Formation of Urea Dipeptides from Carbonyldiimidazole: Application toward the Protease Inhibitors GE 20372 and MAPI

Full text of DOI:10.1021/jo970514p

6420
J . Org. Chem. 1997, 62, 6420-6423
F or m a tion of Ur ea Dip ep tid es fr om
Ca r bon yld iim id a zole: Ap p lica tion tow a r d
th e P r otea se In h ibitor s GE 20372 a n d
MAP I
Xiaowei Zhang, J ason Rodrigues,† Lawrence Evans,†
Becky Hinkle,† Lisa Ballantyne,† and Michael Pen˜a*
Department of Chemistry and Biochemistry, Arizona State
University, Box 871604, Tempe, Arizona 85287-1604
values of the tetrapeptides and showed the relative
importance of the aldehyde: IC50 for Mer-N5075A is 17.8
µM as compared with 1.3 µM for (S)-R-MAPI.4 Other
peptide-based protease inhibitors have been synthesized
and shown to be highly effective in AIDS therapy as
exemplified by Indinavir and Saquinavir.5
Received March 20, 1997
GE 20372 A and B (1a ,b) are tetrapeptides which were
recently isolated and shown to inhibit HIV-protease by
Stefanelli and co-workers.1 Closely related to GE 20372
are the tetrapeptides described as “microbial alkaline
protease inhibitors”, also termed (S)-R- and (R)-â-MAPI
(2a ,b) which were shown to have similar activity.2 Both
GE 20372 A and B were isolated from a culture broth of
a Streptomyces species. The structures were elucidated
from a combination of high-field NMR and chemical
degradations. In this paper, we wish to communicate our
results in preparing model systems 14 and 16 incorporat-
ing a key feature of the natural product, namely, the
carbonyl link between two of the amino acid residues.
This effort should aid in the eventual synthesis of both
GE 20372 and MAPI.
Sch em e 1
In on-going work on the synthesis of naturally occur-
ring compounds which inhibit HIV-protease, we were
attracted to the GE 20372 family because of the unusual
attachment of two of the amino acids. A recent publica-
tion has shown the utility of S,S-dimethyl dithiocarbonate
in the preparation of unsymmetically substituted ureas.6
Their method relied on the condensation of an amine with
the reagent followed by treatment with LDA to prevent
further reactions. The thiocarbamate intermediate was
condensed with a different amine to produce a disubsti-
tuted urea. Unfortunately, this method could not be
employed with amino acids due to the requirement of
LDA which would racemize any stereogenic center.
Triphosgene has recently been employed in preparing
cyclic ureas from a chiral diamine7 and in preparing
simple urea dipeptides.8 Recently, chiral isocyanates
were obtained from protected amino acid derivatives
which were incorportated to form various oligoureas.9
In initial studies employing N,N′-carbonyldiimidazole
(CDI), we discovered that simple urea dipeptides may be
obtained by mixing CDI with an amino acid ester
hydrochloride salt in the presence of an amine base
(Scheme 1). We studied this reaction extensively by
surveying bases and reaction times to find the optimum
conditions to form a “urea” dipeptide. We examined
several amines such as triethylamine, diisopropylethyl-
This family of tetrapeptides has two common fea-
tures: a terminal aldehyde and a carbonyl link between
two of the amino acid residues.2 Structurally related to
the MAPI family is Mer-N5075A (3) which was recently
isolated from a culture broth of Streptomyces chromofus-
cus.3 The structure of Mer-N5075A is similar to (S)-R-
MAPI except for the presence of a terminal alcohol in
place of the aldehyde. The researchers obtained IC50
(4) Kaneto, R.; Chiba, H.; Dobashi, K.; Kojima, I.; Saki, K.; Shiba-
moto, N.; Nishida, H.; Okamoto, R.; Akagawa, H.; Mizuno, S. J .
Antibiot. 1993, 46, 1622-1624.
† Undergraduate research participants.
