Thiopeptide synthesis. α-Amino thionoacid derivatives of nitrobenzotriazole as thioacylating agents

Full text of DOI:10.1021/jo961245q

J . Org. Chem. 1996, 61, 9045-9048
9045
proceeds with about 2% loss of enantiomeric purity, as
demonstrated by HPLC analysis of the reaction product
10a , formed in reaction with R-methylbenzylamine. This
procedure, although superior to previous methods, still
suffers from the formation of benzimidazole 2 as a
significant byproduct, and the overall yield for the four-
step process was only about 20%. Furthermore, our
recent attempt8 to use this procedure failed due to the
limited reactivity of the benzimidazolinone 4 as a thio-
acylating agent. To overcome these limitations, we have
developed a new method for thiopeptide synthesis.
Th iop ep tid e Syn th esis. r-Am in o
Th ion oa cid Der iva tives of
Nitr oben zotr ia zole a s Th ioa cyla tin g Agen ts
M. Ashraf Shalaby, Christopher W. Grote, and
Henry Rapoport*
Department of Chemistry and Materials Sciences Division,
Lawrence Berkeley National Laboratory, University of
California, Berkeley, California 94720
Received J uly 2, 1996
Resu lts a n d Discu ssion
In tr od u ction
Our present work describes the use of R-amino thiono-
acid derivatives of nitrobenzotriazole as thioacyl transfer
reagents in thiopeptide synthesis. The synthetic strategy
is similar to that applied in the previous work7 for the
preparation of benzimidazolinone thioacylating agents.
However, improvement was sought in two areas. First,
an efficient thioacylating agent was needed that pos-
sessed a better leaving group than the benzimidazolinone
group and that could be readily cleaved under mild
conditions, thus allowing shorter reaction time for peptide
segment coupling to take place without racemization.9
Thioacyl halides are not candidates because they are
unstable even at low temperature and only a few are
known.10 Similarly, thioacyl anhydrides are not easily
accessible and are unstable.10 The benzotriazole group
was chosen because the benzotriazole anion is an excel-
lent leaving group11 and has the advantage that its
derivatives are frequently quite stable. Second, it was
important to avoid the formation of the benzimidazole
byproduct 2 with the concomitant purification require-
ment and loss of yield. This avoidance was achieved by
using 4-nitro-1,2-phenylenediamine as a latent triazole
source. The introduction of an electron-withdrawing
group para to the unacylated amino group reduces its
nucleophilicity and its participation for the formation of
any benzimidazole.
There has been considerable interest recently in the
synthesis and properties of thiopeptides in which the
-CSNH- group replaces one or more peptide bonds.1
These modified peptides have demonstrated increased
activity in vivo as biological response modifiers, neuro-
effectors, and immunomodulators due to the stability of
their thioamide bonds toward enzymatic degradation as
compared to that of their oxygenated counterpart.2
Synthetic routes employed to prepare these thiopeptides
included replacement of oxygen by sulfur using P4S10 or
Lawesson’s phosphetane disulfide reagent,3 and thioesters4
or dithioesters5 of N-protected amino acids. Several
procedures also have been reported for monothionation
of peptides using N-protected amino monothioacids and
benzotriazolyloxytris(pyrrolidino) phosphonium hexafluo-
rophosphate (PYBOP) and some of its derivatives.6
Unfortunately, these methods displayed lack of reaction
site specificity, low yields and purity because of side
reactions, and loss of enantiomeric integrity in the final
product, apparently because of racemization induced by
the thioacylating agents.
Recently, a major improvement was described7 for the
site specific incorporation of thioamide linkages into a
growing peptide under mild conditions using thioacyl-
benzimidazolinones of amino acid derivatives as thio-
acylating agents (Scheme 1). In our hands, this method
The procedure for the preparation of benzotriazole
thioacylating agents (9a -d ,f) is illustrated in Scheme 2.
Coupling was effected between 4-nitro-1,2-phenylenedi-
amine and the N-BOC amino acid in THF at 0 °C using
mixed carbonic anhydride methodology for peptide syn-
thesis.12 After isolation, this process, in all examples,
gave the crystalline anilide 6 in 90-92% yield. Direct
thionation of 6 was achieved with a mixture of P4S10 and
anhydrous Na2CO3 in THF. The reaction proceeded
smoothly for a total of 3 h at 0 °C and rt to afford
thioamides 7 in excellent yield (86-88%) with the excep-
tion of 7e which was obtained in 16% yield. In the latter
case, the major product was acylthioimidate 8 (64%
yield), formed in competitive cyclization between the
nucleophilic sulfur of the thioamide group and the
carbonyl oxygen of the â-methylester of aspartic acid. In
general a better yield of 7 was obtained when thionation
(1) Selected references: (a) Clausen, K.; Spatola, A. F.; Lemieux,
C.; Schiller, P. W.; Lawesson, S.-O. Biochem. Biophys. Res. Commun.
1984, 120, 305. (b) Lajoie, G.; Lepine, F.; Lemarie, S.; J olicoeur, F.;
Aube, C.; Turcotte, A.; Belleau, B. Int. J . Pept. Protein Res. 1984, 24,
316. (c) Maziak, L.; Lajoie, G.; Belleau, B. J . Am. Chem. Soc. 1986,
108, 182. (d) Sherman, D. B.; Spatola, A. F.; Wire, W. S.; Burks, T. F.;
Nguyen, T. M. D.; Schiller., P. W. Biochem. Biophys. Res. Commun.
