Studies on novel peptidomimetics having bi-directional dispositions of hydroxylated D-Pro-Gly motifs anchored on a C2-symmetric iminosugar-based foundation

Article abstract of DOI:10.1021/jo010937y

A rigid pyrrolidine based scaffold comprising of 2,5-dideoxy-2,5-imino-D-idaric acid (1) is developed. Attachment of peptide strands to the carboxylic groups at both ends of this novel template led to the peptidomimetics 2 and 3. Conformational analysis by NMR studies revealed that compounds 2b, 3b and 2c, 3c take interesting turn structures (C₂ symmetric for 2c and 3c) in DMSO-d₆ consisting of identical intramolecular hydrogen bonds at two ends between LeuNH → sugar-OH as depicted in structure A, whereas 2a and 3a display structures with regular β-turns with hydrogen bonds between LeuNH → Boc-C=0 in one-half of their molecular frameworks (structure B), characteristic of the turn structures commonly observed in "D-Pro-Gly"-containing peptides. These results suggest that a cis hydroxyl group at the 3-position of the proline residue favors a pseudo β-turn-like nine-membered ring structure in hydroxyproline-containing peptides involving an intramolecular hydrogen bond between the hydroxyl and the i + 2 backbone amide.

Full text of DOI:10.1021/jo010937y

J . Org. Chem. 2002, 67, 2093-2100
2093
Stu d ies on Novel P ep tid om im etics Ha vin g Bi-d ir ection a l
Disp osition s of Hyd r oxyla ted D-P r o-Gly Motifs An ch or ed on a
C2-Sym m etr ic Im in osu ga r -Ba sed F ou n d a tion
T. K. Chakraborty,* P. Srinivasu, S. Kiran Kumar, and A. C. Kunwar*
Indian Institute of Chemical Technology, Hyderabad 500 007, India
chakraborty@iict.ap.nic.in
Received September 21, 2001
A rigid pyrrolidine based scaffold comprising of 2,5-dideoxy-2,5-imino-D-idaric acid (1) is developed.
Attachment of peptide strands to the carboxylic groups at both ends of this novel template led to
the peptidomimetics 2 and 3. Conformational analysis by NMR studies revealed that compounds
2b, 3b and 2c, 3c take interesting turn structures (C2 symmetric for 2c and 3c) in DMSO-d6
consisting of identical intramolecular hydrogen bonds at two ends between LeuNH f sugar-OH as
depicted in structure A, whereas 2a and 3a display structures with regular â-turns with hydrogen
bonds between LeuNH f Boc-CdO in one-half of their molecular frameworks (structure B),
characteristic of the turn structures commonly observed in “D-Pro-Gly”-containing peptides. These
results suggest that a cis hydroxyl group at the 3-position of the proline residue favors a pseudo
â-turn-like nine-membered ring structure in hydroxyproline-containing peptides involving an
intramolecular hydrogen bond between the hydroxyl and the i + 2 backbone amide.
In tr od u ction
in the peptide strands that could participate in an
intramolecular H-bonding to stabilize further any pos-
sible H-bonded structure commonly seen in various
peptidomimetic designs.1,2
For discovering new peptide-based drugs, many struc-
turally rigid nonpeptidic molecular scaffolds have been
designed. Insertion of these moieties in appropriate sites,
a common approach to restrict the conformational de-
grees of freedom in small peptides, produces the specific
three-dimensional structures required for binding to their
receptors.1 Many of these templates contain diamine or
diacid moieties and have been used extensively to nucle-
ate parallel â-sheet structures in peptides.2 In this paper,
we describe the development of a novel structurally
constrained molecular scaffold consisting of 2,5-dideoxy-
2,5-imino-D-idaric acid (1). Bi-directional elongation of the
diacid moieties of 1 with identical peptide strands led to
the formation of peptides 2 and 3. Compound 2 was
specifically chosen to provide an additional amide proton
(1) (a) Recent Advances in Peptidomimetics; Tetrahedron Symposia-
in-Print, No. 83; Aube´, J ., Guest Ed.; Pergamon Press: Oxford, 2000;
Tetrahedron 2000, 56 (50). (b) Robinson, J . A. Synlett 2000, 429-441.
(c) Peptides and Peptidomimetics that Adopt Folded Structures;
Symposia-in-Print, No. 15; Kelly, J . W., Guest Ed.; Pergamon Press:
Oxford, 1999; Bioorg. Med. Chem. 1999, 7 (1) (d) Hanessian, S.;
McNaughton-Smith, G.; Lombart, H.-G.; Lubell, W. D. Tetrahedron
1997, 53, 12789-12854. (e) Giannis, A.; Kolter, T. Angew. Chem., Int.
Ed. Engl. 1993, 32, 1244-1267.
(2) (a) Qiu, W.; Gu, X.; Soloshonok, V. A.; Carducci, M. D.; Hruby,
V. J . Tetrahedron Lett. 2001, 42, 145-148. (b) Nowick, J . S.; Chung,
D. M.; Maitra, K.; Maitra, S.; Stigers, K. D.; Sun, Y. J . Am. Chem.
Soc. 2000, 122, 7654-7661. (c) Chitnumsub, P.; Fiori, W. R.; Lashuel,
H. A.; Diaz, H.; Kelly, J . W. Bioorg. Med. Chem. 1999, 7, 39-59 and
references therein. (d) Ranganathan, D.; Haridas, V.; Kurur, S.;
Thomas, A.; Madhusudanan, K. P.; Nagaraj, R.; Kunwar, A. C.; Sarma,
A. V. S.; Karle, I. L. J . Am. Chem. Soc. 1998, 120, 8448-8460. (e) J ones,
I. G.; J ones, W.; North, M. J . Org. Chem. 1998, 63, 1505-1513. (f)
Hibbs, D. E.; Hursthouse, M. B.; J ones, I. G.; J ones, W.; Malik, K. M.
A.; North, M. J . Org. Chem. 1998, 63, 1496-1504. (g) Winningham,
M. J .; Sogah, D. Y. Macromolecules 1997, 30, 862-876. (h) Holmes,
D. L.; Smith, E. M.; Nowick, J . S. J . Am. Chem. Soc. 1997, 119, 7665-
7669 and references therein. (i) Nowick, J . S.; Smith, E. M.; Noronha,
G. J . Org. Chem. 1995, 60, 7386-7387. (j) Nowick, J . S.; Powell, N. A.;
Martinez, E. J .; Smith, E. M.; Noronha, G. J . Org. Chem. 1992, 57,
3763-3765. (k) Kemp, D. S.; Bowen, B. R.; Muendel, C. C. J . Org.
Chem. 1990, 55, 4650-4657 and references therein.
A closer look at compounds 2 and 3 could identify easily
the occurrence of the “D-Pro-Gly” type motifs on both
sides of the central pyrrolidine dicarboxylic acid frame-
work. It is well established that the type II′ â-turn
conformation adopted by a D-Pro-Xxx segment, with a æ
value of ca. +60° for the pyrrolidine ring, promotes
nucleation of â-hairpin structures in proteins.3 While
induction of proline causes a reversal in the backbone
trajectory in peptides, polyproline-based peptides exhibit
helical structures as often seen, for example, in collag-
enous peptides4 that have repeating units of Gly-X-Y
composed predominantly of proline (X) and hydroxypro-
10.1021/jo010937y CCC: $22.00 © 2002 American Chemical Society
Published on Web 03/03/2002

2094 J . Org. Chem., Vol. 67, No. 7, 2002
Chakraborty et al.
Sch em e 1. Syn th esis of th e Cor e Bu ild in g Block
1 a n d th e P ep tid om im etics 2 a n d 3a
rings using lithium aluminum hydride (LAH) in the
presence of anhydrous AlCl3 led to the formation of the
diol 6 in 60% yield. Selective removal of the N-benzyl
group under catalytic hydrogenation conditions was
followed by N-Boc protection to furnish the intermediate
7 in 86% yield. The secondary hydroxyl groups of 7 were
next protected as benzyl ethers, and primary hydroxyl
groups were deprotected using DDQ to give 8 in 92%
yield from 7. Oxidation of the primary hydroxyls of 8
using an excess of pyridinium dichromate (PDC) in DMF
gave the required diacid. A small amount of the crude
diacid was dissolved in Et2O and esterified by adding an
excess of ethereal solution of CH2N2 to give the corre-
sponding diester that was purified for characterization
purposes.7 The remaining diacid, after aqueous workup,
was directly subjected to the peptide-coupling reaction
following the standard solution-phase conditions using
1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydro-
chloride (EDCI) and 1-hydroxybenzotriazole (HOBt) as
coupling agents and dry DMF and CH2Cl2 as solvents.