(1) Stefanelli, S.; Cavaletti, L.; Sarubbi, E.; Ragg, E.; Colombo, L.;
Selva, E. J . Antibiot. 1995, 48, 332-334.
(2) Watanbe, T.; Murao, S. Agric. Biol. Chem. 1979, 43, 243-250.
Stella, S.; Saddler, G. S.; Sarubb, E.; Colombo, S.; Stefanelli, M.;
Denaro, M.; Selva, E. J . Antibiot. 1991, 44, 1019-1022. Kaneto, R.;
Chiba, H.; Nisshida, H.; Okamoto, R.; Akagawa, H.; Mitsuno, S. J .
Antibiot. 1993, 46, 1622-1624.
(3) Sarubbi, E.; Seneci, P. F.; Angelastro, M. R.; Peet, N. P.; Denaro,
M.; Islam, K. FEBS Lett. 1993, 319, 253-256.
(5) Wilson, E. K. Chem. Eng. News 1996, J uly 29, 42-46.
(6) Leung, M.-k.; Lai, J .-L.; Lau, K.-H.; Yu, H.-h.; Hsiao, H.-J . J .
Org. Chem. 1996, 61, 4175-4179.
(7) Ishii, K.; Aoki, S.; Koga, K. Tetrahedron Lett. 1997, 38, 563-
566.
(8) Majer, P.; Randad, R. S. J . Org. Chem. 1994, 59, 1937-1938.
(9) Burgess, K.; Ibarzo, J .; Linthicum, D. S.; Russel, D. H.; Shin,
H.; Shitangkoon, A.; Totani, R.; Zhang, A. J . J . Am. Chem. Soc. 1997,
119, 1556-1564.
S0022-3263(97)00514-8 CCC: $14.00 © 1997 American Chemical Society

Notes
J . Org. Chem., Vol. 62, No. 18, 1997 6421
Sch em e 2a
Sch em e 3a
a
Conditions: (a) carbonyldiimidazole, CH2Cl2, N-methylmor-
pholine, then phenylalanine methyl ester HCl; (b) carbonyldiimi-
dazole, CH2Cl2, N-methylmorpholine, then phenylalanine tert-
butyl ester HCl; (c) H2, Pd-C.
a
Conditions: (d) DCC, HOBt, (S)-phenylalaninol, CH2Cl2 (85%);
(e) i. Dess-Martin, CH2Cl2 (88%), ii. HC(OMe)3, PTSA, MeOH
(61%); (f) H2/Pd-C (97%); (g) diphenyl phosphorazidate, 8a , DMF,
TEA (60%); (h) TFA (88%).
amine, and N-methylmorpholine (NMM) and found that
NMM gave the best results. The reaction time was
particularly important in that stopping the reaction after
1 h we isolated urea dipeptide 6 and imidazole carbamate
5. We isolated this carbamate by silica gel chromatog-
Sch em e 4a
1
raphy and identified it by H-NMR and mass spectros-
copy. This compound was unstable as it decomposed to
the free amino acid ester and imidazole. This result was
especially useful in that the intermediate imidazole
carbamate 5 was more stable than CDI.
We theorized that we could perform a sequential
displacement of the two imidazoles of CDI with two
different amino acid esters in order to make an unsym-
metrical urea dipeptide. This was exemplified by con-
densing alanine benzyl ester p-toluenesulfonic acid (PTSA)
salt (7) with CDI and phenylalanine methyl ester hydro-
chloride in the presence of N-methylmorpholine which
afforded urea 8a after hydrogenation of the benzyl ester
(Scheme 2). The intermediate urea dipeptide was ob-
tained in ∼95% purity; however, we believe the impurity
was obtained from the CDI. Reductive removal of the
benzyl ester gave acid 8a as a viscous oil which was easily
purified by column chromatography. We discovered that
the base is important for preservation of the optical
purity of the urea dipeptide. If triethylamine was
employed, we observed racemization of the product.