1989, 162, 1126. (e) Seebach, D., Ko, S. Y.; Kessler, H.; Kock, M.;
Reggelin, M.; Schmeider, P.; Walkinshaw, M. D.; Bolsterli, J . J .; Bevec,
D. Helv. Chim. Acta 1991, 74, 1953. (f) Lankiewicz, L.; Bowers, C. Y.;
Reynolds, G. A.; Labroo, V.; Cohen, L. A.; Vonhof, S.; Siren, A.-L.;
Spatola, A. F. Biochem. Biophys. Res. Commun. 1992, 184, 359. (g)
Szokan, G.; Majer, Z.; Kollat, E.; Kajtar, M.; Hollosi, M.; Peredy-Kajtar,
M. J . Liq. Chromatogr. 1995, 18, 941.
(2) (a) Bock, M. G.; DiPardo, R. M.; Williams, P. D.; Pettibone, D.
J .; Clineschmidt, B. V.; Ball, R. G.; Veber, D. F.; Freidinger, R. M. J .
Med. Chem. 1990, 33, 2323. (b) Brown, D. W.; Campbell, M. M.;
Chambers, M. S.; Walker, C. V. Tetrahedron Lett. 1987, 28, 2171.
(3) (a) Pedersen, B. S.; Scheibye, S.; Nilsson, N. H.; Lawesson, S.-
O. Bull. Soc. Chim. Belg. 1978, 87, 223. (b) Cava, M. P.; Levinson, M.
I. Tetrahedron 1985, 41, 5061 and references therein.
(4) (a) Guthrie, D. J . S.; Williams, C. H.; Elmore, D. T. Int. J . Pept.
Protein Res. 1986, 28, 208. (b) Ried, W.; von der Emden, W. Liebigs
Ann. Chem. 1961, 642, 128. (c) Ried, W.; Schmidt, E. Liebigs Ann.
Chem. 1966, 695, 217.
(5) Thorsen, M.; Yde, B.; Pedersen, U.; Clausen, K.; Lawesson, S-O.
Tetrahedron 1983, 39, 3429 and references therein.
(6) Hoeg-J ensen, T.; Holm, A.; Sorensen, H. Synthesis 1996, 383 and
references therein.
(7) (a) Zacharie, B.; Martel, R.; Sauve, G.; Belleau B. Bioorg. Med.
Chem. Lett. 1993, 3, 619. (b) Belleau, B.; Brillon, D.; Sauve, G.;
Zacharie, B. U.S. Patent No. 5,138,061, Aug 11, 1992. (c) Zacharie, B.;
Sauve, G.; Penney, C. Tetrahedron 1993, 49, 10489.
(8) Grote, C. W.; Kim, D. J .; Rapoport, H. J . Org. Chem. 1995, 60,
6987.
(9) J urayj, J .; Cushman, M. Tetrahedron 1992, 48, 8601 and
references therein.
(10) (a) Hartke, K. Phosphorus, Sulfur Silicon Relat. Elem. 1991,
58, 223. (b) Brutsche, A; Hartke, K. Liebigs Ann. Chem. 1992, 921.
(11) (a) Staab, H. A.; Seel, G. Liebigs Ann. Chem. 1958, 612, 187.
(b) Katritzky, A. R.; Rachwal, S.; Hitchings, G. J . Tetrahedron 1991,
47, 2683.
(12) Anderson, G. W.; Zimmerman, J . E.; Callahan, F. M. J . Am.
Chem. Soc. 1967, 89, 5012.
S0022-3263(96)01245-5 CCC: $12.00 © 1996 American Chemical Society

9046 J . Org. Chem., Vol. 61, No. 25, 1996
Sch em e 1. Ben zim id a zolin on e Rou te
Notes
Sch em e 2. Ben zotr ia zole Rou te
of 6 was carried out using limited P4S10 (40-50 mol %);
this reaction gave only the corresponding thioamide.
The structure of 7 was established by combustion
analysis and 13C NMR spectroscopy which showed a
characteristic signal for the thioamide carbon at lower
field (δ 200-213 ppm) which was absent in the starting
compound. Intramolecular diazonium cyclization of 7
using nitrous acid generated in situ with NaNO2 in AcOH
gave benzotriazoles 9 in 72-83% yield. These com-
pounds are stable orange solids and can be stored for
months at 0 °C without decomposition. However, the
aspartic acid benzotriazole derivative 9f is unstable and
should be prepared immediately prior to use.
conditions used for coupling R-methylbenzylamine with
9a and 9b. The product thiopeptides 11c,d ,f were
obtained in 82-85% yield and were determined by HPLC
analysis to be enantiomerically pure. The amino acids
utilized in this study were selected to provide some scope
to the coupling, for example, aliphatic and aromatic side
chain (Ala and Phe), hindered amino acids (Val), and
functionality (Ser and Asp-â-methyl and â-tert-butyl
ester).
In summary we provide a mild, convenient, efficient,
and racemization-free route for incorporation of a thio-
amide linkage into peptides employing inexpensive and
easily prepared reagents.
Having established a suitable procedure for the syn-
thesis of the desired benzotriazoles of the R-amino
thionoacids, we next examined their efficiency and
reactivity as thioacylating reagents. Initial experiments
were carried out with R-methylbenzylamine as the amine
component, coupling with 9a and 9b to optimize the
reaction conditions and to test for enantiomeric integrity.
