Peptides constrained to type VI β-turns. 1. Evidence for an exceptionally stable intramolecular hydrogen bond

Article abstract of DOI:10.1021/jo961180r

The synthesis and conformational analysis of conjugates of amino acids with a type VI β-turn dipeptide mimic (1) is described. The mimic possessed high structural similarity to the central two residues of the turn and was constrained from rotation about t

Full text of DOI:10.1021/jo961180r

J . Org. Chem. 1997, 62, 2847-2852
2847
P ep tid es Con str a in ed to Typ e VI â-Tu r n s. 1. Evid en ce for a n
Excep tion a lly Sta ble In tr a m olecu la r Hyd r ogen Bon d
Kyonghee Kim and J uris P. Germanas*
Department of Chemistry, University of Houston, Houston, Texas 77204-5641
Received J une 24, 1996 (Revised Manuscript Received February 13, 1997X)
The synthesis and conformational analysis of conjugates of amino acids with a type VI â-turn
dipeptide mimic (1) is described. The mimic possessed high structural similarity to the central
two residues of the turn and was constrained from rotation about two of the four single bonds.
Coupling of amino acids to the carboxyl group of the mimic afforded conjugates that were capable
of forming intramolecular hydrogen bonds. In nonpolar solvents, IR and NMR spectra of the
conjugates indicated that the amide hydrogen of the amino acid residue was hydrogen bonded to
the carbonyl group of the N-terminal carbamate functionality, as in typical â-turns. In the hydrogen-
bonding solvent DMSO, the intramolecular hydrogen bond was still present, according to the
temperature dependence of the chemical shift. The presence of the i, i + 3 hydrogen bond in the
conjugates of mimic 1 was substantiated by the spectral properties of conjugates of the isomeric
mimic 3, which showed no evidence for the presence of an intramolecular hydrogen bond. The
results from these studies suggest that the intramolecularly hydrogen-bonded type VIa turn is an
inherently more stable conformation for a peptide than the non-hydrogen-bonded type VIb
conformation, in the absence of other structural constraints.
In tr od u ction
Reverse turns play important roles in polypeptide
function, acting as elements of structure as well as
modulators of bioactivity.1 Among the reverse turns
found in proteins, the â turn is the most prevalent.2 In
this structure, four amino acids arrange themselves in a
manner such that the polypeptide backbone undergoes
a 180° change in its direction. Often, a hydrogen bond
is formed between the carbonyl oxygen of the first, or i
residue, and the amide hydrogen of the fourth, or i + 3
residue of the turn. Several types of â-turns are found
in proteins, where distinct conformations for the indi-
vidual amino acids result from different values of the
intervening torsion angles.2
The type VI turn is a unique member of the â-turn
family because it is the only turn that incorporates an
s-cis peptide bond (Figure 1).3 Natural type VI â-turns
always contain a proline residue at the i + 2 position,
since peptides incorporating this amino acid are the only
ones that can exist substantially in the s-cis configura-
tion. The type VI turn is often found in peptides and
proteins containing the sequence ArProAr, where Ar
represents an amino acid with an aryl side chain.4 Type
VI turns are subdivided into type VIa and VIb turns.5 In
the type VIa structures, an intramolecular hydrogen bond
is formed between the i carbonyl oxygen and the i + 3
amide hydrogen, as in other â-turns; in the type VIb
structures, the orientation of the torsion angle Ψ3 directs
F igu r e 1. Hydrogen-bonded conformations available to pep-
tides and bicyclic lactam amino acid conjugates.
* To whom correspondence should be addressed. Tel.: (713) 743-
3300. Fax: (713) 743-2709. E-mail: germanas@uh.edu.
X Abstract published in Advance ACS Abstracts, April 1, 1997.
(1) Creighton, T. E. Proteins: Structures and Molecular Properties;
Freeman: New York, 1993; pp 225-227.
(2) Rose, G. D.; Gierasch, L. M.; Smith, J . A. Adv. Protein Chem.
1985, 37, 1-109.
the C-terminus of the turn away from the N-terminus
so that the hydrogen bonding groups are improperly
aligned to interact (Figure 1).
(3) Ricahrdson, J . S. Adv. Protein Chem. 1981, 34, 167-339.
(4) (a) Yao, J .; Fehrer, V. A.; Espejo, B. F.; Reymond, M. T.; Wright,
P. E.; Dyson, H. J . J . Mol. Biol. 1994, 243, 736-753. (b) Yao, J .;
Bruschweiler, R.; Dyson, H. J .; Wright, P. E. J . Am. Chem. Soc. 1994,
116, 12051-12052.
(5) Richardson, J . S.; Richardson, D. C. In Prediction of Protein
Structure and the Principles of Protein Conformation; Fasman, G. D.,
Ed.; Plenum Press: New York, 1989; pp 48-53.