N-Methylmorpholine gave the best results as no racem-
ization was seen and it was easily removed by a water
workup.
a
Conditions: (i) diphenylphosphorazidate, 8b, DMF, TEA
(60%); (j) TFA (53% as 2,4-DNP).
lalaninol11 with 11 yielded amide 12. Oxidation with the
Dess-Martin reagent12 followed by acetalation13 (methyl
orthoformate/PTSA/methanol) and reductive removal of
the benzyloxycarbonyl (Cbz) group gave 13b. Acid 8a
was coupled with 13b (DPPA/DMF/TEA), and following
deacetalation (TFA) 14 was obtained as a white solid
which was further purified by preparative HPLC. Analy-
sis of the spectral data for 16 showed an excellent
correlation with GE 20372.
A closer model system to GE 20372 A was prepared
from urea dipeptide 8b where the carboxylic acid of the
phenylalanine portion was blocked as a tert-butyl ester14
(Scheme 4). The acid 8b was prepared according to our
general procedure (CDI condensation of alanine benzyl
ester with phenylalanine tert-butyl ester followed by
hydrogenation). Coupling of 8b with 13b (DPPA/DMF/
TEA)15 afforded 15. Simultaneous deprotection of the
ester and acetal (TFA) gave tetrapeptide 16 which we
discovered to be unstable. We believe the carboxylic acid
The optical purity of the acid 8a was inferred by
comparison with the corresponding diastereomeric acid
10. Condensing racemic alanine 9 with (S)-phenylala-
nine methyl ester hydrochloride followed by hydrogena-
tion gave 10 as a 1:1 mixture of diastereomers. Exami-
nation of both 1H- and 13C-NMR of 10 showed the
presence of both diastereomers. The methyl group of
alanine was especially useful as it appeared as a mutiplet
1
racemized the phenylalaninal portion as judged by H-
NMR analysis. We therefore characterized 16 as a 2,4-
DNP derivative after removing the protecting groups and
quickly treating the residue with dinitrophenylhydrazine.
This problem of racemization may be circumvented in the
1
(1.3 ppm) in the H-NMR of 10, although it appeared as
1
a clean doublet (J ) 7 Hz) in the H-NMR of 8a . In the
13C-NMR spectrum of 10, extra peaks are seen for a
number of the carbons; however, 8a did not show the
presence of the other diastereomer in the 13C-NMR
spectrum.
(11) Hvidt, T.; Szarek, W. A.; Maclean, D. B. Can. J . Chem. 1988,
66, 779-782.
(12) Dess, D. B.; Martin, J . C. J . Org. Chem. 1983, 48, 4155-4156.
(13) Wenkert, E.; Goodwin, T. E. Synth. Commun. 1977, 7, 409-
415.
(14) Roeske, R. J . Org. Chem. 1962, 28, 1251-1253. Thorsen, M.;
Andersen, T. P.; Pedersen, U.; Yde, B.; Lawesson, S.-O. Tetrahedron
1985, 41, 5633-5636.
(15) Shioiri, T.; Ninomiya, K.; Yamada, S.-i. J . Am. Chem. Soc. 1972,
94, 6203-6205.
Completion of the model system study required amine
13b which was prepared from N-(benzyloxycarbonyl)va-
line (11)10 (Scheme 3). Selective coupling of (S)-pheny-
(10) Kruse, C.; Holden, K. G. J . Org. Chem. 1985, 50, 2792-2794.
Chen, F. M. F.; Benoiton, N. L. Can. J . Chem. 1987, 65, 1224-1227.