The best results were obtained when R-methylbenzyl-
amine was added slowly over a 40 min period to a
solution of 9a or 9b in THF at 0 °C. Reaction took place
almost instantly as evidenced by the change of color of
the reaction medium from orange to colorless. After the
addition was completed, the product was easily separated
from the reaction mixture by simple chromatography
through a plug of silica gel, eluting with hexane/EtOAc,
2/1. Compounds 10a and 10b were obtained in 88% and
86% yield, respectively. Their enantiomeric purity was
evaluated by HPLC using racemic samples of 9a and 9b
as controls, from which the corresponding 10a and 10b
were prepared. The HPLC analysis indicated the enan-
tomeric ratio (er) of the products to be in excess of 99/1.
Couplings were then conducted with an amino acid
residue known to be somewhat sensitive to racemization.
Thus, thiobenzotriazole derivatives 9c,d ,f were coupled
with phenylalanine methyl ester in THF under the exact
Exp er im en ta l Section
Gen er a l. Melting points are uncorrected. 1H and 13C NMR
spectra were recorded in solution in the indicated deuterated
solvents at 400 and 100.6 MHz, respectively, and chemical shifts
(δ) are reported relative to either a tetramethylsilane internal
standard or the signals due to the solvent; coupling constants,
J , are reported in hertz. TLC was carried out using precoated
sheets (Merck silica gel 60-F250, 0.2 mm) which, after develop-
ment, were visualized at 254 nm, and/or by spraying with 0.3%
ninhydrin solution in tert-BuOH/AcOH (93/3) and heating.
Merck silica gel 60 (70-230 mesh) was used for column chro-
matography. The enantiomeric purities of the thiopeptide
products were determined by analytical HPLC on a 300 × 3.9
mm normal phase microsorb column utilizing detection at 254
nm. All reactions were conducted under a nitrogen or argon
atmosphere. Solvents were distilled prior to use: THF from Na/
benzophenone; N-methylmorpholine and triethylamine from
CaH and then stored over 4 Å molecular sieves. Organic extracts
were dried over anhydrous Na2SO4 and filtered, and the filtrate
was evaporated under reduced pressure at 30-40 °C unless
otherwise indicated. Microanalysis was performed by the Col-
lege of Chemistry Micro Analytical Laboratory, University of
California, Berkeley.
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
6a -f. Cou p lin g of r-N-BOC L-Am in o Acid s w ith 4-Nitr o-
1,2-p h en ylen ed ia m in e. N-Methylmorpholine (2.2 mL, 20
mmol) was added to a solution of the R-N-BOC L-amino acid

Notes
J . Org. Chem., Vol. 61, No. 25, 1996 9047
(5a -f) in THF (100 mL) at -20 °C, followed by dropwise addition
of isobutyl chloroformate (1.3 mL, 10 mmol). The mixture was
stirred for 10 min, 4-nitro-1,2-phenylenediamine (1.53 g, 10
mmol) was added, and the resulting slurry was stirred at -15
°C for 2 h and at rt overnight. The precipitate was filtered off,
and the filtrate was evaporated to dryness. The residue was
dissolved in EtOAc (250 mL), and the solution washed succes-
sively with 1 M NaHPO4, brine, 5% NaHCO3, and brine, and
then dried and evaporated to dryness. Crystallization of the
residue from EtOAc/hexane afforded pure 6a -f as yellow solids
in 90-92% yield.
mL) and 1 h of cooling, the precipitated yellow solid 7 was
collected, washed with Et2O (∼15 mL), and dried at rt.
r-N-BOC-L-a la n in e 2-a m in o-5-n itr oth ioa n ilid e (7a ): yield
88%; mp 184 °C; [R]22D ) -78.3° (c 1.0, DMSO); 1H NMR (DMSO)
δ 10.2 (s, 1H), 7.95 (dd, 1H, J ) 9.1, 2.5), 7.84 (s, 1H), 7.44 (d,
1H, J ) 5.0), 6.76 (d, 1H, J ) 9.1), 6.46 (s, 1H), 4.42 (m, 1H),
1.38 (s, 9H), 1.37 (d, 3H, J ) 6.7); 13C NMR δ 208.6, 155.7, 150.8,
135.2, 124.9, 124.7, 122.4, 113.7, 78.6, 56.9, 28.2, 20.1. Anal.
Calcd for C14H20N4O4S: C, 49.4; H, 5.9; N, 16.5. Found: C, 49.5;
H, 5.9; N, 16.2.
r-N-BOC-L-p h en yla la n in e 2-a m in o-5-n it r ot h ioa n ilid e
(7b): yield 87%; mp 175 °C; [R]22 ) +48.8° (c 1.95, DMSO); 1H
r-N-BOC-L-a la n in e 2-a m in o-5-n itr oa n ilid e (6a ): mp 187
D
1
°C; [R]22 ) -46.5° (c 1.0, DMSO); H NMR (DMSO) δ 9.33 (s,
NMR (DMSO) δ 10.21 (s, 1H), 7.92 (dd, 1H, J ) 6.4, 2.7), 7.5
(m, 1H), 7.33 (m, 5H), 6.73 (d, 1H, J ) 9.1), 6.4 (s, 2H), 4.56 (m,
1H), 3.06 (m, 2H), 1.35 (s, 9H); 13C NMR δ 207.1, 155.9, 150.7,
137.3, 135.2, 129.5, 128.0, 126.5, 124.4, 122.2, 113.6, 78.6, 28.2.