The resulting compound 2a (and 3a ) was subjected to
catalytic hydrogenation to give 2b (and 3b). Finally, Boc-
deprotection furnished the fully deprotected compound
2c (and 3c).
Con for m a tion a l An a lysis. NMR Stu d ies. NMR
studies of 2 and 3 were carried out in DMSO-d6, except
in case of 3a , which was studied in CDCl3. For others
(2a -c and 3b,c), NMR studies in CDCl3 could not be
carried out either due to poor solubility or exchange
broadening of the spectral lines. Some of the experiments
in apolar solvents were performed in a mixture of CDCl3
and DMSO-d6 (95:5, v/v). The NMR spectra of the
peptides described here are quite well resolved, and most
of the spectral parameters could be obtained easily and
are reported in Tables 1-3 for compounds 2a , 2b, and
2c, respectively. For others (3a -c), the spectral details
are provided in the Experimental Section. While the
assignments were carried out with the help of two-
dimensional double-quantum-filtered correlation spec-
troscopy (DQF COSY) and total correlation spectroscopy
(TOCSY), rotating frame nuclear Overhauser effect spec-
troscopy (ROESY) experiments provided the information
on the proximity of protons, the details of which are
provided in the Experimental Section.
a
Reagents and conditions: (a) MsCl, Et3N, CH2Cl2-DMF, 0 °C,
0.5 h; (b) BnNH2, 130 °C, 6 h; (c) LAH-AlCl3 (3:2), CH2Cl2-Et2O,
0 °C to reflux, 2 h; (d) Pd(OH)2-C, MeOH, rt, 4 h; (e) Boc2O,
MeOH; (f) NaH, BnBr, TBAI (cat.), THF, 0 °C to rt, 12 h; (g) DDQ,
CHCl3-H2O, rt, 45 min; (h) PDC, DMF, 0 °C to rt, 24 h; (i) EDCI,
HOBt, 0 °C, 0.5 h; then TFA-H2N-Gly-Leu-COR (R ) OMe, or
NHMe), DIPEA, DMF-CH2Cl2, 0 °C to rt, 12 h; (j) Pd(OH)2-C,
MeOH, rt, 6 h; (k) TFA-CH2Cl2, 0 °C, 0.5 h.
line (Hyp, Y) residues. The design of the novel molecular
framework of 1 with bi-directional dispositions of “hy-
droxy-D-proline” moieties will enable us to study the
conformational bias conferred by it when inserted in
peptides. It is also expected to provide an insight into
the role of the hydroxyl groups on the structures of
hydroxyproline containing peptides.
Resu lts a n d Discu ssion
Syn th esis of th e Cor e Bu ild in g Block 1 a n d th e
P ep tid om im etics 2 a n d 3. Scheme 1 outlines the
synthesis of the core building block 1, which was finally
transformed into the peptides 2 and 3. Dimesylation of
1,3:4,6-di-O-(p-methoxy)benzylidene-D-mannitol 45 was
followed by the reaction with an excess of benzylamine
at 130 °C to get the pyrrolidine compound 56 in 62% yield
from 4. Reductive opening of the p-methoxybenzylidene
The presence of the Boc group attached to the imine
of the central pyrrolidine ring caused differences in the
behavior of the left and right side peptide chains in 2a ,b
and 3a ,b. For convenience, the right-side peptide chain
is designated as peptide-I, while the left one is termed
as peptide-I′.
Usually, small peptides exist in solution in several
conformations and the NMR parameters show the
weighted average of all such structures. However, inser-
tion of the conformationally constrained scaffolds in the
designs can give rise to organized structures. Variable-
temperature studies allow us to examine such structures
(3) For leading references, see: (a) Fisk, J . D.; Gellman, S. H. J .
Am. Chem. Soc. 2001, 123, 343-344. (b) Fisk, J . D.; Powell, D. R.;
Gellman, S. H. J . Am. Chem. Soc. 2000, 122, 5443-5447. (c) Mad-
alengoitia, J . S. J . Am. Chem. Soc. 2000, 122, 4986-4987. (d) Das, C.;
Raghothama, S.; Balaram, P. J . Am. Chem. Soc. 1998, 120, 5812-
5813. (e) Stanger, H. E.; Gellman, S. H. J . Am. Chem. Soc. 1998, 120,
4236-4237. (f) Raghothama, S. R.; Awasthi, S. K.; Balaram, P. J .
Chem. Soc., Perkin Trans. 2 1998, 137-143. (g) Haque, T. S.; Little,
J . C.; Gellman, S. H. J . Am. Chem. Soc. 1996, 118, 6975-6985.
(4) For some leading references, see: (a) Vitagliano, L., Berisio, R.,
Mazzarella, L., Zagari, A. Biopolymers 2001, 58, 459-464. (b) Babu,
I. R.; Ganesh, K. N. J . Am. Chem. Soc. 2001, 123, 2079-2080. (c)
Persikov, A. V.; Ramshaw, J . A.; Kirkpatrick, A.; Brodsky, B. Bio-
chemistry 2000, 39, 14960-14967. (d) Nagarajan, V.; Kamitori, S.;
Okuyama, K. J . Biochem. 1999, 125, 310-318. (e) Holmgren, K. S.;
Taylor, M.; Bretscher, L. E.; Raines, R. T. Nature 1998, 392, 666-
667. (f) Prockop, D. J .; Kivirikko, K. Annu. Rev. Biochem. 1995, 64,
403-434. (g) Ko, C. Y.; J ohnson, L. D.; Priest, R. E. Biochim. Biophys.
Acta 1979, 581, 252-259. (h) Szymanowicz, A.; Malgras, A.; Randoux,
A.; Borel, J . P. Biochim. Biophys. Acta 1979, 576, 253-262. (i) Gryder,
R. M.; Lamon, M.; Adams, E. J . Biol. Chem. 1975, 250, 2470-2474.
(5) Rampy, M. A.; Pinchuk, A. N.; Weichert, J . P.; Skinner, R. W.
S.; Fisher, S. J .; Wahl, R. L.; Gross, M. D.; Counsell, R. E. J . Med.
Chem. 1995, 38, 3156-3162.
(7) For some earlier works on pyrrolidine-2,5-dicarboxylic acids
see: (a) Na´jera, C.; Yus, M. Tetrahedron: Asymmetry 1999, 10, 2245-
2303. (b) Langlois, N.; Rojas, A. Tetrahedron 1993, 49, 77-82. (c)
Baldwin, J . E.; Hulme, C.; Schofield, J . J . Chem. Res., Synop. 1992,
173-175. (d) Kemp, D. S.; Curran, T. P. J . Org. Chem. 1988, 53, 5729-
5731. (e) Ohta, T.; Hosoi, A.; Kimura, T.; Nozoe, S. Chem. Lett. 1987,
2091-2094. For an earlier reference on polyhydroxylated prolines,
see: (f) Long, D. D.; Frederiksen, S. M.; Marquess, D. G.; Lane, A. L.;
Watkin, D. J .; Winkler, D. A.; Fleet, G. W. J . Tetrahedron Lett. 1998,
39, 6091-6094. For an earlier synthesis of imino sugar based sugar
amino acid, see: (g) McCort, I.; Dure´ault, A.; Depezay, J .-C. Tetrahe-
dron Lett. 1998, 39, 4463-4466.
(6) Masaki, Y.; Oda, H.; Kazuta, K.; Usui, A.; Itoh, A.; Xu, F.
Tetrahedron Lett. 1992, 33, 5089-5092.