6422 J . Org. Chem., Vol. 62, No. 18, 1997
Notes
N′-(Ben zyloxyca r bon yl)va lyl-(S)-p h en yla la n in ol (12). A
tetrahydrofuran solution containing 6.60 g (26.0 mmol) of
N-(benzyloxycarbonyl)valine (11), 3.70 g (24.0 mmol) of (S)-
phenylalaninol, 5.40 g (26.0 mmol) of DCC, and 3.60 g (27.0
mmol) of N-hydroxybenzotriazole (HOBt) was stirred overnight
at room temperature. The solvent was removed in vacuo and
the residue extracted with EtOAc. Filtration and evaporation
of the solution gave a white solid which was further purified by
column chromatography (50% EtOAc/hexane) to give 7.90 g
(85%) of 12 as a white solid: Rf ) 0.15 (50% EtOAc/hexane);
natural product, GE 20372, as the target contains a basic
side chain which may form a zwitterion with the terminal
carboxylic acid.
We are currently expanding the scope of the prepara-
tion of other carbonyl-linked amino acids, such as 8a ,b,
in order to eventually synthsize GE 20372, MAPI, and
Mer-N5075A. Our efforts will be reported in the future.
Exp er im en ta l Section
mp 151-153 °C; [R]24 -45.1 (c 0.63, MeOH); 1H-NMR (CDCl3)
D
δ 0.8 (d, 3H, J ) 6.6 Hz), 0.9 (d, 3H, J ) 6.6 Hz), 2.1 (m, 1H),
2.6 (s, 1H), 2.8 (m, 2H), 3.4-3.6 (m, 2H), 3.9 (dd, 1H, J ) 6.3,
8.5 Hz), 4.2 (m, 1H), 5.1 (s, 2H), 5.3 (br d, 1H, J ) 7.7 Hz), 6.3
(br d, 1H, J ) 7.7 Hz), 7.3-7.1 (m, 10H); 13C-NMR (CDCl3) δ
171.7, 156.6, 137.7, 136.2, 129.3, 128.6, 128.3, 128.1, 126.6, 67.2,
63.5, 60.9, 52.8, 36.9, 30.8, 19.2, 17.8, 14.2. Anal. Calcd for
C22H28N2O4: C, 68.71; H, 7.34; N, 7.29. Found: C, 68.58; H,
7.38; N, 7.20.
Gen er a l Meth od s. All solvents were distilled from calcium
hydride prior to use except for tetrahydrofuran (THF) which was
distilled from molten potassium and ethyl ether which was
distilled from sodium-benzophenone. Methanol and ethanol
were distilled from Mg turnings. All reagents were used as
obtained from commercial suppliers unless otherwise noted. Thin
layer chromatography was performed with glass-backed pre-
coated plates (Si-254F). Column chromatography utilized silica
gel 230-400 mesh, 60 Å. The following deuterated solvents and
their following internal reference points were used: deuterio-
chloroform (CDCl3) with tetramethylsilane (TMS) referenced to
TMS (0.000 ppm, 1H) or chloroform (77.00 ppm, 13C), methanol-
d4 referenced to methanol (3.48 ppm, 1H; 39.00 ppm, 13C), and
Am id e 13a . A dichloromethane solution of 1.0 g (2.60 mmol)
of alcohol 12 and 1.4 g (3.3 mmol, 1.3 equiv) of the Dess-Martin
reagent was stirred for 1 h. The reaction was quenched by the
addition of a saturated solution of Na2S2O3 and NaHCO3. The
organic layer was further washed with aqueous NaHCO3 and
then dried over Na2SO4. Filtration of solvent in vacuo left 870
mg (88%) of a pale yellow solid which was not characterized but
quickly carried on to the next step: Rf ) 0.42 (50% EtOAc/
hexane); 1H-NMR (CDCl3) 0.8 (2d, 6H, J ) 6.8 Hz), 2.1 (m, 1H),
3.1 (d, 2H, J ) 6.6 Hz), 4.0 (m, 1H), 4.7 (q, 1H, J ) 7.4 Hz), 5.1
(s, 2H), 5.2 (br d, 1H, J ) 8.8 Hz), 6.4 (br d, 1H, J ) 6.6 Hz),
7.1-7.4 (m, 10H), 9.6 (s, 1H).