Anal. Calcd for C20H24N4O4S: C, 57.7; H, 5.8; N, 13.4. Found:
C, 57.7; H, 6.0; N, 13.2.
D
1H), 8.08 (d, 1H, J ) 1.9), 7.86 (dd, 1H, J ) 9.0, 2.5), 7.21 (d,
1H, J ) 6.1), 6.74 (d, 1H, J ) 9.1), 6.44 (s, 2H), 4.09 (m, 1H),
1.38 (s, 9H), 1.23 (d, 3H, J ) 7.2); 13C NMR δ 172.4, 155.5, 149.8,
135.4, 123.3, 122.2, 121.2, 113.5, 78.3, 50.3, 28.2, 17.4. Anal.
Calcd for C14H20N4O5: C, 51.9; H, 6.2; N, 17.3. Found: C, 51.9;
H, 6.4; N, 17.4.
r-N-BOC-L-va lin e 2-a m in o-5-n itr oth ioa n ilid e (7c): yield
87%; mp 104-105 °C (crystallized with one molecule of ether);
r-N-BOC-L-p h en yla la n in e 2-a m in o-5-n itr oa n ilid e (6b):
[R]22 ) -87° (c 1.0, DMSO); 1H NMR (DMSO) δ 10.2 (s, 1H),
1
mp 186 °C; [R]22 ) +16.8° (c 1.0, DMSO); H NMR (DMSO) δ
D
D
7.9 (d, 1H, J ) 9.1), 7.7 (s, 1H), 7.4 (d, 1H, J ) 6.4), 6.4 (s, 2H),
3.9-3.5 (m, 1H), 2.05 (m, 1H), 1.4 (s, 9H), 0.97-0.92 (dd, 6H, J
) 14.0, 6.5); 13C NMR δ 208.4, 157.0, 151.4, 135.7, 125.7, 125.5,
125.0, 122.9, 114.2, 79.1, 68.0, 65.4, 31.6, 28.7, 19.7, 15.6. Anal.
Calcd for C16H24N4O4S (C2H5)2O: C, 54.3; H, 7.8; N, 12.7.
Found: C, 54.5; H, 8.0; N, 12.8.
9.41 (br s, 1H), 7.96 (d, 1H, J ) 1.16), 7.87 (dd, 1H, J ) 6.6,
2.4), 7.31 (m, 1H), 6.74 (d, 1H, J ) 9.0), 6.38 (s, 2H), 4.31 (m,
1H), 3.03 (m, 1H), 2.9 (m, 1H), 1.34 (s, 9H); 13C NMR δ 171.3,
155.7, 149.9, 137.8, 135.4, 129.3, 129.3, 128.1, 126.4, 123.4, 122.4,
121.0, 113.5, 78.4, 56.4, 37.0, 28.1. Anal. Calcd for C20H24
-
N4O5: C, 60.0; H, 6.0; N, 14.0. Found: C, 60.1; H, 6.2; N, 13.8.
r-N-BOC-O-ben zyl-L-ser in e 2-a m in o-5-n itr oth ioa n ilid e
(7d ): yield 84%; mp 161-162 °C; [R]22D ) -23.6° (c 1.0, DMSO);
1H NMR (DMSO) δ 10.16 (s, 1H), 7.9 (dd, 1H, J ) 9.0, 2.6), 7.7
(s, 1H), 7.3 (s, 1H), 7.3 (m, 5H), 6.3 (s, 2H), 4.6 (dd, 1H, J )
12.4, 6.0), 4.5 (dd, 2H, J ) 19.0, 11.0), 3.7 (m, 2H), 1.37 (s, 9H);
13C NMR δ 205.3, 156.1, 151.1, 138.8, 128.7, 128.0, 125.5, 125.1,
122.9, 114.2, 79.3, 72.8, 71.9, 61.2, 28.6, 23.6. Anal. Calcd for
r-N-BOC-L-va lin e 2-a m in o-5-n itr oa n ilid e (6c): mp 139-
140 °C; [R]22 ) -63.8° (c 1.0, DMSO); 1H NMR (DMSO) δ 9.37
D
(s, 1H), 8.47 (s, 1H), 7.8 (d, 1H, J ) 9.0), 7.07 (d, 1H, J ) 7.4),
6.7 (d, 1H, J ) 9.0), 6.4 (s, 2H), 3.9 (m, 1H), 1.36 (s, 9H), 0.9
(dd, 6H, J ) 11.5, 5.9); 13C NMR δ 171.0, 156.5, 150.1, 135.9,
123.7, 122.4, 121.6, 114.0, 78.8, 61.2, 60.2, 30.1, 28.6, 19.1, 14.5.
Anal. Calcd for C16H24N4O5: C, 54.5; H, 6.8; N, 15.9. Found:
C, 54.7; H, 7.0; N, 16.0.
r-N-BOC-O-ben zyl-L-ser in e 2-a m in o-5-n itr oa n ilid e (6d ):
mp 149-150 °C; [R]22D ) -21.7° (c 1.0, DMSO); 1H NMR (DMSO)
δ 9.5 (s, 1H), 7.8 (d, 1H, J ) 8.8), 7.2 (m, 5H), 7.15 (d, 1H, J )
6.2), 6.7 (d, 1H, J ) 8.9), 6.4 (s, 2H), 4.5 (s, 2H), 4.3 (d, 1H, J )
5.7), 3.6 (br s, 2H), 1.36 (s, 9H); 13C NMR δ 170.2, 156.0, 150.4,
138.6, 135.9, 128.7, 127.9, 124.0, 122.9, 121.5, 114.0, 79.0, 72.6,
70.0, 55.2, 28.6, 23.6. Anal. Calcd for C21H26N4O6: C, 58.6; H,
6.1; N, 13.0. Found: C, 58.5; H, 6.0; N, 13.0.