Recently, we reported the design and synthesis of a
series of bicyclic lactams (1) as mimics of the type VI turn
(Scheme 1).6 The lactams represented dipeptides of the
formula XaaPro in which the torsion angles of the Ψ bond
of the Xaa residue and the Φ and ω bonds of the Pro
residue were fixed to the values found in type VI turns
S0022-3263(96)01180-2 CCC: $14.00 © 1997 American Chemical Society

2848 J . Org. Chem., Vol. 62, No. 9, 1997
Kim and Germanas
Sch em e 1a
Resu lts
Syn th esis. The synthesis and structural character-
ization of the bicyclic lactam 1 was described previously.6
Coupling of amino acids to the carboxyl group of lactam
1 would be carried out to introduce the amide hydrogen
necessary for forming the i, i + 3 hydrogen bond, as in
the 10-membered ring â-turn conformation IIa . Spec-
troscopic techniques would then be used to assess the
hydrogen-bonding properties of the lactam-amino acid
conjugates. In addition to structure IIa , other intramo-
lecular hydrogen bonding motifs would be available to
structure II, such as the i, i + 2 γ turn conformation IIb,
as well as the eight-membered ring i, i +3 conformation
IIc. To distinguish between these alternatives, bicyclic
lactam-amino acid conjugates whose structures would
allow only a subset of the alternative conformations to
be attained were prepared and spectroscopically charac-
terized.
The synthesis of the mimic-amino acid conjugates is
shown in Scheme 1. Deprotection of the carboxyl group
of lactam 1 was effected by hydrogenolysis of the benzyl
ester function. Coupling of L-amino acid esters to the
resultant acid was achieved with the aid of the BOP
reagent,7 affording peptides 2. In an identical manner
were L-amino acid esters attached to the carboxyl group
of the isomeric trans-lactam 3 to provide peptides 4.
Peptides 5 were prepared by condensing N-protected
amino acid anhydrides with the amino group of mimic
1, obtained by acidolytic removal of the carbamate
function (Scheme 1). Overall yields of conjugates were
excellent (60-90%). No epimerization of the chiral center
adjacent to the lactam carbonyl group in the coupling
products was detected, according to NMR (>95% reten-
tion).
A second set of lactam-amino acid conjugates, peptides
6-8, were prepared from L-amino acids and the enanti-
omers of the mimics 1 and 3 (Scheme 1). These com-
pounds were diastereomers of conjugates 2b, 4b, and 5b,
respectively. Since the stereochemistry of the lactam
moiety of peptide 6 was identical to that of a Gly-D-Pro
dipeptide, it represented a “mirror image” â-turn.8 The
conformational properties of such a compound would
provide insight into the stability of “mirror image” type
VIa turns.
a
Key: (a) (i) H2, Pd-C; (ii) H2NCH(R)CO2Me, BOP; (b) (i)
CF3CO2H, CH2Cl2; (ii) BOCNHCH(R)CO2H.
NMR Sp ectr oscop y. Pertinent features of the NMR
spectra of the mimic-amino acid conjugates are sum-
marized in Tables 1 and 2. A particularly striking
observation was the difference between the chemical
shifts of the amide protons of the cis mimic conjugates 2
and 6 and those of the trans mimic isomers 4 and 7. In
CDCl3, the chemical shifts of the carboxamide hydrogens
(NH1, Figure 2) of the trans mimic conjugates fell into a
region characteristic for non-hydrogen-bonded amide
NH’s.9 For the conjugates with the cis-lactam moiety (2
and 6), however, the chemical shifts of the analogous
NH’s appeared substantially downfield from this region.
For instance, the carboxamide proton NH1 of the cis-
mimic conjugate 2b resonated at 8.3 ppm, 2.0 ppm
downfield from that for the trans isomer 4b. Signifi-
cantly deshielded amide protons often indicate that the
nucleus is involved in a hydrogen bond.9
(Figure 1). The two remaining bonds of the dipeptide,
Φ2 and Ψ3, were unconstrained and were thus capable
of assuming various conformations, including those char-
acteristic of type VIa or VIb structures. Peptides incor-
porating lactam 1 would be capable of adopting intramo-
lecular hydrogen-bonded conformations, as in structures
II (Figure 1). The conformational properties of these
compounds would afford the opportunity to investigate
the effect of constraint of a peptide on its propensity to
adopt a type VIa or alternative conformations. In this
paper, we provide evidence that constraint of the central
three dihedral angles of a peptide to the type VI turn
conformation results in the formation of an exceptionally
stable i, i + 3 intramolecular hydrogen bond as found in
type VIa turns.
(6) Kim, K.; Dumas, J .-P.; Germanas, J . P. J . Org. Chem. 1996, 61,
3138-3144. Dumas, J .-P.; Germanas, J . P. Tetrahedron Lett. 1994,
35, 1493-1496. The synthesis of a structurally similar compound was
also reported by another group; see: Gramberg, D.; Robinson, J . A.
Tetrahedron Lett. 1994, 35, 861-864.
(7) Castro, B.; Dormoy, G.; Evin, G; Castro, B Tetrahedron Lett.
1975, 14, 1219-1222.
(8) Sibanda, B. L.; Thornton, J . M. Nature 1985, 316, 170-174.
(9) Liang, G. B.; Rito, C. J .; Gellman, S. H. J . Am. Chem. Soc. 1992,
114, 4440-4442.