1
DMSO-d6 referenced to DMSO (2.50 ppm, H; 39.51 ppm, 13C).
Melting points are uncorrected. Elemental analyses were
performed by Atlantic Microlab (Norcross, GA). Mass spectros-
copy (FAB) was performed at the Nebraska Center for Mass
Spectroscopy.
Acid 8a . A dichloromethane solution of 1.25 g (7.70 mmol,
1.10 equiv) of N,N′-carbonyldiimidazole (CDI) was stirred at
room temperature before adding via cannula a dichloromethane
solution of 2.45 g (7.00 mmol) of alanine benzyl ester p-
toluenesulfonate (7) and 3.0 mL (2.50 mmol) of N-methylmor-
pholine (NMM). The reaction mixture was stirred for 5 min
before adding a second dichloromethane solution of 1.50 g (7.00
mmol) of phenylalanine methyl ester hydrochloride salt and 3.0
mL (2.50 mmol) of NMM. The entire sequence of events was
maintained under a positive pressure of N2. The resultant
reaction mixture was stirred overnight at room temperature. The
reaction mixture was poured into a separatory funnel containing
dilute aqueous NaHCO3. The organic layer was washed several
times with water and dried over Na2SO4. Filtration and
evaporation of solvent in vacuo left a viscous oil which was
placed under vacuum for 2 h to remove all traces of NMM. The
residue was further purified by column chromatography (75%
EtOAc/hexane) to give 2 g of the urea diester as a viscous oil:
Rf ) 0.60 (75% EtOAc/hexane); 1H-NMR (CD3OD) δ 1.3-1.34
(m, 3H), 2.9-3.1 (m, 2H), 3.65 (s, 3H), 4.3 (m, 1H), 4.5 (m, 1H),
5.2 (m, 2H), 7.1-7.3 (m, 10H); 13C-NMR (CD3OD) δ 173.6, 172.8,
157.9, 136.4, 135.8, 129.0, 128.2, 128.1, 127.8, 127.7, 126.4, 66.3,
54.2, 51.1, 48.6, 37.7, 16.9.
The product was not fully characterized but carried on to the
next step as the 1H-NMR spectrum indicated a purity of about
95%. A methanol solution of the diester and 35 mg of Pd-C
was hydrogenated for 40 min. Filtration and evaporation of the
solvent in vacuo left a viscous oil which was further purified by
column chromatography (20% MeOH/chloroform) to give 1.65 g
(73% from 7) of 8a as a foamy white solid: Rf ) 0.50 (20% MeOH/
chloroform); [R]24D +27.6 (c 0.81, MeOH); mp 50-53 °C; 1H-NMR
(CD3OD) δ 1.33 (d, 3H, J ) 6.9 Hz), 2.95 (dd, 1H, J ) 7.2, 13.8
Hz), 3.05 (dd, 1H, J ) 6, 13.8 Hz), 3.6 (s, 3H), 4.2 (q, 1H, J )
7.2 Hz), 4.6 (t, 1H, J ) 6 Hz), 7.2 (m, 5H); 13C-NMR (CDCl3) δ
177.0, 174.5, 159.6, 137.9, 130.5, 129.6, 128.0, 55.7, 52.7, 49.8,
39.3, 18.8; FAB-HRMS for C14H18N2O5 294, found (M + H) m/z
295.1283 (3.7 ppm deviation).
