starting material (Rf ) 0.19, 20% EtOAc/hexane) was con-
sumed. The solvent was removed under vacuum, and the
residue was redissolved in CH2Cl2 (10 mL) followed by the
addition of an aqueous Na2CO3 solution (10 mL) and acetyl
chloride (0.045 mL). The reaction mixture was stirred over-
night under argon at room temperature. The organic phase
was separated, dried over anhydrous Na2SO4, filtered, con-
centrated, and purified by flash chromatography eluting with
70% EtOAc/hexane to give the acetamide 7 (0.10 g, 75%). 7:
42.0, 33.0, 24.0, 23.0, 21.0; [R]25 -22.9 (c ) 1, CHCl3).
D
N-(2-P h en ylet h yl)-N-(h yd r oxy)-Nr-b en zoyl-L-leu cin -
a m id e (3). Compound 1 (0.21 g, 0.36 mmol) was dissolved in
DMF (3.30 mL) followed by the addition of DEA (0.38 mL).
The reaction mixture was stirred for 4 h at room temperature
under an inert atmosphere. The reaction was monitored by
TLC (Rf ) 0.30, 20% EtOAc/hexane). The mixture was
concentrated under vacuum and purified by flash chromatog-
raphy eluting with 40% EtOAc/hexane to give benzamide 3
1
Rf ) 0.20 in 70% EtOAc/hexane; H NMR (CDCl3) δ 9.90 (s,
1H), 7.20 (m, 5H), 6.35 (d, 1H), 4.95 (q, 1H), 3.90 (m, 2H), 2.95
(t, 2H), 2.00 (s, 3H), 1.50 (broad m, 3H), 0.90 (m, 6H); 13C NMR
δ 172.0, 170.0, 138.5, 129.0, 128.5, 126.2, 48.5, 47.5, 39.5, 33.0,
25.0, 23.0, 22.5. Anal. Calcd for C16H24N2O3‚ 0.4 H2O: C, 64.15;
H, 8.34; N, 9.35. Found: C, 64.54; H, 8.30; N, 8.95.
1
(0.13 g, 90%). 3: Rf ) 0.30, in 40% EtOAc/hexane; H NMR
(CDCl3) δ 10.10 (s, 1H), 7.70 (d, 2H), 7.25 (m, 8H), 5.30 (m,
1H), 4.00 (m, 1H), 3.65 (m, 1H), 2.80 (t, 2H), 1.70 (broad m,
3H), 0.95 (m, 6H); 13C NMR δ 170.5, 168.5, 138.2, 133.0, 132.0,
129.0, 128.5, 128.0, 127.0, 126.0, 49.0, 48.0, 40.0, 32.5, 25.0,
N-(2-P h en yleth yl)-N-(ben zoyloxy)th ioa ceta m id e (8).
Compound C11 (0.10 g, 0.35 mmol) was dissolved in anhydrous
THF (1 mL) followed by the addition of 2,4-bis(4-methoxyphen-
yl)-1,3-dithia-2,4-diphosphetane-2,4-disulfide (Lawesson’s re-
agent, 0.072 g, 0.18 mmol) portionwise. The reaction mixture
was covered with aluminum foil and stirred for 4 h at room
temperature until the starting material (Rf ) 0.26, 20% EtOAc/
hexane) was consumed. The solvent was removed under
vacuum, and the residue was purified by flash chromatography
eluting with 20% EtOAc/hexane to give the desired thioacet-
.
23.0, 22.2. Anal. Calcd for C21H26N2O3 0.5 H2O: C, 69.40; H,
7.49; N, 7.71. Found: C, 69.37; H, 7.32; N, 7.48.
N -(2-P h e n yle t h y l)-N -(h y d r o xy )-N r-F m o c -L -le u c in -
a m id e (4). Compound 1 (1.50 g, 2.6 mmol) was dissolved in
10% NH4OH/MeOH (3 mL) and stirred for 30 min under argon.