Studies on Novel Peptidomimetics
J . Org. Chem., Vol. 67, No. 7, 2002 2095
Ta ble 1. 1H Ch em ica l Sh ifts (δ in p p m ), Cou p lin g Con sta n ts (J in Hz), a n d Tem p er a tu r e Coefficien ts of th e Am id e
P r oton Ch em ica l Sh ifts (∆δ/∆T in p p b/K) of 2a (500 MHz, DMSO-d 6)
protons
NH
Gly(I)
Leu(I)
Gly(I′)
Leu(I′)
8.65 (t, J NH-RH ) 5.5)
7.42 (d, J NH-RH ) 8.3)
8.48 (dd, J NH-R′H ) 5.3,
J NH-RH ) 6.0)
7.86 (d, J NH-RH ) 8.5)
CRH
3.72 (dd, J R-R′ ) 17.8)
4.22 (dt, J R-â ) 7.4,
J R-â′′ ) 7.4)
3.82 (dd, J R-R′ ) 16.6,
J NH-RH ) 6.0)
4.27 (ddd, J R-â ) 5.4,
J R-â′ ) 10.0)
CR′H
CâH
3.70 (dd)
3.69 (dd, J NH-R′H ) 5.3)
1.39-1.54 (m)
1.45-1.57 (m)
}
CγH
CδH
0.85 (d, J γ-δ ) 6.7)
0.81 (d, J γ-δ ) 6.3)
3.8
0.84 (d, J γ-δ ) 6.3)
0.81 (d,J γ-δ ) 6.3)
6.4
Cδ′H
-∆δ/∆T
5.0
6.4
others: 7.61 (q, J ) 4.7 Hz, NH-Me(I)), 2.51 (d, NH-Me(I)), 7.85 (q, J ) 4.7 Hz, NH-Me(I′)), 2.54 (d, NH-Me(I′));
-∆δ/∆T (in ppb/K) ) 7.9 for NHMe(I), 6.7 for NHMe(I′)
pyrrolidine ring:a 7.25-7.20 (m, 10 H, aromatic), 4.83 and 4.53 (m, 4 H, O-CH2Ph), 4.64 (d, J C2H-C3H ) 7.8 Hz, C2-H),
4.67 (d, J C5H-C4H ) 7.5 Hz, C5-H), 4.31 (m, J C3H-C4H ) 7.8 Hz, C3-H), 4.32 (m, C4-H), 1.30 (s, Boc)
a
Couplings obtained by simulating the spectrum with the VNMR software.
Ta ble 2. 1H Ch em ica l Sh ifts (δ in p p m ), Cou p lin g Con sta n ts (J in Hz), a n d Tem p er a tu r e Coefficien ts of th e Am id e
P r oton Ch em ica l Sh ifts (∆δ/∆T in P p b/K) of 2b (500 MHz, DMSO-d 6)
protons
NH
Gly(I)
Leu(I)
Gly(I′)
Leu(I′)
8.42 (t, J NH-RH ) 5.9)
7.62 (d, J NH-RH ) 8.8)
8.32 (dd, J NH-RH ) 5.7,
J NH-R′H ) 6.3)
7.78 (d, J NH-RH ) 8.8)
CRH
3.80 (dd, J R-R′ ) 16.9)
4.22 (ddd, J R-â ) 4.4,
J R-â′ ) 10.3)
3.58 (dd, J R-R′ ) 16.9,
J NH-RH ) 5.7)
4.25 (ddd, J R-â ) 4.9,
J R-â′ )10.4)
CR′H
CâH
CγH
CδH
Cδ′H
-∆δ/∆T
3.56 (dd)
3.82 (dd, J NH-R′H ) 6.3)
1.44-1.54 (m)
1.43-1.53 (m)
0.85 (d, J γ-δ ) 6.4)
3.5
0.85 (d, J γ-δ ) 6.4)
0.80 (d, J γ-δ′ ) 6.4)
3.4
0.80 (d, J γ-δ′ ) 6.4)
5.5
6.4
others: 7.72 (q, J ) 4.5 Hz, NH-Me(I)), 2.55 (d, NH-Me(I)), 7.58 (q, J ) 4.5 Hz, NH-Me(I′)), 2.54 (d, NH-Me(I′)),
-∆δ/∆T (in ppb/K) ) 6.6 for both NHMe(I) and NHMe(I′)
pyrrolidine ring:a 5.75 (d, J OH-C3H ) 4.8 Hz, C3-OH), 5.83 (d, J OH-C4H ) 4.8 Hz, C4-OH), 4.33 (d, J C2H-C3H ) 8.1 Hz,
C2-H), 4.19 (m, J C3H-C4H ) 8.1 Hz, C3-H), 4.31(d, J C4H-C5H ) 7.8 Hz, C5-H), 4.20 (m, C4-H), 1.27 (s, Boc)
a
Couplings obtained by simulating the spectrum with the VNMR software.
Ta ble 3. 1H Ch em ica l Sh ifts (δ in p p m ), Cou p lin g Con sta n ts (J in Hz), a n d Tem p er a tu r e Coefficien ts of th e Am id e
P r oton Ch em ica l Sh ifts (∆δ/∆T in p p b/K) of 2c (500 MHz, DMSO-d 6)
protons
Gly
Leu
NH
8.35 (dd,J NH-RH ) 6.2,
J NH-R′H ) 5.6)
7.79 (d, J NH-RH ) 8.5)
CRH
3.79 (dd, J R-R′ ) 16.7,
J NH-RH ) 6.2)
4.19 (ddd, J R-â(pro-S) ) 4.9,
J R-â(pro-R) ) 9.9)
CR′H
CâH(pro-R)
3.67 (dd, J NH-R′H ) 5.6)
1.39 (ddd, J γ-â(pro-R) ) 5.2,
J â(pro-R)-â(pro-S) ) 12.9)a
1.45 (ddd, J γ-â(pro-S) ) 8.2,
J â(pro-S)-â(pro-R) ) 12.9)a
1.52 (m)
CâH(pro-S)
CγH
CδH(pro-S)
CδH(pro-R)
-∆δ/∆T
0.80 (d, J γ-δ(pro-S) ) 6.3)
0.85 (d, J γ-δ(pro-R) ) 6.5)
3.9
3.9
others: 7.71 (q, J ) 4.6 Hz, NH-Me), 2.55 (d, NH-Me); -∆δ/∆T ) 5.0 (NHMe)
pyrrolidine ring:a 3.18 (bs, NH), 3.96 (bs, 2 H, C2(C5)-H), 4.18 (t, J ) 3.4 Hz,
2 H, C3(C4)-H), 5.35 (d, J ) 3.5 Hz, 2 H, C3(C4)-OH).
a
Couplings obtained by simulating the spectrum with the VNMR software.
in detail where the temperature coefficients of amide
molecules causing substantial downfield shifts in their
NMR signals. The intramolecularly hydrogen bonded
amide proton chemical shifts, on the other hand, show
relatively smaller solvent dependence.
The cross-peak intensities in the ROESY spectra of 2a
and 2b, as shown schematically in Figure 1, were used
for obtaining the restraints in the MD calculations. The
proton chemical shifts (∆δ/∆Τ) are used to measure their
involvement in intramolecular hydrogen bonds. While the
LeuNH protons showed medium values of the tempera-
ture coefficients in both 2 and 3, all other amide protons
had larger values of ∆δ/∆Τ as shown in Tables 1-3 and
in the Experimental Section. The variations in the
chemical shifts of the amide protons in nonpolar (CDCl3)-
polar solvent (DMSO-d6) titration also provide informa-
tion on their participation in the intramolecular hydrogen
bondings.8 The addition of strongly hydrogen-bonding
solvents such as DMSO-d6 to aCDCl3 solution of the
peptides perturbs all the exposed amide protons, which
start forming H-bonds with the added polar solvent
(8) For leading references on intramolecular hydrogen bonding in
small peptides, see refs 2c and 3b and the following references: (a)
Gung, B. W.; Zhu, Z. J . Org. Chem. 1996, 61, 6482-6483. (b) Nowick,
J . S.; Abdi, M.; Bellamo, K. A.; Love, J . A.; Martinez, E. J .; Noronha,
G.; Smith, E. M.; Ziller, J . W. J . Am. Chem. Soc. 1995, 117, 89-99. (c)
Tsang, K. Y.; Diaz, H.; Graciani, N.; Kelly, J . W. J . Am. Chem. Soc.
1994, 116, 3988-4005. (d) Gellman, S. H.; Dado, G. P.; Liang, G.-B.;
Adams, B. R. J . Am. Chem. Soc. 1991, 113, 1164-1173.

2096 J . Org. Chem., Vol. 67, No. 7, 2002
Chakraborty et al.
minimized and superimposed aligning the hydrogen
bonded parts clearly reveal type II′ â-turns in one-half
of the molecule while the other side peptide strand
displays random structures (Figure 2). The twist confor-
mation of the pyrrolidine ring is clearly brought out by
these studies.
The solution structure of 2a was also investigated in
CDCl3 containing 5% DMSO-d6. Solvent titration again
showed participation of Leu(I)NH in intramolecular
hydrogen bonding as indicated by small change in its
chemical shift by adding up to 30% v/v of DMSO-d6.