Into a methanol solution of 220 mg (0.76 mmol) of the
aldehyde were added 3.0 mL (27.4 mmol) of methyl orthoformate
and 1 mg (catalytic) of p-toluenesulfonic acid, and the mixture
was heated to reflux for 4 h. The solvent was removed in vacuo
which left a viscous oil that was further purified by column
chromatography to give 590 mg (61%) of 13a as a white solid:
Rf ) 0.67 (50% EtOAc/hexane); mp 161-163 °C; [R]24 -48.0 (c
D
1
0.5, MeOH); H-NMR (CDCl3) δ 0.8 (d, 3H, J ) 6.6 Hz), 0.9 (d,
3H, J ) 6.6 Hz), 2.1 (m, 1H), 2.7 (dd, 1H, J ) 8.2, 13.7 Hz), 2.9
(dd, 1H, J ) 6.3, 13.9 Hz), 3.3 (s, 3H), 3.4 (s, 3H), 3.9 (dd, 1H, J
) 7.5, 8.5 Hz), 4.2 (d, 1H, J ) 3.3 Hz), 4.4 (m, 1H), 5.1 (s, 2H),
5.2 (br d, 1H, J ) 8.2 Hz), 5.9 (br d, 1H, J ) 9.3 Hz), 7.1-7.4
(m, 10H); 13C-NMR (CDCl3) δ 170.9, 156.3, 137.7, 136.3, 129.3,
128.6, 128.4, 128.2, 128.1, 126.4, 104.5, 67.0, 60.6, 55.6, 55.5,
51.5, 35.7, 31.1, 19.1, 17.7; Anal. Cald for C24H32N2O5: C, 67.25;
H, 7.53; N, 6.54. Found: C, 67.32; H, 7.54; N, 6.56.
Am in e 13b. A methanol solution of 340 mg (0.785 mmol) of
acetal 13a and 30 mg of Pd-C was hydrogenated in a Parr
hydrogenator for 2 h. Filtration and removal of solvent in vacuo
gave 224 mg (97%) of 13b as a white solid: Rf ) 0.06 (50%
EtOAc/hexane); 1H-NMR (CDCl3) δ 0.75 (d, 3H, J ) 6.6 Hz), 0.85
(d, 3H, J ) 6.6 Hz), 1.8 (m, 1H), 2.7 (m, 1H), 2.9 (m, 2H), 4.2 (d,
1H, J ) 4.4 Hz), 3.42, 3.40 (2s, 6H), 4.25 (m, 1H), 7.2 (m, 5H);
13C (CD3OD) δ 175.2, 138.3, 128.9, 127.9, 125.9, 105.2, 60.1, 54.6,
54.1, 51.5, 34.9, 31.6, 18.4, 16.0. This compound was not fully
characterized but quickly carried on to the next step.
Tetr a p ep tid e 14. A dimethylformamide solution of 230 mg
(0.790 mmol) of 8a , 223 mg (0.760 mmol) of 13b, 379 mg (1.38
mmol) of diphenyl phosphorazidate, and 0.3 mL (2.16 mmol) of
triethylamine was stirred at 0 °C for 4 h and then warmed to
room temperature overnight. The solution was poured into a
separatory funnel containing water and ethyl acetate. The
organic layer was washed with several portions of water and
dried over Na2SO4. Filtration and evaporation of solvent in
vacuo left a solid which was further purified by column chro-
matography (10% MeOH/chloroform) to give 258 mg (60%) of
the tetrapeptide acetal 14 as a white solid: Rf ) 0.24 (50%
Acid 8b. This compound was prepared according to the same
procedure as 8a . From 1.52 g (4.30 mmol) of alanine benzyl ester
p-toluenesulfonate (7), 800 mg (4.90 mmol) of CDI, 1.16 g (4.50
mmol) of phenylalanine tert-butyl ester hydrochloride salt, and
1.50 mL (13.0 mmol) of N-methylmorpholine was obtained 1.10
g of 8b (79%) as a white solid: Rf ) 0.43 (20% MeOH/
chloroform); [R]24D +22.0 (c 0.35, MeOH); mp 55-57 °C; 1H-NMR
(CD3OD) δ 1.3 (m, 12H), 3.0 (d, 2H, J ) 6.6 Hz), 4.2 (q, 1H, J )
7.1 Hz), 4.4 (t, 1H, J ) 6.6 Hz), 7.2-7.3 (m, 5H); 13C-NMR
(CD3OD) δ 176.0, 173.0, 159.4, 137.4, 130.5, 129.2, 127.7, 82.7,
56.0, 49.6, 39.4, 28.2, 18.7; FAB-HRMS for C17H24N2O5 336.1685,
found (M + H) m/z 337.1757 (1.8 ppm deviation).