C
21H26N4O5S: C, 56.5; H, 5.9; N, 12.5. Found: C, 56.4; H, 5.9;
N, 12.5.
r-N-BOC-L-a sp a r tic a cid â-m eth yl ester r-2-a m in o-5-
n itr oth ioa n ilid e (7e) a n d a cylth ioim id a te (8) were obtained
as a mixture (1.6 g) from 6e following the general procedure
described above. Separation by column chromatography, eluting
with hexane/EtOAc (2/1), afforded first pure acylthioimidate 8
(1.2 g, 3.27 mmol, 64%) as a yellow solid: mp 200 °C dec; Rf )
0.29 (hexane/EtOAc, 1/1); [R]22 ) -33.5° (c 0.89, DMSO); 1H
D
NMR (DMSO) δ 8.0 (dd, 1H, J ) 9.0, 2.5), 7.9 (d, 1H, J ) 2.4),
7.8 (d, 1H, J ) 8.0), 6.7 (d, 1H, J ) 9.1), 6.5 (s, 1H), 4.4 (m, 1H),
3.2 (dd, 1H, J ) 18.0, 9.0), 2.7 (dd, 1H, J ) 18.0, 3.7), 1.35 (s,
1H); 13C NMR δ 212.6, 177.2, 156.2, 151.7, 136.0, 127.0, 126.7,
r-N-BOC-L-a sp a r tic a cid â-m eth yl ester r-2-a m in o-5-
n itr oa n ilid e (6e): mp 103 °C; [R]22 ) -39.3° (c 1.0, DMSO);
D
1H NMR (DMSO) δ 9.5 (s, 1H), 8.0 (s, 1H), 7.8 (dd, 1H, J ) 9.0,
2.5), 7.3 (d, 1H, J ) 7.0), 6.7 (d, 1H, J ) 9.0), 6.4 (s, 2H), 4.4 (m,
1H), 3.5 (s, 3H), 2.8 (dd, 1H, J ) 16.0, 5.9), 2.6 (dd, 1H, J )
16.0, 8.0), 1.36 (s, 9H); 13C NMR δ 171.3, 170.7, 155.9, 150.6,
135.8, 124.0, 123.2, 121.4, 113.9, 79.1, 52.0, 51.8, 36.3, 28.6, 25.9.
Anal. Calcd for C16H22N4O7: C, 50.2; H, 5.8; N, 14.7. Found:
C, 50.4; H, 5.9; N, 14.6.
118.4, 114.7, 80.0, 57.2, 36.7, 28.6. Anal. Calcd for C15H18
-
N4O5S: C, 49.2; H, 4.9; N, 15.3. Found: C, 49.3; H, 4.9; N, 14.9.
Further elution with hexane/EtOAc (2/1) gave thioanilide 7e
(300 mg, 0.76 mmol, 16% yield): mp 146-147 °C after two
crystallizations from THF; Rf ) 0.20 (hexane/EtOAc, 1/1); [R]22
D
) -87.5° (c 0.88, DMSO); 1H NMR (DMSO) δ 10.17 (s, 1H), 7.9
(dd, 1H, J ) 9.0, 2.5), 7.8 (s, 1H), 7.5 (d, 1H, J ) 5.9), 6.7 (d, 1H,
J ) 9.0), 6.4 (s, 2H), 4.7 (dd, 1H, J ) 13.8, 6.2), 3.5 (s, 3H), 3.0
(dd, 1H, J ) 16.6, 5.8), 2.8 (dd, 1H, J ) 16.6, 8.0), 1.3 (s, 9H);
13C NMR δ 205.9, 171.0, 156.1, 151.2, 135.7, 125.5, 125.2, 122.9,
114.2, 79.3, 57.7, 52.0, 28.6. Anal. Calcd for C16H22N4O6S: C,
48.2; H, 5.6; N, 14.1. Found: C, 48.3; H, 5.5; N, 14.1.
r-N-BOC-L-a sp a r tic a cid â-ter t-bu tyl ester r-2-a m in o-5-
n itr oa n ilid e (6f): mp 187 °C; [R]22 ) -39.9° (c 1.0, DMSO);
D
1H NMR (DMSO) δ 9.38 (s, 1H), 8.0 (s, 1H), 7.8 (dd, 1H, J )
9.0, 2.5), 7.2 (d, 1H, J ) 7.2), 6.7 (d, 1H, J ) 9.0), 6.4 (s, 2H), 4.3
(m, 1H), 2.7 (dd, 1H, J ) 16.0, 6.3), 2.5 (dd, 1H, J ) 16.0, 7.7),
1.36 (s, 18H); 13C NMR δ 170.7, 169.9, 155.8, 150.5, 135.8, 123.9,
123.1, 121.5, 114.0, 80.7, 79.0, 60.2, 51.9, 28.6, 28.2, 21.2, 14.5.