Peptides Constrained to Type VI â-Turns. 1
J . Org. Chem., Vol. 62, No. 9, 1997 2849
Ta ble 1. 1H NMR P a r a m eter s for Mim ic-Am in o Acid
Additional support for an i, i + 3 hydrogen bond in
structures 2 and 6 came from the values of the H6-
carbamate hydrogen coupling constants of the conjugates.
All conformations of the conjugates described in this
study are assumed to interconvert rapidly on the NMR
time scale; as a result, the NMR-derived parameters are
weighted averages of the values for the individual
conformers.12 In the trans-mimic conjugates 4 and 7, as
well as in conjugates 5 and 8, where amino acids were
coupled to the amino group of the cis-mimic 1, the values
of J H6-NH were approximately 6 Hz, consistent with the
presence of several populated rotamers about the C6-
NBOC bond (Figure 2). For cis-lactam conjugates 2 and
6, however, the value of J H6-NH was over 8 Hz; the large
value in these structures signified substantial population
of a rotamer about the C6-NBOC bond with a dihedral
angle between the N-H and C6-H bonds close to 0 or
180°.13 An intramolecular hydrogen bond between the
carboxamide hydrogen and the carboxyl oxygen in the
cis-mimic conjugates required restriction of the NH-C6H
torsion angle to approximately 0°.
a
Con ju ga tes in CDCl3
2d
stereo-
δ NH1 d δ NH2 d J NH
δ NH3 d
(ppm)
compd chemistryc
R
(ppm)
(ppm)
(Hz)
cisb
1
2a
2b
6
(6R,8aR)
(6R,8aR)
(6R,8aR) CH2Ph
5.2
5.4
5.4
5.1
e
H
8.3
8.3
7.4
6.9
7.2
5.4
(6S,8aS)
CH2Ph
transb
3
(6S,8aR)
(6S,8aR)
(6S,8aR) CH2Ph
(6R,8aS) CH2Ph
5.6
5.4
5.4
5.4
e
e
4.5
4.8
4a
4b
7
H
7.0
6.3
6.3
cisb
5a
5b
8
(6R,8aR)
(6R,8aR) CH2Ph
(6S,8aS) CH2Ph
H
6.5
6.5
6.4
4.8
4.8
4.8
5.3
5.0
5.4
a
Sample concentrations were 1-2 mM, sample temperature
b
was rt. Relationship between amino and carboxyl groups on six-
membered ring of lactam. c Absolute stereochemistry of lactam
moiety. Designations as defined in Figure 2. e Not determined
d
due to line broadening or obscuration by other resonances.
IR Sp ectr oscop y. The NH stretch regions of the IR
spectra of the isomeric monoamino acid conjugates 4b
and 2b in CH2Cl2 are shown in Figure 4. At moderate
concentrations (15-30 mM) both compounds displayed
When DMSO-d6 was used as the solvent for the NMR
experiments, the chemical shifts for both the carboxamide
and carbamate hydrogens of the trans-lactam conjugates
4 and 7 moved appreciably downfield (Table 2). Such
behavior is typical for amides that experience a change
in state of hydrogen bonding when placed into the
strongly hydrogen bond accepting solvent DMSO.10 The
carboxamide proton chemical shifts for the corresponding
cis-lactam isomers, on the other hand, were essentially
identical to their values in CDCl3, indicating that their
hydrogen-bonding state had not been influenced by the
change in solvent.
bands attributable to hydrogen-bonded (3300-3350 cm-1
)
and non-hydrogen-bonded (3400-3500 cm-1) NH stretch-
ing vibrations.14 At lower concentrations (<10 mM) only
the band at 3420 cm-1 was visible in the spectrum of the
trans-mimic conjugate 4b (Figure 4a). The spectrum of
the cis-mimic conjugate 2b displayed hydrogen-bonded
and free NH stretches at 3320 and 3455 cm-1, respec-
tively, at moderate concentrations. Upon dilution, the
relative intensities of the two bands did not change
appreciably; in fact, the hydrogen-bonded NH stretch was
still intense at a concentration of 1 mM (Figure 4b).
Since the intensity of the 3350 cm-1 band for the trans-
mimic conjugate 4b was strongly concentration depend-
ent, the hydrogen bonding observed at high concentra-
tions of this compound was likely due to intermolecular
interaction. The spectral properties of the cis-lactam
isomer 2b, however, were consistent with intramolecular
hydrogen bonding that was unaffected by dilution.