The disappearance of starting material was monitored by TLC
(Rf ) 0.30, 20% EtOAc/hexane). After the reaction was
completed, the solvent was removed under vacuum. The crude
was purified by flash chromatography eluting with 20% EtOAc/
hexane to give hydroxamic acid 4 (1.30 g, 87%). 4: Rf ) 0.19,
1
amide 8 (0.08 g, 76%). 8: Rf ) 0.4 in 20% EtOAc/hexane; H
NMR (CDCl3) 7.92 (dd, 2H), 7.70 (t, 1H), 7.50 (t, 2H), 7.25 (m,
5H), 4.60 (t, 2H), 3.20 (t, 2H), 2.50 (s, 3H); high-resolution mass
spectrum (FAB) theory for (C17H17N1O2S) M + 1 ) 300.1058,
found M + 1 ) 300.1058.
1
20% EtOAc/hexane; H NMR (CDCl3) δ 8.90 (s, 1H), 7.70 (d,
2H), 7.50(d, 2H), 7.20 (m, 9H), 5.65 (d, 1H), 4.90 (q, 1H), 4.10

Synthesis of N-(Hydroxy)amide-Containing Peptides
J . Org. Chem., Vol. 65, No. 5, 2000 1447
N-(2-P h en ylet h yl)-N-(b en zoyloxy)-Nr-F m oc-L-leu cin -
(th io)a m id e (9). 1 (4.52 g, 7.8 mmol) was dissolved in
anhydrous THF (20 mL) followed by the addition of 2,4-bis-
(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane-2,4-disul-
fide (Lawesson’s reagent, 1.59 g, 3.9 mmol) portionwise. The
reaction mixture was covered with aluminum foil and stirred
for 72 h at room temperature. The solvent was removed under
vacuum, and the residue was purified by flash chromatography
eluting with 15% EtOAc/hexane to give the desired N-benzoyl-
oxythioamide 9 (0.9 g, 53% taking into account recovered
starting material). 9: Rf ) 0.22 in 15% EtOAc/hexane; 1H NMR
(CDCl3) δ 7.95 (d, 2H), 7.75 (d, 2H), 7.60 (t, 2H), 7.30 (broad
m, 12H), 5.75 (d, 1H), 4.90 (broad m, 1H), 4.60 (m, 1H), 4.30
(m, 4H), 3.15 (m, 2H), 1.60 (broad m, 1H), 0.75 (dd, 6H); 13C
NMR δ 202.0, 163.0, 156.0, 144.0, 143.8, 141.2, 137.8, 135.0,
130.0, 128.0, 127.9, 127.8, 127.5, 127.0, 126.8, 125.2, 125.0,
120.0, 67.0, 56.0, 54.0, 47.0, 45.5, 32.0, 24.5, 23.5, 21.5. Anal.
derivative 12 (0.11 g, 45%). 12: Rf ) 0.30 in 30% EtOAc/
hexane; 1H NMR (CDCl3) δ 9.80 (s, 1H), 7.25 (m, 5H), 5.40 (d,
1H), 5.00 (q, 2H), 3.90 (m, 3H), 2.90 (m, 2H), 2.00 (m, 1H),
1.60 (m, 3H), 1.40 (s, 9H), 0.90 (m, 12H); 13C NMR δ 173.8,
170.0, 156.0, 138.2, 129.1, 129.0, 128.5, 126.5, 80.0, 59.6, 49.2,
47.8, 39.1, 33.9, 32.8, 31.1, 24.9, 24.8, 22.9, 22.7, 22.1, 19.1,
18.3; [R]25 -22.4 (c ) 1, CHCl3). Anal. Calcd for C24H40N3O5‚
D
0.5 H2O: C, 62.72; H, 8.99; N, 9.14. Found: C, 62.88; H, 8.88;
N, 9.06.