Con for m a tion a l An a lysis of 2b a n d 3b. The struc-
tures of 2b and 3b in DMSO-d6 were found to be very
similar to that of a sugar diacid, 2,5-dideoxy-D-idaric acid,
containing peptidomimetic, which had bi-directionally
elongated Phe-Leu residues.12 They take an unusual
structure with a pseudo-â-turn, consisting of a LeuNH
f pyrrolidineC3-OH hydrogen bond, reminiscent of the
main-chain NH f side-chain OH type H-bond seen
sometimes in serine- and threonine-containing peptides.13
However, the magnitudes of the temperature coefficients
of the amide proton chemical shifts (∆δ/∆T) for LeuNH
(-3.4 and -3.5 ppb/K for 2b; -3.4 and -3.3 ppb/K for
3b) imply that these hydrogen bonds are only of medium
strength. The turn structure is further supported by the
proximity of GlyNH, LeuNH, and LeuδH to pyrrolidine-
OH protons on either side of the ring as shown in the
ROESY spectra. For the Leu side chains in 2b on both
sides, the couplings between R and â protons correspond
to about 75% predominance of a single conformation
about Leu ø1.14 Very similar populations for the rotamers
were obtained about the Leu ø1 of 3b also. The sugar ring
conformation for both 2b and 3b are similar to those
observed in 2a and 3a . A twist conformation for the
pyrrolidine ring is deduced from the couplings.
The structures sampled during the restrained MD
calculations on the basis of the ROESY cross-peaks found
for 2b in DMSO-d6 reveal an ensemble of “scorpion”-like
structures as shown in Figure 3, where the two peptide
chains form cyclic conformations at both ends involving
hydrogen bonds between LeuNH and pyrrolidine-OH.
The central portions of these structures are fairly con-
served with the variations localized mainly at the Leu
side-chains. Like in 2a , here also the pyrrolidine ring
structures take twist conformations that are in agree-
ment with the NMR data.
F igu r e 1. Schematic representation of the hydrogen-bonded
ring structures of 2a and 2b with the long-range ROEs seen
in their ROESY spectra.
two-spin approximation was used to obtain the intermo-
lecular distances.9 The upper and lower bounds of these
distance restraints were fixed at (15% of the derived
distances. Several long-range (more than four bonds)
distance constraints and torsional restraints (all ω
dihedrals and the H-C3-C4-H torsion) were used in
the energy calculations and MD studies. The results
derived from the 100 ps MD runs for compound 2a and
2b are shown in Figures 2 and 3, respectively. The MD
structures of 2c were very similar to those of 2b.
Con for m a tion a l An a lysis of 2a a n d 3a . Detail NMR
studies of compounds 2a (in DMSO-d6) and 3a (in CDCl3),
having their hydroxyl groups protected as benzyl ethers,
revealed that they behave like true “D-Pro-Gly”-contain-
ing peptides,3 which nucleate type II′ â-turns. The
medium value of ∆δ/∆T for Leu(I)NH in 2a (-3.8 ppb/
K) and its downfield shift in 3a that changed very little
in solvent titration studies (∆δ ) 0.3 ppm, 0-30% v/v of
DMSO-d6 in CDCl3) indicate the propensity of Leu(I)NH
f BocCdO hydrogen bond in these molecules. In addi-
tion, the Leu(I)NH-Gly(I)NH and Leu(I)NH-pyrrolidi-
neC2-H ROE cross-peaks, as shown in Figure 1, confirm
the presence of such turn structures. The other peptide
chain, peptide-I′, consisting of Leu(I′) and Gly(I′) residues,
does not show any well-defined structure in both these
molecules. The couplings between the protons in the
pyrrolidine ring are consistent with a twist conformation
The structures of 2b and 3b could not be obtained in
pure CDCl3 due to their poor solubility. However, addi-
tion of 5% DMSO-d6 rendered clear solutions. Solvent
titrations performed on these solutions by adding se-
quentially increased amounts of DMSO-d6 (upto 30% v/v)
showed that the chemical shifts of LeuNH change by
small amounts during the titrations. This indicates that
the hydrogen bonding patterns are similar to those
noticed in DMSO-d6 solutions and the structures of 2b
and 3b are probably similar in both solvents.
(3 T, where 3 and 4 refer to C3 and C4, respectively) for
4
both 2a and 3a .10 The Leu(I′) side chain takes a single
predominant conformation about the CR-Câ bond.11 As
against this, Leu(I) shows the presence of substantial
populations of more than one isomer about CR-Câ.
Several long-range restraints derived from the ROESY
cross-peaks of 2a in DMSO-d6 were used in the MD
calculations. The superimposed display of the 10 struc-
tures out of 20 samples collected at regular intervals of
5 ps during a 100 ps MD run, subsequently energy-
Con for m a tion a l An a lysis of 2c a n d 3c. Compounds
2c and 3c, with unprotected hydroxyl and imine groups
(12) (a) Chakraborty, T. K.; Ghosh, S.; J ayaprakash, S.; Sharma, J .
A. R. P.; Ravikanth, V.; Diwan, P. V.; Nagaraj, R.; Kunwar, A. C. J .
Org. Chem. 2000, 65, 6441-6457. (b) Chakraborty, T. K.; Ghosh, S.;
RamanaRao, M. H. V.; Kunwar, A. C.; Cho, H.; Ghosh, A. K.
Tetrahedron Lett. 2000, 41, 10121-10125.
(13) (a) Marraud, M.; Aubry, A. Int. J . Peptide Protein Res. 1984,
23, 123; Perczel, A.; (b) Hollosi, M.; Foxman, B. M.; Fasman, G. D. J .
Am. Chem. Soc., 1991, 113, 9772.
(9) Lian, L. Y.; Barsukov, I. L.; Sutcliffe, M. J .; Sze, K. H.; Roberts,
G. C. K. Methods Enzymol. 1994, 239, 657-700.
(10) Haasnoot, C. A. G.; Leeuw, F. A. A. M. de; Leeuw, H. P. M. de;
Altona, C. Biopolymers 1981, 20, 1211-1245.
(11) Demarco, A.; Ilinas, M.; Wuthrich, K. Biopolymers 1978, 17,
617-636.

Studies on Novel Peptidomimetics
J . Org. Chem., Vol. 67, No. 7, 2002 2097
F igu r e 2. Stereoview of the 10 superimposed structures taken from the 20 samples collected at 5 ps intervals during 100 ps MD
simulations of 2a , subsequently energy-minimized and superimposed aligning the hydrogen-bonded parts, revealing clearly type
II′ â-turns in one-half of the molecule with the other side peptide strand displaying random structures.
F igu r e 3. Stereoview of the 20 superimposed structures, sampled at 5 ps intervals during 100 ps MD simulations of 2b,
subsequently energy-minimized and superimposed aligning the hydrogen bonded parts, revealing an ensemble of “scorpion”-like
structures where the two peptide chains form cyclic conformations at both ends involving hydrogen bonds between LeuNH and
pyrrolidine-OH with fairly conserved structures observed in the central portion of the molecule and the variations localized mainly
at the Leu side chains.
show the presence of C2 symmetries in them. The
medium strengths of the LeuNH hydrogen bonds (∆δ/
∆T ) -3.9 ppb/K, for both 2c and 3c) along with the rOe
cross-peaks between pyrrolidine-OH and LeuNH, LeuâH-
(pro-R), LeuγH, LeuδH(pro-R) and NHMe (in 2c) support
the presence of nine-membered pseudo â-turn structures
similar to those seen in 2b and 3b. The temperature
coefficients of the GlyNH protons in these molecules were
comparable to those of the LeuNH protons (∆δ/∆T ) -3.9
ppb/K in 2c and -4.2 ppb/K in 3c) indicating that they
may be involved in some GlyNH f pyrrolidine-OH type
H-bonds similar to those seen earlier by others in serine,
threonine containing peptides.13 The rOe cross-peaks
between GlyNH and pyrrolidine-OH further support
these possibilities. The Leu side-chains of these molecules
are quite rigid and show the rotamer populations of g-
about ø1 nearly 71% and 69% for 2c and 3c, respectively.
The predominance of the g- rotamers about CR-Câ bond
is further supported by much stronger rOe cross-peaks
between LeuNH-LeuâH(pro-R) than LeuNH-LeuâH(pro-
S) and the LeuâH(pro-S)-NHMe cross-peak. The rigidity
of Leu side-chain is also observed between Câ-Cγ (ø2)
bond with about 55% and 62% rotamer populations
having anti relationships between LeuâH(pro-S) and
LeuγH for 2c and 3c, respectively. The cross-peaks
between LeuRH-LeuδH(pro-S) and LeuNH-LeuγH pro-
vide additional evidences of the rigidity of the Leu side
chains.
consistent with a twist conformation of 43T (where 4 and
3 refer to C3 and C4, respectively) for the five-membered
ring with C4 and C3 both being exo to the peptide chains
I and I′, respectively.10
Discu ssion
The ROESY spectra of 2b and 3b showed exchange
peaks between the resonances for two peptide chains as
a result of the cis-trans isomerism involving amide bond
containing the Boc group. For 2a and 3a , the H-bonded
structures resulted in these molecules locking into one
predominant conformer. However, the presence of other
minor conformers contributed to some exchange peaks
between the peptide chains in these molecules as well.