The temperature dependence of the chemical shifts
(∆δ/∆T) of amide hydrogens is often used to assess
intramolecular hydrogen bonding in peptides. When an
amide is involved in a stable intramolecular hydrogen
bond, its hydrogen experiences only a small change in
its chemical shift in DMSO upon variation of tempera-
ture.11 For the trans-mimic conjugates 4 and 7, both
carbamate (NH2) and carboxamide (NH1) hydrogens
displayed sizable values for the temperature gradients
of their chemical shifts (∆δ/∆T < -4.0 ppb/K, Table 2),
indicating that these hydrogens were not involved in
intramolecular hydrogen bonds. The temperature de-
pendence of δ for the cis-mimic conjugates 2 and 6,
however, was uniformly small (∆δ/∆T g -3.0 ppb/K,
Table 2). These values fell within the range indicative
of intramolecularly hydrogen-bonded NH’s, according to
the criterium of Kessler (0 > ∆δ/∆T g -3.0 ppb/K).11
Two-dimensional NMR spectroscopy provided further
evidence for the presence of a stable conformation
incorporating an intramolecular hydrogen bond in the cis-
mimic conjugates. The NOESY spectrum of conjugate
2a displayed crosspeaks between the resonances assigned
to H7â and H3â of the lactam ring and the hydrogen atom
of the angular carboxamide (Figure 3). Crosspeaks
between the amide hydrogen and the H1 or H8 protons,
however, were absent. These results were indicative of
significant population of a stable conformer about the
C8a-CO bond, in which the amide hydrogen of the
angular carboxamide (NH1) was positioned over the six-
membered lactam ring of the mimic.
Discu ssion
To assess the conformational propensities of peptides
incorporating type VI turn mimics, the spectroscopic
features of select amino acid adducts of lactams 1 and 3
were analyzed. The improper stereochemical relation-
ship between the carboxyl and amino groups on the six-
membered ring of the lactam moiety prevented the
adoption of the type VIa â-turn conformation IIa by the
trans lactam conjugates 4 and 7; the γ-turn conformation
IIb, however, was conceivable for both cis- and trans-
lactam conjugates (Figure 1). The absence of intramo-
lecular hydrogen bonding in trans mimic amino acid
conjugates 4 and 7, however, suggested instability of
γ-turn IIb for the trans lactam addcuts. The NMR
spectral properties of monoamino acid mimic conjugates
2 and 6 suggested that their carboxmide hydrogens (NH1,
Figure 2) were involved in a strong intramolecular
(12) Feeney, J . J . Magn. Reson. 1976, 21, 473-481.
(13) Bystrov, V. F.; Arseniev, A. S.; Gavrilov, Y. D. J . Magn. Reson.
1978, 30, 151-160.
(14) Gellman, S. H.; Dado, G. P.; Liang, G.-B.; Adams, B. R. J . Am.
Chem. Soc. 1991, 113, 1164-1166.
(10) Raj, P. A.; Soni, S. D., Ramasubbu, N.; Bhandary, K. K.; Levine,
M. J . Biopolym. 1990, 30, 73-85.
(11) Kessler, H. Angew. Chem., Int. Ed. Engl. 1982, 21, 512-523.

2850 J . Org. Chem., Vol. 62, No. 9, 1997
Kim and Germanas
Ta ble 2. 1H NMR P a r a m eter s for La cta m -Am in o Acid Con ju ga tes in DMSO-d 6
a
∆δ NH1 d,e
CDCl3-DMSO
(ppm)
∆δ NH2 d,e
stereo-
∆δ/∆T NH1
(ppb/K)
CDCl3-DMSO
∆δ/∆T NH2
(ppb/K)
compd
chemistryc
R
(ppm)
cisb
1
2a
2b
2c
transb
3
4a
4b
7
(6R,8aR)
(6R,8aR)
(6R,8aR)
(6S,8aS)
H
-0.4
-0.1
-0.6
-3.0
-2.7
-3.0
-1.9
-1.4
-2.1
-5.3
-6.0
-5.2
CH2Ph
CH2Ph
(6S,8aR)
(6S,8aR)
(6S,8aR)
(6R,8aS)
H
-1.3
-2.1
-2.1
-4.5
-4.7
-4.0
-1.3
-1.6
-1.7
-8.6
-9.0
-9.0
CH2Ph
CH2Ph
a
b
Sample concentrations were 1-2 mM. Relationship between amino and carboxyl groups on six-membered ring of lactam. c Absolute
stereochemistry of lactam moiety. Chemical shift difference for hydrogen in CDCl3 and DMSO-d6. e Designations as defined in Figure
d
2.
F igu r e 2. C and NH numberings in lactam-amino acid
conjugates.
F igu r e 3. Graphical representation of observable NOE’s in
conjugate 2b.
hydrogen bond. Both the â-turn structure IIa and the
γ-turn structure IIb would be consistent with these
results (Figure 1). NOESY spectra and coupling con-
stants, however, supported the â-turn conformation IIa .
The absence of the γ-turn conformation IIb for the trans-
lactam adducts 4 also indirectly discounted the presence
of the γ-turn conformation for analogs 2 and 6 as well,
F igu r e 4. NH stretch region of IR spectrum of select lactam
since the formation of IIb was not dependent on the
amino acid conjugates at 2 mM concentrations in CH2Cl2: (a)
presence of an N-terminal hydrogen-bonding group.
conjugate 4b; (b) conjugate 2b.
According to the spectral properties, the â-turn con-
formation IIa was present in all C-terminal amino acid
adducts of the cis-lactam carboxyl group, regardless of
the amino acid coupled to the carboxyl function (2a vs
2b) or the stereochemical relationship between the bi-
cyclic lactam moiety and the pendant amino acid (2b vs
6). Thus, the positioning of the carboxyl and amino
groups on the six-membered ring of the lactam moiety
of all adducts 2 and 6 was sufficient to induce formation
of the type VIa turn conformation. The low NH chemical
shift temperature dependence and other NMR properties
of conjugates of 1 indicated that the intramolecularly
hydrogen-bonded conformation was highly populated in
solution.