Nr-Am id o[Nr-BOC-L-va lin yl]-N-(2-p h en yleth yl)-N-(h y-
d r oxy)-D-leu cin a m id e (13). Fmoc-protected D-hydroxamic
acid 5 (0.50 g, 1.0 mmol) was dissolved in a solution containing
DMF (14.4 mL) and DEA (1.6 mL) and stirred until the
starting material (Rf ) 0.19, 20% EtOAc/hexane) was con-
sumed. The solvent was removed under vacuum, and the
residue was redissolved in CH2Cl2 (10 mL) followed by the
addition of DCC (0.26 g, 1.30 mmol) and BOC-L-valine (0.28
g, 1.30 mmol). The reaction mixture was stirred overnight
under argon at room temperature. After filtration, the filtrate
was extracted and separated, dried over anhydrous Na2SO4,
filtered, and concentrated. The residue was purified by flash
chromatography eluting with 30% EtOAc/hexane to give the
L,D derivative 13 (0.30 g, 63%). 13: Rf ) 0.30 in 30% EtOAc/
hexane; 1H NMR (CDCl3) δ 9.80 (s, 1H), 7.25 (m, 5H), 5.40 (d,
1H), 5.00 (q, 2H), 3.90 (m, 3H), 2.90 (m, 2H), 2.00 (m, 1H),
1.60 (m, 3H), 1.40 (s, 9H), 0.90 (m, 12H); 13C NMR (50 MHz)
δ 173.5, 170.6, 155.9, 138.6, 129.3, 129.0, 128.9, 128.7, 128.6,
126.5, 80.3, 60.0, 49.4, 49.3, 47.7, 41.0, 40.8, 39.5, 35.8, 34.0,
Calcd for
Found: C, 72.57; H, 6.30; N, 4.69.
C36H36N2O3S‚H2O: C, 72.69; H, 6.44; N, 4.71.
N-(2-P h en ylet h yl)-N-(h yd r oxy)-Nr-b en zoyl-L-leu cin -
(th io)a m id e (10). 9 (0.13 g, 0.22 mmol) was dissolved in DEA
(0.23 mL) and DMF (2.0 mL) and stirred for 4 h at room
temperature. The reaction vessel was covered with aluminum
foil. The disappearance of the starting material was monitored
by TLC (Rf ) 0.22 in 15% EtOAc/hexane). Then the solvent
was removed under vacuum at room temperature, and the
residue was purified by flash chromatography eluting with
15% EtOAc/hexane to give the benzamide 10 (0.032 g, 39%).
1
10: Rf ) 0.44 in 30% EtOAc/hexane; H NMR (CDCl3) δ 8.01
32.9, 31.2, 25.1, 25.0, 24.9, 23.2, 22.9, 22.2, 19.4, 17.8; [R]25
D
(broad s, 1H), 7.80 (m, 2H), 7.35 (m, 8H), 4.70 (q, 1H), 4.00
(m, 2H), 3.00 (m, 2H), 1.60 (m, 3H), 0.90 (m, 6H); high-
resolution mass spectrum (FAB) theory for (C21H26N2O2S) M
+ 1 ) 371.1793, found M + 1 ) 371.1783.
-0.6 (c ) 1, CHCl3). Anal. Calcd for C24H40N3O5‚0.5H2O: C,
62.72; H, 8.99; N, 9.14. Found: C, 62.88; H, 8.88; N, 9.06.
Nr-Am id o[Nr-BOC-L-va lin yl]-N-(2-p h en yleth yl)-N-(h y-
d r oxy)-L-leu cin a m id e (14). 4 (0.17 g, 0.36 mmol) was dis-
solved in a solution containing EtOAc (3.6 mL) and DEA (0.4
mL) and stirred until the starting material (Rf ) 0.19, 20%
EtOAc/hexane) was consumed. The solvent was removed under
vacuum, and the residue was redissolved in acetonitrile (5.4
mL). BOC-L-valine (0.09 g, 0.41 mmol) and benzotriazol-1-
yloxytris(dimethylamino)phosphonium hexafluorophosphate
(BOP reagent, 0.16 g, 0.36 mmol) were added, followed by the
addition of TEA (0.05 mL). Stirring was continued under argon
at room temperature overnight. A saturated sodium chloride
solution (brine) was added, and the product was extracted with
ethyl acetate. The organic phase was separated, dried over
anhydrous Na2SO4, filtered, and concentrated. The residue was
purified by flash chromatography eluting with 30% EtOAc/
hexane to give the L,L derivative 14 (0.11 g, 70%). 14: Rf )