The possibility of two isomers did not exist for Boc-
deprotected compounds 2c and 3c, both of which showed
C2 symmetric structures. The bulky benzyl protective
groups on the hydroxyls of the pyrrolidine rings pre-
vented the nucleation of the LeuNH f pyrrolidineC3-
OBn hydrogen bonds in compounds 2a and 3a , which
were seen in free hydroxyl containing peptides 2b and
3b. Such an observation was also made earlier on benzyl-
protected sugar diacid containing peptide,12 and this
steric crowding forced the peptide chains in 2a and 3a
to fold in opposite direction and adopt regular type II′
â-turns involving 10-membered hydrogen bonded struc-
tures between Leu(I)NH and the carbonyl of N-Boc group.
It is remarkable that protection-deprotection of the
hydroxyl groups can make these peptidomimetic mol-
ecules switch from one conformation to another that has
the potential to lead to many useful applications.
The small values of the coupling constants of the
pyrrolidine ring protons in 2c and 3c imply change in
the ring puckering compared to 2a , 3a and 2b, 3b. The
J H2-H3 ) J H4-H5 ) 3.4 Hz and J H3-H4 ≈ 0 Hz are

2098 J . Org. Chem., Vol. 67, No. 7, 2002
Chakraborty et al.
and temperature coefficients (∆δ/∆T) of amide proton chemical
shifts of 2a , 2b, and 2c are given in Tables 1, 2, and 3,
respectively. For compounds 3a -c, these data are provided
in the Experimental Section. To obtain the temperature
coefficients of NH-chemical shifts, the spectra were recorded
between 30 and 70 °C.
Con clu sion
A novel pyrrolidine-based scaffold comprising 2,5-
dideoxy-2,5-imino-D-idaric acid (1) is developed for the
first time. The various functional groups present on this
designed scaffold, especially the hydroxyl groups on the
pyrrolidine ring, can play important roles in designing
new molecular entities. It is noteworthy that the protected/
unprotected hydroxyl groups not only control the hydro-
phobic/hydrophilic nature of such molecular assemblies,
they can also influence the three-dimensional structures
such assemblies will adopt. We hope that the studies
described here will help to understand the structures of
hydroxyproline containing peptides. The nonproteino-
genic properties of the design will probably make com-
pounds built on this scaffold physiologically more stable
as well. This iminosugar based diacid compound is
expected to find wide ranging applications in peptidomi-
metic studies, especially in designing bioactive conforma-
tions of small peptides and in creating de novo molecular
entities with well-defined structures and useful proper-
ties.
Molecu la r Dyn a m ics. All molecular mechanics/dynamics
calculations were carried out using Sybyl 6.7 program on a
Silicon Graphics O2 workstation. The Tripos force field with
default parameters was used throughout the simulations. A
dielectric constant of 47 D was used in all minimizations as
well as MD runs. A number of interatomic distances, all the
ω dihedrals, all of which were fixed as trans, and the H-C3-
C4-H torsional angle as 150 ( 20° were used as restraints in
the minimization as well as during the MD runs.17 Force
constants of 15 kcal/Å and 5 kcal/rad were employed for ROE
cross-peak-derived distance and dihedral angle constraints,
respectively. An additional H-bond distance constraint of 2.2
Å with a force constant of 30 kcal/Å was initially used to derive
the starting energy-minimized structures of these molecules.
Minimizations were done first with steepest decent, followed
by conjugate gradient methods for
a maximum of 2000
iterations each or root mean square (RMS) deviation of 0.05
kcal/mol, whichever was earlier. The energy-minimized struc-
tures were then subjected to MD simulations. All the above-
mentioned restraints, excluding the H-bond restraint, were
employed during the MD simulations. The energy-minimized
structures were initially equilibrated for 10 ps and subse-
quently subjected to molecular dynamics at 300 K for 100 ps
with a step size of 1 fs, sampling the trajectory at equal
intervals of 5 ps. The samples collected were next minimized
using the above-mentioned energy minimization protocol with
the same set of restraints used during simulations. The energy-
minimized structures thus obtained were compared and
superimposed as shown in Figures 2 and 3.18 For the sake of
clarity in viewing, only 10 best matching structures are
included in Figure 2.
Exp er im en ta l Section
Gen er a l P r oced u r es. All reactions were carried out in
oven- or flame-dried glassware with magnetic stirring under
nitrogen atmosphere using dry, freshly distilled solvents,
unless otherwise noted. Reactions were monitored by thin-
layer chromatography (TLC) carried out on 0.25 mm silica gel
plates with UV light, I2, 7% ethanolic phosphomolybdic acid-
heat and 2.5% ethanolic anisaldehyde (with 1% AcOH and
3.3% concd H2SO4)-heat as developing agents. Silica gel finer
than 200 mesh was used for flash column chromatography.
Yields refer to chromatographically and spectroscopically
homogeneous materials unless otherwise stated. Melting
points are uncorrected. IR spectra were recorded as neat
liquids or KBr pellets. Mass spectra were obtained under liquid
secondary ion mass spectrometric (LSIMS) technique.
NMR Sp ectr oscop y. NMR spectra were recorded on 500
MHz spectrometers at 30 °C with 7-10 mM solutions in
appropriate solvents using tetramethylsilane as internal
standard or the solvent signals as secondary standards and
the chemical shifts are shown in δ scales. Multiplicities of NMR
signals are designated as s (singlet), d (doublet), t (triplet), q
(quartet), br (broad), m (multiplet, for unresolved lines), etc.
13C NMR spectra were recorded with complete proton decou-
pling. The assignments were carried out with the help of two-
dimensional total correlation spectroscopy (TOCSY).15 For
some cases, the rotating frame nuclear Overhauser effect
spectroscopy (ROESY) experiments,15 which provide the in-
formation on the proximity of protons, were additionally used
to confirm the assignments made. All the experiments were
carried out in the phase-sensitive mode using the procedure
of States et al.16 The spectra were acquired with 2 × 256 or 2
× 192 free induction decays (FID) containing 8-16 transients
with relaxation delays of 1.5-2.0 s. The ROESY experiments
were performed with mixing time of 0.3 s. For ROESY
experiments, a spin-locking field of about 2 kHz was used. The
TOCSY experiments were performed with the spin locking
fields of about 10 kHz and a mixing time of 0.08 s. The two-
dimensional data were processed with Gaussian apodization
in both the dimensions. The chemical shifts, coupling constants
Syn th esis of 5. To a solution of 4 (10 g, 23.9 mmol) in dry
CH2Cl2 (50 mL) and dry DMF (25 mL) was added Et3N (9.9
mL, 71.7 mmol) at 0 °C. After 5 min, methanesulfonyl chloride
(4.07 mL, 52.6 mmol) was added and the mixture stirred for
0.5 h. It was then diluted with CHCl3, washed with saturated
NH4Cl solution, water, and brine, dried (Na2SO4), filtered, and
concentrated in vacuo. The resulting crude dimesylated prod-
uct was thoroughly dried under high vacuum, dissolved in dry
benzylamine (78 mL, 717 mmol), and heated at 130 °C for 6
h. The excess benzylamine was distilled off under reduced
pressure. The resulting crude was diluted with EtOAc, washed
with saturated NH4Cl solution, water, and brine, dried (Na2-
SO4), filtered, and concentrated in vacuo. Purification by
column chromatography (SiO2, 20% EtOAc in petroleum ether
eluant) afforded 5 (7.25 g, 62%) as a white solid: Rf ) 0.5 (silica
gel, 30% EtOAc in petroleum ether); [R]20D 72.9 (c 1.48, CHCl3);
mp 144-145 °C; IR (KBr) νmax 2846, 1615, 1492, 1276, 1123
1
cm-1; H NMR (CDCl3, 500 MHz) δ 7.53 (d, J ) 7.5 Hz, 2 H,
NCH2-Ar2H and 6H), 7.44 (d, J ) 8.6 Hz, 4 H, PMP-H), 7.32
(t, J ) 7.5 Hz, 2 H, NCH2-Ar3H and 5H), 7.22 (t, J ) 7.5 Hz,
1 H, NCH2-Ar4H), 6.92 (d, J ) 8.6 Hz, 4 H, PMP-H), 5.47 (s,
2 H, -OCHO- of p-methoxybenzylidene), 4.46 (d, J ) 16.1
Hz, 1 H, NCHPh), 4.38 (d, J ) 2.4 Hz, 2 H, C2-H, C5-H), 4.24
(d, J ) 16.1 Hz, 2 H, C1-H, C6-H), 4.06 (d, J ) 16.1 Hz, 1 H,
NCH′Ph), 3.94 (dd, J ) 16.1, 2.4 Hz, 2 H, C1-H′, C6-H′), 3.82
(s, 6 H, OCH3), 3.49 (bs, 2 H, C3-H, C4-H); 13C NMR (CDCl3,
125 MHz): δ 160.13, 141.79, 130.97, 128.39, 127.53, 127.31,
126.52, 113.70,113.64, 99.49, 79.40, 66.74, 57.63, 55.26, 51.80;
MS (LSIMS) m/z 490 (40) [M+ + H], 512 (8) [M+ + Na]; HRMS
(LSIMS) calcd for C29H31NO6 [M+] 489.2151, found 489.2152.