EtOAc/hexane); mp 225-226 °C; [R]24 -21.1 (c 0.18, MeOH),
D
1H-NMR (DMSO-d6) δ 0.7 (dd, 6H, J ≈ 2, 6.8 Hz), 1.0 (d, 3H, J
) 7.1 Hz), 1.8 (m, 1H), 2.6 (dd, 1H, J ) 10, 14 Hz), 2.8-2.9 (m,
3H), 3.2 (s, 3H), 3.3 (s, 3H), 3.6 (s, 3H), 4.0-4.1 (m, 4H), 4.3 (q,
1H, J ) 7.8 Hz), 6.4 (br d, 2H, J ) 7.7 Hz), 7.1-7.3 (m, 10H),
7.6 (br d, 1H, J ) 9.3 Hz), 7.8 (br d, 1H, J ) 8.8 Hz); 13C-NMR
(DMSO-d6) δ 172.8, 172.5, 170.3, 156.7, 138.6, 136.9, 129.1,
128.9, 128.2, 127.9, 126.5, 125.8, 104.9, 57.6, 55.1, 54.0, 51.6,
51.2, 48.4, 37.5, 34.4, 30.6, 19.1, 18.9, 17.9. Anal. Calcd for

Notes
J . Org. Chem., Vol. 62, No. 18, 1997 6423
C30H42N4O7: C, 63.14; H, 7.42; N, 9.82. Found: C, 63.24; H,
7.46; N, 9.76.
Tetr a p ep tid e 16. A trifluoroacetic acid solution of 15 (230
mg, 0.380 mmol) was stirred at 0 °C for 2 h. The solvent was
removed in vacuo leaving a white solid which was first washed
with water and then ether. The solid was placed under vacuum
for 2 h. The crude product was treated with a saturated solution
of 2,4-DNP to give 140 mg (53% from 15) of a pale orange solid:
Rf ) 0.14 (25% MeOH/chloroform); mp 215 °C (dec.); [R]24D -16.6
(c 0.3, DMSO); 1H-NMR (DMSO-d6) δ 0.75 (2d, 6H, J ) 6.6 Hz),
1.0 (d, 3H, J ) 7.2 Hz), 1.95 (m, 1H), 2.8-3.1 (m, 4H), 4.1 (m,
2H), 4.25 (m, 1H), 4.8 (m, 1H), 6.2 (d, 1H, J ) 8.2 Hz), 6.4 (d,
1H, J ) 7.7 Hz), 7.1-7.25 (m, 10H), 7.6 (d, 1H, J ) 8.8 Hz), 7.8
(d, 1H, J ) 9.3 Hz), 8.0 (d, 1H, J ) 4.9 Hz), 8.3 (m, 2H), 8.8 (d,
1H, J ) 2.7 Hz); 13C-NMR (DMSO-d6) δ 175.0, 174.1, 171.8,
158.3, 153.8, 146.1, 138.9, 138.6, 138.2, 130.8, 130.5, 129.5, 129.4,
127.7, 127.6, 124.3, 107.9, 58.8, 55.2, 52.8, 49.8, 38.8, 31.9, 28.9,
20.6, 20.2, 19.2; FAB-HRMS for C33H38N8O9 690.2761, found (M
+ Na) m/z 713.2648 (1.5 ppm deviation).