Several mimics of the central dipeptide of other â-turn
types have been designed.15 A number of these have
shown the ability to adopt intramolecular hydrogen-
bonded conformations analogous to IIa .15c,e,h-l,m In cer-
tain cases, the mimic was incorporated into a cyclic
peptide,15c,e,i which restricted the number of possible
conformations. In other cases, the mimics were analyzed
in a non-hydrogen-bonding solvent.15h,j In a few cases
the mimic was dissolved in polar solvent, and in many,

Peptides Constrained to Type VI â-Turns. 1
J . Org. Chem., Vol. 62, No. 9, 1997 2851
directly before use. Tetrahydrofuran (THF) and ether (Et2O)
were distilled just prior to use from sodium/benzophenone, and
dichloromethane (CH2Cl2) was distilled from P2O5. Benzene
was distilled from sodium, and methanol was distilled from
magnesium. Reactions were carried out under a dry nitrogen
atmosphere in glassware that had been flame or oven dried
(T > 100 °C) overnight.
1H and 13C NMR spectra were recorded at 300 or 600 MHz,
using (CH3)4Si as an internal standard. COSY and NOESY
spectra were recorded with 2048 by 512 data points and were
zero filled to 1K × 1K sizes. A mixing time of 350 ms was
used for the NOESY spectra. Chemical shift temperature
dependences were determined in DMSO-d6 by varying the
sample temperature between 278 and 328 K. At least five
temperatures were determined for each sample, and the value
of ∆δ/∆T was obtained by a linear least-squares fit of the data.
Column chromatography was performed on silica gel (Merck
reagents silica gel 60, 230-400 mesh ASTM). Thin-layer
chromatography (TLC) was carried out on Analtech Uniplate
silica gel plates with a 0.25 mm coating containing fluorescent
indicator. Spots were visualized using ninhydrin, phospho-
molybdic acid, iodine, or UV light.
F igu r e 5. Stereoview of the molecular mechanics lowest
energy conformation of 2a .
but not all, of those instances the hydrogen-bonded
conformation was largely absent.15h Stable â-sheets have
been observed in aqueous solution when relatively long
peptide chains were attached to a â-turn template; factors
besides hydrogen bonding, however, likely contribute to
the stability of the secondary structure in this case.15j,m
The intramolecularly hydrogen-bonded conformation for
the cis-mimic adducts 2 and 6, on the other hand,
persisted in DMSO. The stability of conformation IIa
for such peptides should prove useful as a model for
antiparallel â-sheets in polar solvents.
The exceptional stability of the 10-membered-ring
hydrogen-bonded conformation IIa in compounds 2 and
6 likely arises from the unique structural features of
mimic 1. First, the lactam moiety of compound 1
possesses considerable structural homology to the analo-
gous dipeptide from a typical type VI turn. Second, the
mimic 1 incorporates a high degree of conformational
constraint (three of five rotatable bonds restricted).
Third, according to the NMR spectral properties and
molecular mechanics calculations, the bicyclic lactam
moiety of compound 1 is fairly rigid. Fourth, the length
and geometry of the hydrogen bond appears to be close
to ideal. The minimum energy structure of 2a , calculated
with the CHARMM force field, revealed a length of 2.75
Å for the hydrogen bond and a 120° angle between the
COBOC and NHGly bonds (Figure 5). The ability of the
cis-lactam 1 to enhance the stability of more complex
peptide secondary structures, such as â-ladders, will be
reported in due course.
Molecular mechanics calculations and theoretical and struc-
tural analyses were performed with the Quanta package using
the CHARMM force field.
Gen er a l P r oced u r e for Con ju ga tion of 1 (or 3) w ith
Am in o Acid Ester s. A solution of ester 1 or 3 (0.6 g, 1.7
mmol) in EtOH (5 mL) containing 10% Pd/C (10 mg) was
stirred under an atmosphere of hydrogen gas provided by a
balloon fitted with a syringe needle. After 1 h, the solution
was filtered and solvent removed under reduced pressure to
afford the crude carboxylic acid (0.5 g, 100%). To a stirred
solution of the carboxylic acid of compound 1 (or 3) (0.5 g, 1.7
mmol) and BOP reagent (1.0 g, 2.3 mmol) in anhydrous DMF
was added L-amino acid ester hydrochloride (3 equiv) followed
by DIEA (1.9 mL, 10 mmol). After 5 h, the reaction mixture
was concentrated under reduced pressure and extracted with
CHCl3. The organic layer was washed with saturated NaHCO3
NaCl and then dried with MgSO4. Excess DMF was evapo-
rated under reduced pressure to afford the crude product of
conjugated peptidomimetic. Column chromatography on silica
eluting with a solvent mixture of hexane:ethyl acetate:diethyl
ether:ethyl alcohol (1:5:4:0.5) afforded the peptide product,
which was homogeneous by RPLC.