(14) Chakraborty, T. K.; J ayaprakash, S.; Diwan, P. V.; Nagaraj,
R.; J ampani, S. R. B.; Kunwar, A. C. J . Am. Chem. Soc. 1998, 120,
12962-12963.
(15) (a) Cavanagh, J .; Fairbrother, W. J .; Palmer, A. G., III; Skelton,
N. J . Protein NMR Spectroscopy; Academic Press: San Diego, 1996.
(b) Wu¨thrich, K. NMR of Proteins and Nucleic Acids; Wiley: New York,
1986.
(17) Kessler, H.; Griesinger, C.; Lautz, J .; Mu¨ller, A.; F. van
Gunsteren, W.; Berendsen, H. J . C. J . Am. Chem. Soc. 1988, 110,
3393-3396.
(18) MD simulations, using all the specified restraints including
those for the H-bond, at 1000 K for 100 ps following the same protocol
described in the Experimental Section gave structures that after
annealing and minimization appeared similar to those shown in
Figures 2 and 3.
(16) States, D. J .; Haberkorn, R. A.; Ruben, D. J . J . Magn. Reson.
1982, 48, 286-292.

Studies on Novel Peptidomimetics
J . Org. Chem., Vol. 67, No. 7, 2002 2099
Syn th esis of 6. To a solution of 5 (7 g, 14.2 mmol) in dry
CH2Cl2 (140 mL) and dry diethyl ether (140 mL) was added
LiAlH4 (6.5 g, 171.5 mmol) portionwise at 0 °C. The mixture
was slowly heated to reflux temperature, after which time
anhydrous AlCl3 (15.2 g, 114.3 mmol) in dry diethyl ether (140
mL) was added slowly to the hot solution over a 45 min period
and reflux was continued for another 1.5 h. The mixture was
cooled to 0 °C, and the excess LiAlH4 was decomposed carefully
with slow addition of EtOAc (70 mL), followed by the addition
of water (210 mL). The solution was decanted from the
precipitated solid. To the residual slurry, again EtOAc (200
mL) was added, stirred for 15 min, allowed to settle, and then
decanted. From the combined decanted solutions, organic layer
was separated, washed with water, brine, dried (Na2SO4),
filtered, and concentrated in vacuo. The crude was purified
by column chromatography (SiO2, 60% EtOAc in petroleum
ether eluant) to afford 6 (4.2 g, 60%) as a syrupy liquid: Rf )
eluant) afforded 8 (2.42 g, 96%): Rf ) 0.3 (silica gel, 30% EtOAc
in petroleum ether); [R]20 -11.9 (c 1.7, CHCl3); IR (neat) νmax
D
3438, 2915, 1684, 1392, 1100 cm-1; 1H NMR (CDCl3, 500 MHz)
δ 7.37-7.30 (m, 10 H, ArH), 4.76 and 4.66 (ABq, 2 H,
PhCH2O), 4.67 (s, 2 H, PhCH2O), 4.34 (m, 2 H, C3-H, C4-H),
4.11 (m, 1 H, C5-H), 4.01 (m, 1 H, C6-H), 3.95 (m, 1 H, C2-H),
3.84 (m, 1 H, C1-H), 3.78 (m, 1 H, C1-H′), 3.69 (m, 1 H, C6-
H′), 3.26 (m, 1 H, C6-OH), 2.29 (t, J ) 7.2 Hz, 1 H, C1-OH),
1.47 (s, 9 H, Boc); 13C NMR (CDCl3, 125 MHz) δ 154.98, 137.36,
128.56, 128.06, 127.77, 127.68, 81.95, 81.32, 80.85, 73.49,
73.42, 61.92, 61.20, 58.63, 57.47, 28.37; MS (LSIMS) m/z 344
(30) [M+ + H - C5H8O2]; HRMS (LSIMS) calcd for C25H34NO6
[M+ + H] 444.2386, found 444.2387.
Syn th esis of 2a . To a solution of 8 (1 g, 2.25 mmol) in dry
DMF (30 mL), PDC (8.4 g, 22.5 mmol) was added at 0 °C. The
reaction mixture was stirred at room temperature for 24 h
under nitrogen atmosphere. It was then diluted with EtOAc,
washed with saturated CuSO4 solution, water, and brine, dried
(Na2SO4), filtered, and concentrated in vacuo to get the diacid.
A small amount of the crude diacid (1/9 portion, 0.25 mmol)
was dissolved in Et2O (1 mL) and treated with an ethereal
solution of CH2N2 until all the acids were converted into the
diester. It was then concentrated in vacuo, and purification
by column chromatography (SiO2, 14% EtOAc in petroleum
ether eluant) afforded the dimethyl diester (89 mg, 71%) as a
white solid: Rf ) 0.45 (silica gel, 20% EtOAc in petroleum
0.5 (silica gel, 80% EtOAc in petroleum ether); [R]20 42.9 (c
D
1.0, CHCl3); IR (neat) νmax 3415, 2846, 1600, 1508, 1238 cm-1
;
1H NMR (CDCl3, 500 MHz) δ 7.27-7.19 (m, 5 H, NCH2-ArH),
7.22 (d, J ) 8.6 Hz, 4 H, PMP-H), 6.87 (d, J ) 8.6 Hz, 4 H,
PMP-H), 4.4 (ABq, 4 H, PMPCH2O-), 4.29 (m, 2 H, C2-H, C5-
H), 3.80 (s, 8 H, OCH3, NCH2Ph), 3.58 (dd, J ) 10.1, 4.3 Hz,
2 H, C1-H, C6-H), 3.49 (dd, J ) 10.1, 2.5 Hz, 2 H, C1-H′, C6-
H′), 3.30 (m, 2 H, C3-H, C4-H), 2.85 (bs, 2 H, C3-OH, C4-
OH); 13C NMR (CDCl3, 125 MHz): δ 159.28, 129.97, 129.31,
128.16, 128.0, 126.72, 113.83, 78.93, 73.08, 67.41, 61.55, 55.21,
52.68; MS (LSIMS) m/z 492 (8) [M+ - H], 516 (2) [M+ + Na];
HRMS (LSIMS) calcd for C29H36NO6 [M+ + H] 494.2543, found
494.2544.
ether); [R]20 -114.2 (c 1.025, CHCl3); mp 111-113 °C; IR
D
(neat) νmax 2930, 1746, 1700, 1384, 1353, 1192, 1115 cm-1; 1H
NMR (CDCl3, 500 MHz) δ 7.31-7.22 (m, 10 H, ArH), 4.74 (d,
J ) 8.0 Hz, 1 H, C5-H), 4.73 and 4.60 (two d, J ) 11.5 Hz, 2
H, PhCH2O), 4.71 and 4.60 (two d, J ) 11.5 Hz, 2 H, PhCH2O),
4.68 (d, J ) 8.0 Hz, 1 H, C2-H), 4.44 (t, J ) 8.0 Hz, 1 H, C3-
H), 4.40 (t, J ) 8.0 Hz, 1 H, C4-H), 3.70 and 3.69 (two s, 6 H,
Syn th esis of 7. To a solution of 6 (4.0 g, 8.1 mmol) in MeOH
(30 mL) was added Pd(OH)2 on C (20%, 450 mg). It was
hydrogenated under atmospheric pressure using a H2 balloon.
After 4 h, the reaction mixture was filtered through a short
pad of Celite, and the filter cake was washed with MeOH. The
filtrate and the washings were combined and concentrated in
vacuo. The residue was dissolved in dry MeOH (30 mL), and
Boc2O (2.79 mL, 12.15 mmol) was added and stirred overnight
at room temperature. The reaction mixture was concentrated
in vacuo. Purification by column chromatography (SiO2, 50%
EtOAc in petroleum ether eluant) afforded 7 (3.5 g, 86%): Rf
OCH3), 1.40 (s, 9 H, Boc); 13C NMR (CDCl3, 125 MHz):
δ
170.81, 170.69, 153.18, 137.50, 128.32, 127.73, 127.44, 81.39,
80.69, 80.13, 73.35, 59.92, 59.03, 52.23, 52.04, 28.11; MS
(LSIMS) m/z 500 (4) [M+ + H], 400 (46) [M+ + H - C5H8O2].