Anal. Calcd for C19H28N4O7: C, 53.8; H, 6.7; N, 13.2. Found:
C, 53.9; H, 6.7; N, 12.9.
r-N-BOC-L-a sp a r tic a cid â-ter t-bu tyl ester r-2-a m in o-5-
n itr oth ioa n ilid e (7f): yield 85%; mp 204 °C; [R]22D ) -89.4° (c
1.0, DMSO); 1H NMR (DMSO) δ 10.21 (s, 1H), 7.9 (dd, 1H, J )
9.1, 6.5), 7.7 (s, 1H), 7.4 (d, 1H, J ) 6.1), 6.7 (d, 1H, J ) 9.0), 6.4
(s, 2H), 4.6 (m, 1H), 2.9 (dd, 1H, J ) 16.0, 7.4), 2.7 (dd, 1H, J )
16.0, 7.4), 1.38 (s, 9H); 13C NMR δ 210.8, 174.6, 160.7, 156.0,
140.5, 130.2, 130.0, 127.7, 119.0, 85.6, 84.0, 62.5, 33.0, 26.0, 19.3.
Anal. Calcd for C19H28N4O6S: C, 51.8; H, 6.4; N, 12.7. Found:
C, 51.8; H, 6.5; N, 12.8.
Gen er a l P r oced u r e for t h e P r ep a r a t ion of Ben zot r i-
a zoles 9a -d ,f. To a solution of thioanilide 7 (2 mmol) dissolved
by gentle warming at 40 °C and then cooled to 0 °C in glacial
acetic acid (diluted with 5% water, 15 mL) was added NaNO2
(0.21 g, 3 mmol) in portions over 5 min with stirring. After 30
min, ice water (∼100 mL) was added, and the precipitated
product was filtered and washed with water. The orange solid
was dried in vacuo at rt overnight and then at 50 °C for 4 h to
afford benzotriazoles 9a -d and 9f as amorphous solids of
sufficient purity for use in the next step.
Gen er a l P r oced u r e for th e P r ep a r a tion of Th ioa n ilid es
7a -f. Under a flow of argon, P4S10 (1.1 g, 2.5 mmol)13 was mixed
with Na2CO3 (0.27 g, 2.5 mmol) in THF (100 mL). The mixture
was stirred for 1 h at 25 °C and then cooled to 0 °C. To this
clear solution was added anilide 6 (5 mmol), and the reaction
mixture was stirred at 0 °C for 30 min and at rt for 2.5 h. The
mixture was filtered through Celite, and the filtrate was
evaporated to dryness. The residue was dissolved in EtOAc/
heptane (2/1, 75 mL) and washed with 5% NaHCO3 (2 × 30 mL),
and the aqueous layers were back-extracted with EtOAc/heptane
(75 mL). The combined organic layers were washed with brine,
dried, and evaporated to an oil. Upon addition of Et2O (∼50
(13) Commercial P4S10 was purified by Soxhlet extraction with
carbon disulfide. The crystalline P4S10 which formed in the boiler flask
was used in these procedures.

9048 J . Org. Chem., Vol. 61, No. 25, 1996
Notes
1-(N-BOC-L-th ion oa la n in yl)-6-n itr oben zotr ia zole (9a ):
N-BOC-L-a la n in e r-m eth ylben zylth ioa m id e (10a ): oil; Rf
yield 79%; mp 124 °C (ether); [R]22 ) +20.7° (c 1.0, CHCl3); 1H
1
) 0.54 (hexane/EtOAc, 1/1); [R]22 ) +84.8° (c 1.3, CHCl3); H
D
D
NMR (CDCl3) δ 9.6 (s, 1H), 8.44 (d, 1H, J ) 9.0), 8.30 (d, 1H, J
) 9.0), 6.19 (m, 1H), 5.4 (d, 1H, 8.6), 1.63 (d, 3H, J ) 6.8), 1.4
(s, 9H); 13C NMR δ 212.0, 149.5, 148.7, 131.4, 122.2, 121.5, 112.9,
80.1, 56.7, 28.4, 22.5. Anal. Calcd for C14H17N5O4S: C, 47.9;
H, 4.9; N, 19.9. Found: C, 48.0; H, 5.0; N, 19.9.
NMR (CHCl3) δ 8.7 (s, 1H), 7.2 (s, 5H), 5.6 (m, 1H), 5.3 (s, 1H),
1.5 (d, 3H, J ) 6.9), 1.4 (d, 3H, J ) 6.8), 1.3 (s, 9H); 13C NMR δ
204.0, 155.7, 141.3, 128.7, 127.5, 126.4, 80.2, 54.1, 28.3, 21.4,
20.5. Anal. Calcd for C16H24N2O2S: C, 62.3; H, 7.8; N, 9.1.
Found: C, 62.2; H, 8.0; N, 9.1.
1-(N -BOC-L -t h ion op h e n yla la n in yl)-6-n it r ob e n zot r i-
a zole (9b): yield 81%; mp 127-128 °C (ether); [R]22D ) +223.2°
(c 1.0, CHCl3); 1H NMR (CDCl3) δ 9.6 (s, 1H), 8.4 (d, 1H, J )
8.6), 8.2 (d, 1H, J ) 8.4), 7.2 (s, 5H), 6.5 (s, 1H), 5.4 (m, 1H), 3.3
(m, 1H), 3.0 (m, 1H), 1.39 (s, 9H); 13C NMR δ 209.0, 155.1, 149.6,
149.0, 153.3, 131.7, 129.4, 128.5, 127.3, 122.2, 121.5, 112.7, 80.6,
62.1, 42.8, 28.3. Anal. Calcd for C20H21N5O4S: C, 56.2; H, 5.0;
N, 16.4. Found: C, 56.3; H, 5.0; N, 16.4.