A trifluoroacetic acid solution containing 256 mg (0.45 mmol)
of the tetrapeptide acetal 14 was stirred at 0 °C for 4 h. The
solvent was removed in vacuo and the residue extracted with
chloroform and washed with several portions of aqueous NaH-
CO3. The organic layer was dried over Na2SO4. Filtration and
evaporation of solvent in vacuo left a light tan solid which was
initially purified by column chromatography (10% MeOH/
chloroform) and then further purified by preparative HPLC (90:
10 acetonitrile: water, 4 mL/min; 220 nm, C-18 preparative
column; tR ) 20 min) to give 207 mg (88%) of 14 as a white
solid: Rf ) 0.45 (10% MeOH/chloroform); mp 205-207 °C; [R]24
D
-32.8 (c 0.20, MeOH); 1H-NMR (DMSO-d6) δ 0.8 (t, 6H, J ) 6.5
Hz), 1.1 (d, 1H, J ) 7.2 Hz), 1.9-2.2 (m, 1H), 2.8-3.0 (m, 3H),
3.1 (dd, 1H, J ) 5.0, 14.8 Hz), 3.6 (s, 3H), 4.5 (m, 2H), 4.7 (m,
2H), 6.4 (br d, 2H, J ) 7.8 Hz), 7.2-7.3 (m, 10H), 7.6 (br d, 1H,
J ) 8.7 Hz), 8.4 (br d, 1H, J ) 7.8 Hz), 9.4 (s, 1H); 13C-NMR
(DMSO-d6) δ 201.3, 174.2, 174.1, 172.7, 158.1, 138.8, 138.2,
130.4, 130.3, 129.6, 129.5, 127.9, 127.6, 60.8, 58.6, 55.3, 53.0,
49.7, 38.8, 34.6, 31.8, 20.5, 20.3, 19.1; FAB-HRMS for C28H36N4O6
524.2635, found (M + H) m/z 525.2718 (-1.0 ppm deviation).
P r otected Tetr a p ep tid e 15. This compound was prepared
according to the same procedure as for 14. From 270 mg (0.80
mmol) of acid 8b, 200 mg (0.67 mmol) of amine 13b, 250 mg
(0.90 mmol) of diphenyl phosphorazidate, and 0.1 mL (1.00
mmol) of triethylamine was obtained a white solid which was
further purified by column chromatography (10% MeOH/
chloroform) to give 240 mg (60%) of 15 as a white solid: Rf )
Ack n ow led gm en t. We warmly thank Reagents
Professor Therese Markow and the MARC (Minority
Access to Research Careers) program (L.E.) and also the
Western Alliance to Expand Student Opportunities
(WAESO) for continued support (J .R., B.H., L.B.). The
principal investigator is especially grateful to the Na-
tional Science Foundation for a Career Award (CHE-
9503260).
0.66 (10% MeOH/chloroform); mp 229-230 °C dec; [R]24 -23.6
D
(c 0.20, MeOH); 1H-NMR (CD3OD) δ 0.8 (2d, 6H, J ) 6.6 Hz),
1.2 (d, 3H, J ) 7.1 Hz), 1.4 (s, 9H), 1.95 (m, 1H), 2.65 (dd, 1H,
J ) 9.3, 14.3 Hz), 2.9 (m, 3H), 3.4 (2s, 6H), 4.1 (d, 1H, J ) 6.6
Hz), 4.1-4.3 (m, ∼3H), 4.4 (t, 1H, J ) 4.4 Hz), 7.2-7.4 (m, 10H);
13C-NMR (CD3OD) δ 174.1, 171.6, 158.1, 138.1, 136.6, 129.1,
128.8, 127.9, 127.9, 127.8, 126.3, 125.7, 105.0, 81.4, 58.6, 54.7,
54.4, 53.9, 51.9, 49.3, 37.9, 34.7, 30.5, 26.7, 18.1, 17.2, 16.9. Anal.
Calcd for C33H48N4O7: C, 64.68; H, 7.90; N, 9.14. Found: C,
64.61; H, 7.89; N, 9.15.
Su p p or tin g In for m a tion Ava ila ble: Spectra for com-
pounds 8, 14, and 16 (31 pages). This material is contained
in libraries on microfiche, immediately follows this article in
the microfilm version of the journal, and can be ordered from
the ACS; see any current masthead page for ordering
information.
J O970514P