To a solution of BocNH-Gly-Leu-CONHMe (1.32 g, 4.4
mmol) in dry CH2Cl2 (6 mL) was added trifluoroacetic acid (3
mL) at 0 °C and the mixture stirred for 1 h. The reaction
mixture was then concentrated in vacuo to give TFA.H2N-Gly-
Leu-CONHMe.
) 0.4 (silica gel, 50% EtOAc in petroleum ether); [R]20 37.82
D
(c 1.7, CHCl3); IR (neat) νmax 3408, 2923, 1677, 1500, 1384,
In another round-bottom flask, the remaining portion of the
diacid (2 mmol), prepared above and dissolved in DMF-CH2-
Cl2 (1:2, 9 mL), was sequentially treated with HOBt hydrate
(541 mg, 4.0 mmol) and EDCI (767 mg, 4.0 mmol) at 0 °C.
After 0.5 h, TFA‚H2N-Gly-Leu-CONHMe, prepared above and
dissolved in dry DMF (4 mL), was added to the reaction
mixture followed by the addition of DIPEA (1.53 mL, 8.8
mmol). After being stirred for 12 h at room temperature, the
reaction mixture was diluted with EtOAc, washed with
saturated NH4Cl solution, water, and brine, dried (Na2SO4),
filtered, and concentrated in vacuo. Purification by column
chromatography (SiO2, 7% MeOH in CHCl3 eluant) afforded
2a (804 mg, 48%) as a white solid: Rf ) 0.45 (silica gel, 8%
1
1238 cm-1; H NMR (CDCl3, 500 MHz) δ 7.19 (d, J ) 8.7 Hz,
4 H, PMP-H), 6.86 (d, J ) 7.4 Hz, 4 H, PMP-H), 4.43 (d, J )
11.7 Hz, 1 H, PMPCHO), 4.42 (s, 2 H, PMPCH2O), 4.40 (d, J
) 11.7 Hz, 1 H, PMPCH′O), 4.30 (m, 2 H, C3-H, C4-H), 4.01
(m, 2 H, C5-H, C6-H), 3.89 (m, 1 H, C2-H), 3.79 (s, 6 H, OCH3),
3.77 (m, 1 H, C1-H), 3.66 (m, 2 H, C1-H′, C6-H′), 2.75 (d, J )
8.2 Hz, 1 H, C4-OH), 2.66 (d, J ) 8.2 Hz, 1 H, C3-OH), 1.39
(s, 9 H, Boc); 13C NMR (CDCl3, 125 MHz) δ 159.33, 159.26,
153.70, 129.90, 129.65, 129.12, 113.92, 113.85, 79.92, 77.32,
73.12, 67.24, 66.37, 57.21, 57.11, 55.19, 28.37; MS (LSIMS)
m/z 504 (28) [M+ + H], 526 (38) [M+ + Na]; HRMS (LSIMS)
calcd for C27H38NO8 [M+ + H] 504.2597, found 504.2596.
Syn th esis of 8. To a solution of 7 (3 g, 5.9 mmol) in dry
THF (18 mL) under nitrogen atmosphere at 0 °C was added
portionwise NaH (60% dispersion in oil, 714 mg, 17.8 mmol).
After the mixture was stirred for 10 min at the same temper-
ature, BnBr (1.5 mL, 13.1 mmol) followed by TBAI (220 mg,
0.59 mmol) were added, and stirring was continued from 0 °C
to room temperature for 12 h. The mixture was then diluted
with EtOAc, washed with saturated NH4Cl solution, water,
and brine, dried (Na2SO4), filtered, and concentrated in vacuo.
Purification by column chromatography (SiO2, 16% EtOAc in
petroleum ether eluant) afforded the Bn-protected intermedi-
ate (3.99 g, 98%) that was used directly in the next step
without characterization.
MeOH in CHCl3); [R]20 -80.65 (c 0.15, MeOH); mp 251-253
D
°C; IR (KBr) νmax 3294, 2958, 1641, 1528, 1388, 1122 cm-1; 1H
NMR (DMSO-d6, 500 MHz) see Table 1; 13C NMR (DMSO-d6,
125 MHz) δ 172.10, 171.92, 169.87, 169.51, 168.28, 168.01,
153.40, 137.96, 128.10, 127.57, 127.44, 127.37, 80.15, 79.88,
79.40, 79.12, 72.14, 72.02, 59.63, 50.86, 50.73, 42.57, 42.15,
41.07, 40.43, 27.75, 25.48, 24.03, 22.97, 21.47, 21.30; MS
(LSIMS) m/z 738 (100) [M+ + H - C5H8O2]; HRMS (LSIMS)
calcd for C38H56N7O8 [M+ + H - C5H8O2] 738.4190, found
738.4202.
Syn th esis of 2b. To a solution of 2a (250 mg, 0.29 mmol)
in MeOH (3 mL) was added Pd(OH)2 on C (20%, 50 mg), and
the mixture was hydrogenated under atmospheric pressure
using an H2-filled balloon for 6 h. It was then filtered through
a short pad of Celite, and the filter cake was washed with
MeOH. The filtrate and the washings were combined and
concentrated in vacuo. Purification by column chromatography
(SiO2, 10% MeOH in CHCl3 eluant) afforded 2b (156 mg,
To a solution of the above Bn-protected intermediate (3.9
g, 5.7 mmol) in CHCl3 (132 mL) and water (6.6 mL) was added
DDQ (3.23 g, 14.2 mmol) at room temperature and the mixture
stirred for 45 min. The mixture was then diluted with CHCl3,
washed with saturated NaHCO3, water, and brine, dried (Na2-
SO4), filtered, and concentrated in vacuo. Purification by
column chromatography (SiO2, 50% EtOAc in petroleum ether
80%): Rf ) 0.5 (silica gel, 12% MeOH in CHCl3); [R]20 10.6 (c
D
0.5, MeOH); mp 220-222 °C; IR (KBr) νmax 3328, 2960, 1657,

2100 J . Org. Chem., Vol. 67, No. 7, 2002
Chakraborty et al.
1
1545, 1407, 1254, 1169, 1113 cm-1; H NMR (DMSO-d6, 500
(I)NH), 7.96 (d, J ) 8.3 Hz, 1 H, Leu(I)NH), 7.81 (d, J ) 7.9
Hz, 1 H, Leu(I′)NH), 5.97 (d, J ) 4.6 Hz, 1 H, C3-OH), 5.85
(d, J ) 4.7 Hz, 1 H, C4-OH), 4.35 (ddd, J ) 10.3, 8.3, 4.8 Hz,
1 H, Leu(I)RH), 4.31 (d, J ) 8.1 Hz, 1 H, C2-H), 4.31 (d, J )
8.1 Hz, 1 H, C5-H), 4.27 (m, 1 H, Leu(I′)RH), 4.20 (ddd, J )
8.6, 8.1, 4.6 Hz, 1 H, C3-H), 4.19 (ddd, J ) 8.6, 8.1, 4.7 Hz, 1
H, C4-H), 3.82 (dd, J ) 17.1, 5.9 Hz, 1 H, Gly(I)RH), 3.78 (dd,
J ) 17.1, 6.1 Hz, 1 H, Gly(I′)RH), 3.61 (s, 3 H, Leu(I′)-OCH3),
3.59 (s, 3 H, Leu(I)-OCH3), 3.59 (dd, J ) 17.1, 6.1 Hz, 1 H,
Gly(I′)RH′), 3.54 (dd, J ) 17.1, 5.9 Hz, 1 H, Gly(I)RH′), 1.60
(m, 1 H, Leu(I′)γH), 1.55 (m, 1 H, Leu(I)γH), 1.44 (m, 2 H,
Leu(I′)âH2), 1.43 (m, 2 H, Leu(I)âH2), 1.28 (s, 9 H, Boc), 0.87
(d, J ) 6.4 Hz, 3 H, Leu(I)δH), 0.87 (d, J ) 6.4 Hz, 3 H, Leu-
(I′)δH), 0.81 (d, J ) 6.4 Hz, 3 H, Leu(I)δ′H), 0.81 (d, J ) 6.4
Hz, 3 H, Leu(I′)δ′H); -∆δ/∆T (in ppb/K) ) 7.0 for Gly(I)NH,
MHz) see Table 2; 13C NMR (DMSO-d6, 125 MHz) δ 172.14,
172.06, 170.42, 170.38, 168.66, 168.46, 153.38, 79.51, 74.11,
73.40, 61.50, 61.05, 50.91, 50.80, 42.67, 42.53, 40.72, 40.36,
27.84, 25.59, 25.58, 24.01, 23.95, 23.08, 21.43, 21.33; MS
(LSIMS) m/z 680 (39) [M+ + Na], 558 (22) [M+ + H - C5H8O2];
HRMS (LSIMS) calcd for C29H52N7O10 [M+ + H] 658.3776,
found 658.3746.