1-(N-BOC-L-th ion ovalin yl)-6-n itr oben zotr iazole (9c): yield
77%; amorphous; [R]22D ) +43.9° (c 1.0, CHCl3) 1H NMR (CHCl3)
δ 9.7 (s, 1H), 8.4 (d, 1H, J ) 8.8), 8.3 (d, 1H, J ) 8.9), 6.1 (m,
1H), 5.4 (d, 1H, J ) 9.6), 2.3 (m, 1H), 1.4 (s, 9H), 1.08 (d, 3H, J
) 6.6), 0.97 (d, 3H, J ) 6.8); 13C NMR δ 210.0, 155.6, 149.5,
149.1, 131.8, 122.2, 121.5, 112.8, 80.4, 65.7, 34.3, 28.3, 20.2, 17.0.
Anal. Calcd for C16H21N5O4S: C, 50.7; H, 5.6; N, 18.5. Found:
C, 50.8; H, 5.6; N. 18.4.
1-(N-BOC-O-b e n zyl-L-t h ion ose r in yl)-6-n it r ob e n zot r i-
a zole (9d ): yield 83%; amorphous; [R]22D ) -6.2° (c 1.0, CHCl3);
1H NMR (CDCl3) δ 9.6 (s, 1H), 8.4 (d, 1H, J ) 7.6), 8.2 (d, 1H,
J ) 8.9), 7.2 (s, 1H), 7.1 (m, 5H), 6.3 (m, 1H), 5.8 (d, 1H, J )
8.7), 4.5 (dd, 2H, J ) 31.7, 12.0), 3.9 (m, 2H), 1.6 (s, 9H); 13C
NMR δ 205.7, 155.3, 149.6, 148.9, 137.0, 131.9, 128.3, 127.8,
127.6, 122.1, 121.4, 112.8, 80.6, 73.2, 71.8, 61.0, 28.4. Anal.
Calcd for C21H23N5O4S: C, 55.1; H, 5.1; N, 15.3. Found: C, 55.3;
H, 5.0; N, 15.1.
N-BOC-L-ph en ylalan in e r-m eth ylben zylth ioam ide (10b):
mp 112 °C; Rf ) 0.6 (hexane/EtOAc, 1/1); [R]22 ) +115° (c 1.0,
D
CHCl3); 1H NMR (CDCl3 δ 7.5 (s, 1H), 7.29-7.1 (m, 10H), 5.5
(s, 1H), 4.5 (dd, 1H, J ) 14.0, 6.2), 3.2 (dd, 1H, J ) 12.9, 5.8),
3.1 (m, 1H), 1.7 (m, 1H), 1.49 (s, 9H), 13C NMR δ 201.4, 155.2,
141.0, 136.9, 129.3, 128.7, 127.7, 126.4, 80.3, 63.0, 54.2, 41.9,
28.3, 19.9. Anal. Calcd for C22H28N2O2S: C, 68.7; H, 7.4; N,
7.3. Found: C, 68.9; H, 7.5; N, 7.2.
Gen er a l P r oced u r e for Cou p lin g Th ioa cyla tin g Re-
a gen ts 9c,d ,f w ith L-P h en yla la n in e Meth yl Ester . F or m a -
tion of Th iop ep tid es 11c,d ,f. To a cooled solution (0 °C) of
thioacylating reagent 9c,d ,f (2 mmol) in 30 mL of THF was
added dropwise a solution of L-phenylalanine methyl ester (2
mmol, generated by treating L-phenylalanine methyl ester
hydrochloride with 100 mol % of triethylamine at 0 °C) in 10
mL of THF over a period of 40 min. After the addition was
completed, evaporation of the solvent and purification of the
residue by chromatography (hexane/EtOAc, 2/1) afforded thio-
peptides 11c,d ,f in 82-85% yield.
N-BOC-L-th ion ovalyl-L-ph en ylalan in e m eth yl ester (11c):
oil; Rf ) 0.8 (hexane/EtOAc, 1/1); [R]22D ) +44.4° (c 0.8, CHCl3);
1H NMR (CHCl3) δ 8.2 (d, 1H, J ) 6.6), 7.25 (m, 3H), 7.1 (d, 2H,
J ) 6.5), 5.4 (m, 1H), 5.2 (s, 1H), 4.0 (m, 1H), 3.7 (s, 3H), 3.3
(dd, 1H, J ) 14.0, 6.1), 3.1 (dd, 1H, J ) 14.0, 5.1), 1.43 (s, 9H),
0.9 (d, 6H, J ) 6.4); 13C NMR δ 205.6, 170.9, 155.6, 135.3, 129.3,
128.3, 127.3, 58.3, 52.4, 36.5, 33.1, 28.3, 19.6, 17.9. Anal. Calcd
for C20H30N2O4S: C, 60.9; H, 7.7; N, 7.1. Found: C, 60.6; H,
7.5; N, 7.4.