The (6S,8aS)-diastereomers of the conjugates were obtained
by reaction of amino acid anhydrides with the racemic lactams
1 or 3. The individual diastereomers were obtained after
separation by column chromatography, eluting with a solvent
mixture of hexane:ethyl acetate:diethyl ether:ethyl alcohol (1:
5:4:0.5).
(6R ,8a R )-N -(t er t -Bu t oxyca r b on yl)-6-a m in o-8a -ca r -
boxyin d olizin -5-on e-glycin e m eth yl ester (2a ): yield 84%;
1H NMR (CDCl3) δ 8.23 (br s, 1H), 5.33 (d, J ) 6.9 Hz, 1H),
4.01 (d, J ) 7.2 Hz, 2H), 3.78 (m, 1H), 3.71 (s, 3H), 3.56 (m,
2H), 2.73 (dd, J ) 6.0, 11.7 Hz, 1H), 2.50 (ddd, J ) 3.3, 3.3,
13.8 Hz, 1H), 2.36 (m, 1H), 2.15-1.87 (m, 3H), 1.63-1.50 (m,
2H), 1.40 (s, 9H); 13C NMR (CDCl3) δ 174.1, 170.6, 168.7, 156.1,
80.8, 71.7, 52.5, 52.6, 46.3, 41.9, 38.7, 31.7, 28.8, 26.6, 21.4;
HR FAB MS calcd for C17H27N3O6 369.1825, found 369.1829.
Exp er im en ta l Section
Gen er a l Meth od s. Unless otherwise noted, all starting
materials and solvents were obtained from commercial sup-
pliers and used without further purification. Dimethylform-
amide (DMF) was distilled from CaH2 under reduced pressure
(6R ,8a R )-N -(t er t -Bu t oxyca r b on yl)-6-a m in o-8a -ca r -
boxyin d olizin -5-on e-L-p h en yla la n in e m eth yl ester (2b):
yield: 67%; 1H NMR (CDCl3) δ 8.28 (d, J ) 8.4 Hz, 1H), 7.28-
7.20 (m, 5H), 5.39 (d, J ) 7.2 Hz, 1H), 4.68 (ddd, J ) 3.9, 8.1,
12.0 Hz, 1H), 3.75 (s, 3H), 3.70 (m, 1H), 3.55 (m, 2H), 3.37
(dd, J ) 4.2, 13.8 Hz, 1H), 2.97 (m, 1H), 2.57 (dd, J ) 6.3,
11.7 Hz, 1H), 2.40 (ddd, J ) 2.7, 2.7, 13.8 Hz, 1H), 1.98 (m,
1H), 1.73-1.52 (m, 2H), 1.48 (s, 9H), 1.42-1.23 (m, 2H), 1.05
(m, 1H); 13C NMR (CDCl3) δ 173.6, 172.1, 168.5,156.0, 137.7,
129.7, 129.4, 127.3, 80.7, 71.5, 54.8, 52.6, 46.0, 38.3, 37.0, 31.6,
30.1, 28.9, 25.7, 21.1.
(15) (a) Kemp, D. S.; Sun, E. T. Tetrahedron Lett. 1982, 23, 3759-
3760. (b) Nagai, U.; Sato, K. Tetrahedron Lett. 1985, 26, 647-650. (c)
Feigel, M. J . Am. Chem. Soc. 1986, 108, 181-183. (d) Kemp, D. S.;
Stites, W. E. Tetrahedron Lett. 1988, 29, 5057-5060. (e) Brandmeier,
V.; Feigel, M.; Bremer, M. Angew. Chem., Int. Ed. Engl. 1989, 28, 486-
488. (f) Ernst, I.; Kalvoda, J .; Rihs, G.; Mutter, M. Tetrahedron Lett.
1990, 28, 4011-4014. (g) Diaz, H.; Kelly, J . W. Tetrahedron Lett. 1991,
32, 5725-5728. (h) Genin, M. J .; J ohnson, R. L. J . Am. Chem. Soc.
1992, 114, 8778-8783. (i) Ripka, W. C.; DeLucca, G. V.; Bach, A. C.;
Pottorf, R. S.; Blane, J . M. Tetrahedron 1993, 49, 3593-3608. (j) Diaz,
H.; Tsang, K. Y.; Choo, D.; Espina, J . R.; Kelly, J . J . Am. Chem. Soc.
1993, 115, 3790-3791. (k) Gramberg, D.; Robinson, J . A. Tetrahedron
Lett. 1994, 35, 861-864. (l) Kemp, D. S.; Li, Z. Q. Tetrahedron Lett.
1995, 36, 4175-4178. (m) Schneider, J . P.; Kelly, J . W. J . Am. Chem.
Soc. 1995, 117, 2533-2546. (n) Cornille, F.; Slomczynska, U.; Smythe,
M. L.; Beusen, D. D.; Moeller, K. D.; Marshall, G. R. J . Am. Chem.
Soc. 1995, 117, 909-917.