N-(2-P h en yleth yl)-N-(h ydr oxyl)-Nr-Fm oc-L-leu cin (th io)-
a m id e (11). 9 (0.40 g, 0.67 mmol) was dissolved in 10% NH4-
OH/MeOH (1 mL) and stirred for 30 min under argon. The
reaction vessel was covered with aluminum foil. The disap-
pearance of starting material was monitored by TLC (Rf )
0.22, 15% EtOAc/hexane). After the reaction was complete, the
solvent was removed under vacuum. The residue was purified
by flash chromatography eluting with 20% EtOAc/hexane to
give the unstable N-hydroxythioamide 11 (0.18 g, 55%). 11:
1
Rf ) 0.22 and 0.31, 20% EtOAc/hexane. The mixture 11: H
NMR (CDCl3) δ 10.75 (s, 1H), 7.78 (d, 2H), 7.58 (d, 2H), 7.15
(m, 9H), 5.38 (d, 1H), 4.70 (m, 1H), 4.40 (m, 2H), 4.20 (m, 2H),
3.20 (m, 2H), 1.50 (m, 3H), 0.90 (m, 6H); 13C NMR δ 204.5,
176.0, 144.0, 140.5, 138.0, 128.5, 127.8, 127.0, 126.8, 125.0,
120.0, 67.0, 60.0, 47.0, 46.5, 45.0, 34.0, 25.0, 23.0, 22.5; high-
resolution mass spectrum (FAB) theory for (C29H32N2O2S) M
+ 1 ) 473.2263, found M + 1 ) 473.2248. Anal. Calcd for
1
0.30 in 30% EtOAc/hexane; H NMR (CDCl3) δ 9.80 (s, 1H),
7.25 (m, 5H), 5.25 (d, 1H), 5.00 (q, 2H), 3.90 (m, 3H), 2.90 (m,
2H), 2.00 (m, 1H), 1.60 (m, 3H), 1.40 (s, 9H), 0.90 (m, 2H); 13
C
C
29H32N2O3S‚0.3H2O: C, 70.50; H, 6.65; N, 5.67. Found: C,
NMR δ 173.8, 170.0, 156.0, 138.2, 129.1, 129.0, 128.5, 126.5,
70.65; H, 6.74; N, 5.61. Note: the TLC spot with Rf ) 0.31
rapidly equilibrated to form a mixture of two spots (Rf ) 0.22
and 0.31). When the spot with Rf ) 0.31 was isolated from
the TLC plate and re-eluted up a new plate two spots were
again observed (Rf ) 0.22 and 0.31, respectively).
80.0, 60.0, 49.1, 47.5, 39.2, 32.8, 31.0, 28.3, 24.8, 22.6, 22.2,
19.1, 18.0; [R]25 -33.6 (c ) 1, CHCl3). Anal. Calcd for
D
C
24H40N3O5: C, 63.97; H, 8.95; N, 9.33. Found: C, 64.27; H,
8.93; N, 9.30.
Ack n ow led gm en t. We thank Dr. David Powell in
the Department of Chemistry at the University of
Florida for providing mass spectral services. The au-
thors also thank Mr. J im Rocca at the Advanced
Magnetic Resonance Imaging and Spectroscopy Center
for Structural Biology at the University of Florida for
acquiring the NMR spectra of peptides 13 and 14.
Nr-Am id o[Nr-BOC-L-va lin yl]-N-(2-p h en yleth yl)-N-(h y-
d r oxy)-L-leu cin a m id e (12). 4 (0.26 g, 0.55 mmol) was dis-
solved in a solution containing DMF (5.4 mL) and diethylamine
(DEA, 0.6 mL) and stirred at room temperature until the
starting material (Rf ) 0.19, 20% EtOAc/hexane) was con-
sumed. The solvent was removed under vacuum, and the
residue was redissolved in CH2Cl2 (5 mL) followed by the
addition of dicyclohexylcarbodiimide (DCC, 0.11 g, 0.53 mmol)
and BOC-L-valine (0.12 g, 0.55 mmol). The reaction mixture
was stirred overnight under argon at room temperature. After
filtration, the filtrate was extracted with brine, and the organic
layer was separated, dried over anhydrous Na2SO4, filtered,
and concentrated. The residue was purified by flash chroma-
tography eluting with 30% EtOAc/hexane to give the L,L
Su p p or t in g In for m a t ion Ava ila b le: 1H and 13C NMR
and 13C NMR APT spectra for compounds 13 and 14 and a
2:3 mixture of 13 and 14. This material is available free of
charge via the Internet at http://pubs.acs.org.
J O991589R