1-(N-BOC-O-â-ter t-bu tyl-L-r-th ion oa sp a r tyl)-6-n itr oben -
zotr ia zole (9f). To a solution of thioanilide 7f (330 mg, 0.75
mmol) dissolved in THF/AcOH (1/1, 5 mL) at rt and then cooled
to 0 °C was added NaNO2 (77 mg, 1.1 mmol) with stirring. After
30 min, EtOAc/heptane (3/1, 60 mL) was added, and the solution
was washed successively with H2O (3 × 30 mL), NaHCO3 (3 ×
30 mL), and brine, was dried, and then was evaporated to
dryness, to give a pale yellow foam that was immediately used
HPLC analysis established the er of the product to be >99/1
by comparison with a sample prepared from 9c with racemic
DL-phenylalanine methyl ester. For N-BOC-L-thionovalyl-DL-
phenylalanine methyl ester: mobile phase hexane/EtOAc, 8/1;
flow rate 1 mL/min; for S,S diastereomer, tR 11.4 min; for S,R
diastereomer, tR 16.2 min.
1
in the next step: yield 72%; H NMR (CDCl3) δ 9.6 (s, 1H), 8.4
(d, 1H, J ) 7.2), 8.3 (d, 1H, J ) 9.0), 6.3 (m, 1H), 5.9 (br s, 1H),
3.0 (dd, 1H, J ) 15.0, 5.3), 2.8 (dd, 1H, J ) 14.5, 6.8), 1.37 (s,
9H), 1.36 (s, 9H); 13C NMR δ 210.0, 168.5, 146.6, 146.3, 126.3,
122.3, 121.6, 117.2, 116.6, 112.7, 82.3, 58.1, 41.1, 28.3, 28.2, 28.0,
27.9.
N-BOC-O-ben zyl-L-th ion oser in yl-L-p h en yla la n in e m eth -
yl ester (11d ): mp 79-80 °C (hexane); Rf ) 0.79 (hexane/EtOAc,
1
1/1); [R]22 ) +116.8° (c 1.0, CHCl3); H NMR (CDCl3) δ 8.6 (s,
D
P r oced u r e for Cou p lin g Th ioa cyla tin g Rea gen ts 9a a n d
9b w ith (R)-r-Meth ylben zyla m in e a n d HP LC An a lysis. To
a cooled solution (0 °C) of thioacylating reagent 9a or 9b (2
mmol) in 30 mL of THF was added dropwise a solution of
R-methylbenzylamine (0.26 mL, 2 mmol) in 10 mL of THF over
a period of 40 min. After the addition was completed, insoluble
material was removed by filtration, and the filtrate was evapo-
rated and the residue chromatographed (hexane/EtOAc, 2/1) to
give 10a or 10b in 88% and 86% yield, respectively. To
determine the enantiomeric purities of the products, crude
samples of 10a and 10b were analyzed by HPLC, and the results
were compared with those obtained from samples prepared from
treatment of racemic 9a and 9b with (R)-R-methylbenzylamine.
The HPLC analysis revealed that the er’s of 10a and 10b were
>99/1. For N-BOC-DL-alanine (R)-R-methylbenzylthioamide:
mobile phase hexane/EtOAc (8/1); flow rate 1 mL/min; for S,R
diastereomer, tR 17.4 min; for R,R diastereomer, tR 20.6 min.
For N-BOC-DL-phenylalanine (R)-R-methylbenzylthioamide: mo-
bile phase hexane/EtOAc (8/1); flow rate 1 mL/min; for S,R
diastereomer, tR 11.4 min; for R,R diastereomer, tR 12.2 min.
1H), 7.3 (m, 5H), 7.2 (m, 3H), 7.1 (d, 2H, J ) 2.9), 5.3 (m, 1H),
4.5 (d, 2H, J ) 4.7), 4.0 (m, 1H), 3.6 (s, 3H), 3.3 (dd, 1H, J )
14.0, 60), 3.1 (dd, 1H, J ) 13.8, 5.0), 1.4 (s, 9H); 13C NMR δ
200.0, 170.6, 155.0, 137.3, 135.4, 129.3, 128.5, 128.0, 127.2, 80.6,
58.6, 52.4, 36.3, 28.2, 28.0. Anal. Calcd for C25H32N2O5S: C,
63.5; H, 5.9; N, 5.9. Found: C, 63.7; H, 6.7; N, 5.9.
(N-BOC-O-â-ter t-bu tyl-L-r-th ion oa sp a r tyl)-L-p h en yla la -
n in e m eth yl ester (11f): oil; Rf ) 0.57 (hexane/EtOAc, 3/1);
[R]22 ) +71.4° (c 0.77, CHCl3); 1H NMR (CDCl3) δ 8.79 (br s,
D
1H), 7.32-7.25 (m, 3H), 7.15 (d, 2H, J ) 6.7), 5.8 (br s, 1H),
5.29 (m, 1H), 4.7 (br s, 1H), 3.7 (d, 3H, J ) 4.5), 3.29 (dd, 1H, J
) 13.8, 6.2), 3.18 (dd, 1H, J ) 13.9, 5.4), 3.05 (d, 1H, J ) 12.9),
2.7 (m, 1H), 1.43 (s, 9H), 1.42 (s, 9H); 13C NMR δ 202.3, 171.0,
170.6, 155.1, 135.4, 129.3, 129.2, 128.6, 127.3, 81.8, 80.5, 58.8,
58.6, 56.7, 52.4, 40.4, 36.5, 28.3, 28.0. Anal. Calcd for
C
23H34N2O6S: C, 59.2; H, 7.4; N, 6.0. Found: C, 58.7; H, 7.4;
N, 6.3.
J O961245Q