(6R,8a S)-N-(ter t-Bu toxyca r bon yl)-6-a m in o-8a -ca r boxy-
1
in d olizin -5-on e-glycin e m eth yl ester (4a ): yield 80%; H
NMR (CDCl3) δ 6.72 (br s, 1H), 5.41 (d, J ) 5.7 Hz, 1H), 4.10
(m, 2H), 3.96 (dd, J ) 5.1, 17.7 Hz, 1H), 3.81 (m, 1H), 3.75 (s,
3H), 3.58 (m, 1H), 2.58-2.49 (m, 2H), 2.40 (m, 1H), 1.97-1.85

2852 J . Org. Chem., Vol. 62, No. 9, 1997
Kim and Germanas
(m, 5H), 1.44 (s, 9H); 13C NMR (CDCl3) δ 174.1, 171.1, 170.4,
155.6, 79.78, 70.4, 52.4, 49.7, 46.73, 41.3, 39.2, 30.3, 28.35, 26.8,
21.5.
The (6S,8aS)-diastereomers of the conjugates were obtained
by reaction of amino acid anhydrides with the racemic lactam
1 followed by diastereomer separation by silica gel chroma-
tography eluting with hexane:ethyl acetate:diethyl ether:ethyl
alcohol (1:7:6:0.5).
(6R,8a R)-N-[(ter t-Bu toxyca r bon yl)glycyl]-6-a m in o-8a -
ca r boxyin d olizin -5-on e ben zyl ester (5a ): yield 80%; IR
(CHCl3), 3650, 2980, 1738, 1700, 1650 cm-1; 1H NMR (CDCl3)
δ 7.38-7.29 (m, 5H), 6.72 (d, J ) 7.5 Hz, 1H), 5.18 (s, 2H),
5.15 (br s, 1H), 4.30 (ddd, J ) 11.4, 6.3, 6.3 Hz, 1H), 3.90 (dd,
J ) 4.8, 14.4 Hz, 1H), 3.78 (dd, J ) 5.7, 14.4 Hz, 1H), 3.62-
3.52 (m, 2H), 2.54 (ddd, J ) 14.1, 3.3, 3.3 Hz, 1H), 2.45 (m,
2H), 1.89 (m, 1H), 1.80-1.67 (m, 3H), 1.52 (m, 1H), 1.45 (s,
9H); 13C NMR (CDCl3) δ 172.8, 169.9, 167.5, 154.3, 135.1,
128.7, 128.3, 80.0, 70.1, 67.6, 50.7, 45.1, 44.0, 37.5, 30.6, 28.3,
26.0, 20.6; HR FAB MS calcd for C23H32N3O6 446.2291, found
446.2293.
(6a S ,8a R )-N -(t er t -Bu t oxyca r b on yl)-6-a m in o-8a -ca r -
boxyin d olizin -5-on e-L-p h en yla la n in e m eth yl ester (4b):
yield 75%; 1H NMR (CDCl3) δ 7.30-7.21 (m, 3H), 7.14 (d, J )
6.6 Hz, 2H), 6.28 (d, J ) 8.4 Hz, 1H), 5.42 (d, J ) 4.5 Hz, 1H),
4.84 (ddd, J ) 5.1, 8.7, 8.7 Hz, 1H), 4.07 (dd, J ) 6.0, 12.6 Hz,
1H), 3.82 (s, 3H), 3.71 (m, 1H), 3.30 (dd, J ) 5.4, 14.4 Hz, 1H),
3.19 (m, 1H), 2.96 (dd, J ) 8.8, 14.1 Hz, 1H), 2.53-2.19 (m,
3H), 1.72-1.62 (m, 4H), 1.43 (s, 9H), 1.23 (m, 1H); 13C NMR
(CDCl3) δ 172.7, 171.6, 170.4, 155.4, 136.1, 128.6, 128.7, 127.7,
79.6, 70.5, 53.0, 52.6, 49.4, 46.5, 38.8, 37.1, 30.2, 28.3, 26.7,
21.0.
(6S,8a S)-N-(ter t-Bu toxyca r bon yl)-6-a m in o-8a -ca r boxy-
in d olizin -5-on e-L-p h en yla la n in e m eth yl ester (6): yield
1
67%; H NMR (CDCl3) δ 7.36 (d, J ) 6.6 Hz, 1H), 7.21-7.11
(m, 5H), 5.06 (d, J ) 5.4 Hz, 1H), 4.72 (dd, J ) 7.8, 14.1 Hz,
1H), 3.73 (s, 3H), 3.65 (m, 1H), 3.54 (m, 1H), 3.46 (m, 1H),
3.27 (dd, J ) 6.0, 14.1 Hz, 1H), 3.07 (dd, J ) 8.4, 14.1 Hz,
1H), 2.60 (dd, J ) 6.0, 11.7 Hz, 1H), 2.39 (ddd, J ) 3.3, 3.3,
13.8 Hz, 1H), 1.89-1.76 (m, 2H), 1.39 (s, 9H), 1.65-1.52 (m,
3H); 13C NMR (CDCl3) δ 172.3, 171.2, 168.2, 155.3, 136.0,
128.5, 128.2, 128.1, 126.6, 79.7, 70.8, 53.4, 51.8, 51.8, 45.4, 38.1,
36.7, 30.4, 28.0, 24.9, 20.4.