Syn th esis of 2c. To a solution of 2b (100 mg, 0.15 mmol)
in dry CH2Cl2 (1 mL) was added trifluoroacetic acid (1 mL) at
0 °C and the mixture stirred under nitrogen atmosphere for
0.5 h. The reaction mixture was concentrated in vacuo. The
resulting crude was dissolved in MeOH, cooled to 0 °C, and
then slowly neutralized with aqueous ammonia. It was then
concentrated in vacuo. Purification by column chromatography
(SiO2, 14% MeOH in CHCl3 eluant) afforded 2c (80 mg, 95%):
7.4 for Gly(I′)NH, 3.4 for Leu(I)NH and 3.3 for Leu(I′)NH; 13
C
Rf ) 0.4 (silica gel, 16% MeOH in CHCl3); [R]20 35.5 (c 0.63,
NMR (DMSO-d6, 125 MHz): δ 172.68, 172.61, 170.25, 169.95,
168.84, 168.56, 153.26, 79.36, 73.96, 73.25, 61.21, 60.93, 51.81,
51.77, 49.99, 49.87, 42.22, 42.16, 27.79, 23.83, 22.78, 21.13;
MS (LSIMS) m/z 682 (25) [M+ + Na], 660 (11) [M+ + H], 560
(100) [M+ + H - C5H8O2]; HRMS (LSIMS) calcd for C29H50N5O12
[M+ + H] 660.3456, found 660.3463.
D
MeOH); mp 145-148 °C; IR (KBr) νmax 3415, 2923, 1646, 1423
cm-1 1H NMR (DMSO-d6, 500 MHz) see Table 3; 13C NMR
;
(DMSO-d6, 125 MHz) δ 172.17, 171.66, 168.88, 78.31, 64.10,
50.96, 42.31, 40.63, 25.57, 24.13, 22.97, 21.44; MS (LSIMS)
m/z 558 (10) [M+ + H]; HRMS (LSIMS) calcd for C24H44N7O8
[M+ + H] 558.3251, found 558.3233.
Syn th esis of 3c. It was synthesized from 3b following the
same method as described for the synthesis of 2c: Rf ) 0.4
Syn th esis of 3a . Compound 3a was synthesized following
the same method as described above for the synthesis of 2a :
(silica gel, 10% MeOH in CHCl3); [R]20 30.8 (c 0.44, MeOH);
D
Rf ) 0.5 (silica gel, 6% MeOH in CHCl3); [R]20 -74.4 (c 1.8,
mp 95-98 °C; IR (KBr) νmax 3307, 2938, 1738, 1653, 1530 cm-1
;
D
MeOH); mp 222-224 °C; IR (KBr) νmax 3323, 2946, 1723, 1653,
1H NMR (DMSO-d6, 500 MHz) δ 8.27 (d, J ) 5.9 Hz, 2 H,
GlyNH), 8.03 (d, J ) 7.6 Hz, 2 H, LeuNH), 5.29 (d, J ) 3.6
Hz, 2 H, OH), 4.27 (ddd, J ) 9.7, 7.6, 5.3 Hz, 2 H, LeuRH),
4.17 (m, 2 H, C3-H, C4-H), 3.76 (dd, J ) 17.2, 5.9 Hz, 2 H,
GlyRH), 3.73 (dd, J ) 14.0, 6.0 Hz, 2 H, GlyRH′), 3.92 (bs, 2
H, C2-H, C5-H), 3.61 (s, 6 H, Leu-OCH3), 1.59 (m, 2 H, LeuγH),
1.51 (ddd, J ) 13.4, 9.7, 5.2 Hz, 2 H, LeuâH′), 1.47 (ddd, J )
13.4, 8.9, 5.3 Hz, 2 H, LeuâH), 0.87 (d, J ) 6.6 Hz, 6 H, LeuδH),
0.82 (d, J ) 6.5 Hz, 6 H, Leuδ′H); -∆δ/∆T (in ppb/K) ) 4.2 for
GlyNH, and 3.9 for LeuNH; 13C NMR (DMSO-d6, 125 MHz) δ
172.69, 171.07, 169.14, 78.14, 64.00, 51.76, 50.17, 41.81, 39.66,
24.01, 22.64, 21.26; MS (LSIMS) m/z 560 (37) [M+ + H]; HRMS
(LSIMS) calcd for C24H42N5O10 [M+ + H] 560.2931, found
560.2916.
1515, 1361 cm-1; H NMR (CDCl3, 500 MHz) δ 7.2-7.25 (m,
1
10 H, aromatic), 6.95 (d, J ) 8.1 Hz, 1 H, Leu(I)NH), 6.63 (d,
J ) 8.4 Hz, 1 H, Leu(I′)NH), 6.15 (dd, J ) 6.9, 5.9 Hz, 1 H,
Gly(I)NH), 6.10 (dd, J ) 5.7, 5.3 Hz, 1 H, Gly(I′)NH), 4.81 (m,
2 H, PhCH2O), 4.72 (m, 2 H, PhCH2O), 4.55 (ddd, J ) 8.9, 8.1,
5.4 Hz, 1 H, Leu(I)RH), 4.54 (dd, J ) 8.1, 7.8 Hz, 1 H, C4-H),
4.51 (dd, J ) 8.1, 8.0 Hz, 1H, C3-H), 4.44 (d, J ) 8.0 Hz, 1 H,
C2-H), 4.29 (d, J ) 7.8 Hz, 1 H, C5-H), 4.27 (ddd, J ) 9.4, 8.4,
5.1 Hz, 1 H, Leu(I′)RH), 4.07 (dd, J ) 17.0, 5.7 Hz, 1 H, Gly-
(I′)RH′), 3.97 (dd, J ) 17.3, 5.9 Hz, 1 H, Gly(I)RH), 3.84 (dd, J
) 17.3, 6.9 Hz, 1 H, Gly(I)RH′), 3.79 (dd, J ) 17.0, 5.3 Hz, 1
H, Gly(I′)RH), 3.71 (s, 3 H, Leu(I′)-OCH3), 3.66 (s, 3 H, Leu-
(I)-OCH3), 1.66 (m, 1 H, Leu(I)γH), 1.59 (m, 1 H, Leu(I′)γH),
1.58 (m, 2 H, Leu(I)âH), 1.43 (m, 2 H, Leu(I′)â-H), 1.33 (s, 9
H, Boc), 0.92 (d, J ) 6.4 Hz, 3 H, Leu(I′)δH), 0.92 (d, J ) 6.4
Hz, 3 H, Leu(I′)δH′), 0.91 (d, J ) 6.4 Hz, 3 H, Leu(I)δH′), 0.89
(d, J ) 6.4 Hz, 3 H, Leu(I)δH); 13C NMR (DMSO-d6, 125
MHz): δ 173.14, 172.97, 170.11, 169.60, 169.05, 168.31, 154.19,
137.53, 137.34, 128.54, 128.41, 128.02, 127.88, 81.72, 81.06,
80.93, 73.90, 73.77, 60.87, 60.70, 52.13, 52.01, 50.90, 50.70,
43.14, 42.99, 40.81, 40.23, 28.15, 28.04, 24.60, 22.80, 21.57,
Ack n ow led gm en t. We thank Drs. M. Vairamani
and R. Srinivas for mass spectrometric assistance; CSIR
and UGC New Delhi for research fellowships (S.K.K.
and P.S., respectively); and DST, New Delhi, for finan-
cial support.
Su p p or tin g In for m a tion Ava ila ble: 1H, 13C, TOCSY,
ROESY, and expansion of ROESY NMR spectra of 2a -c and
3a -c, 1H VT NMR spectra of 2a -c, and a listing of the
distance constraints data used in the MD studies. This
material is available free of charge via the Internet at
http://pubs.acs.org.
21.47; MS (LSIMS) m/z 862 (40) [M+ + Na], 840 (12) [M+
+
H], 740 (50) [M+ + H - C5H8O2].
Syn th esis of 3b. Compound 3b was synthesized from 3a
following the same method as described for the synthesis of
2b: Rf ) 0.4 (silica gel, 8% MeOH in CHCl3); [R]20 5.23 (c
D
0.78, MeOH); mp 120-123 °C; IR (KBr) νmax 3338, 2961, 1746,
1653, 1546, 1415 cm-1; H NMR (DMSO-d6, 500 MHz) δ 8.45
1
(d, J ) 6.1 Hz, 1 H, Gly(I′)NH), 8.42 (t, J ) 5.9 Hz, 1 H, Gly-
J O010937Y