(6a R ,8a S )-N -(t er t -Bu t oxyca r b on yl)-6-a m in o-8a -ca r -
boxyin d olizin -5-on yl-L-p h en yla la n in e m eth yl ester (7):
yield 75%; 1H NMR (CDCl3) δ 7.30-7.21 (m, 3H), 7.10 (d, J )
6.6 Hz, 2H), 6.37 (d, J ) 8.4 Hz, 1H), 5.37 (d, J ) 4.8 Hz, 1H),
4.80 (ddd J ) 5.7, 8.7, 8.7 Hz, 1H), 3.75 (s, 3H), 3.69 (m, 2H),
3.39 (m, 1H), 3.18 (dd, J ) 5.4, 13.8 Hz, 1H), 3.03 (dd, J )
7.8, 13.8 Hz, 1H), 2.47 (m, 1H), 2.29 (m, 1H), 2.04 (m, 1H),
1.87-1.75 (m, 4H), 1.51 (m, 1H), 1.46 (s, 9H), 1.23 (m, 1H);
13C NMR (CDCl3) δ 173.3, 172.1, 170.7, 155.9, 135.9, 129.6,
129.4,129.4, 127.9, 80.2, 70.7, 53.3, 52.9, 50.2, 46.9, 39.5, 37.9,
30.2, 28.8, 26.9, 21.6.
(6R,8a R)-N-(ter t-Bu t oxyca r b on yl)-L-p h en yla la n yl-6-
a m in o-8a -ca r boxyin d olizin -5-on e ben zyl ester (5b): yield
80%; 1H NMR (CDCl3) δ 7.41-7.12 (m, 10H), 6.35 (d, J ) 5.1
Hz, 1H), 5.17 (d, J ) 14.8 Hz, 1H), 5.07 (d, J ) 14.8 Hz, 1H),
5.00 (br s, 1H), 4.47 (m, 1H), 4.38 (m, 1H), 3.68-3.51 (m, 3H),
3.19 (dd, J ) 8.4, 14.7 Hz, 1H), 3.02 (m, 1H), 2.62-2.40 m,
3H), 1.97-1.60 (m, 3H), 1.43 (s, 9H), 1.23 (m, 1H); 13C NMR
(CDCl3) δ 173.3, 171.9, 167.7, 137.2, 135.6, 129.7, 129.1, 128.9,
128.6, 127.2, 80.5, 70.6, 68.0, 56.3, 51.0, 45.5, 38.0, 31.0, 28.7,
26.3, 21.0.
(6S,8a S)-N-(ter t-Bu t oxyca r b on yl)-L-p h en yla la n yl-6-
a m in o-8a -ca r boxyin d olizin -5-on e ben zyl ester (8): yield
80%; 1H NMR (CDCl3) δ 7.40-7.18 (m, 10H), 6.41 (br s, 1H),
5.17 (s, 2H), 4.95 (br s, 1H), 4.38 (m, 1H), 4.21 (m, 1H), 3.61-
3.55 (m, 3H), 3.14 (dd, J ) 8.4, 14.7 Hz, 1H), 3.06 (m, 1H),
2.51-2.42 (m, 3H), 1.91-1.57 (m, 3H), 1.39 (s, 9H), 1.23 (m,
1H); 13C NMR (CDCl3) δ 173.3, 171.9, 167.7, 137.2, 135.6,
129.7, 129.1, 128.9, 128.6, 127.2, 80.5, 70.6, 68.0, 56.3, 51.0,
45.5, 38.0, 31.0, 28.7, 26.3, 21.0.
Gen er a l P r oced u r e for th e Con ju ga tion of 1 w ith
An h yd r id es of N-BOC-a m in o Acid s. To a solution of ester
1 (0.21 g, 0.59 mmol) in CH2Cl2 (10 mL) was added trifluoro-
acetic acid (1 mL, 5.9 mmol) and the solution was stirred at
rt. After 2 h, CH2Cl2 and trifluoroacetic acid were removed
under reduced pressure to give a yellowish oil. The resultant
amine salt was dissolved in THF (10 mL) and Et3N (0.5 mL).
The preformed anhydride of the N-BOC amino acid (2 equiv)
was dissolved in THF (5 mL) and cooled with an ice bath. To
the solution of the anhydride was added the amino acid salt
solution dropwise and the mixture allowed to stir at 4 °C. After
2 h, the reaction mixture was warmed to rt and quenched with
water. The resulting mixture was diluted with CHCl3 and
washed with 0.1 N HCl. The organic layer was dried with
MgSO4 and evaporated under reduced pressure to give the
amides as colorless oils. Individual diastereomers were sepa-
rated by silica gel column chromatography, eluting with
hexane:ethyl acetate:diethyl ether:ethyl alcohol (1:7:6:0.5).
Ack n ow led gm en t. We are grateful to the R. A.
Welch Foundation, the American Heart Association-
Texas Affiliate (Grant 95G-448), American Cyanamid,
and the University of Houston for financial support.
Mass spectrometry was provided by the Washington
University Mass Spectrometry Resource, an NIH Re-
search Resource (Grant No. P41RR0954).
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra of
2a , 4a , 5a ,b, 6, and 8, 13C spectra of 2b, 4b, and 7, and NOESY
spectrum of 2a (10 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 O961180R