The structure and conformation of the tryptophanyl diketopiperazines cyclo(Trp-Trp)·C2H6SO and cyclo(Trp-Pro)

Article abstract of DOI:10.1023/A:1009567127868

The structure and conformation of the cyclic dipeptides [cyclo(L-Trp-L-Trp)·C₂H₆SO] and cyclo(L-Trp-L-Pro) have been investigated with X-ray crystallographic and spectroscopic methods. Cyclo(L-tryptophanyl-L-tryptophanyl)·DMSO solvate crystallized in the space group P2₁2₁2₁ with cell dimensions a = 6.193(2), b = 11.545(3), c = 31.117(4) A. The crystal structure is stabilized by four hydrogen bonds (three intermolecular hydrogen bonds and one intramolecular bond). The first intermolecular bond is between the oxygen of DMSO and the nitrogen of indole ring 2, in contrast to the second intramolecular hydrogen bond between the nitrogen of indole ring 1 and the oxygen of DMSO. The two remaining intermolecular hydrogen bonds are between the nitrogens of the DKP ring and the carbonyl oxygens of the DKP ring. The values of X^1A₁ (-45.764) and X^1A₂ (67.437) indicate an extended side chain conformation for Trp residue 1 (E_N) and a folded conformation for Trp residue 2. The DKP ring is more planar than in other cyclic dipeptide compounds (φ₁ = 11.414, ψ₁ = -7.516, φ₂ = 12.471, and ψ₂ = -8.256). In cyclo(L-Trp-L-Trp) the Cβ resonance of L-tryptophan (29.88 ppm) is shifted upfield 0.82 ppm when compared with the same resonance in cyclo(L-Trp-L-Gly) (30.7 ppm) and cyclo(L-Leu-L-Trp) (30.7 ppm). Two conformations of cyclo(Trp-Pro) crystallized in the space group P1 with cell dimensions a = 5.422(1), b = 9.902(1), c = 13.443(2) A, α = 80.42(1), β = 78.61(1), and γ = 89.13(1)°. The conformation of the backbone and the orientation of the aromatic side chains for these conformers are very similar. The DKP rings for both conformers adopt a typical boat conformation in contrast to the flattened chair conformation observed for cyclo(Tyr-Pro) and cyclo(Phe-F-Pro). The tryptophan side chains of these conformers are folded towards the diketopiperazine (DKP) ring. The pyrrolidine ring for conformer 1 can be described as an envelope (Cs-Cβ-endo) conformation in contrast to the pyrrolidine ring symmetry for conformer 2 which is an intermediate between C_s and C₂ rather than pure C_s for the proline ring with Cβ-endo and Cγ-exo with respect to C′. The two prolyl rings are puckered at the β-carbon atoms which deviate from the best planes defined by the four remaining atoms. The crystal structures are stabilized by four intermolecular hydrogens bonds. An intermolecular bond between the nitrogen of the indole ring (conformer 1) and the carbonyl oxygen of the DKP ring (conformer 2) was observed. The second hydrogen bond is between the nitrogen of the indole ring (conformer 2) and the carbonyl oxygen of the DKP ring (conformer 1). The last two hydrogens involve the carbonyl oxygens of the DKP rings and the nitrogens of the DKP rings [carbonyl oxygen of DKP ring (conformer 1) - nitrogen of DKP ring (conformer 2); nitrogen of DKP ring (conformer 1) - carbonyl oxygen of DKP ring (conformer 2)].

Full text of DOI:10.1023/A:1009567127868

Journal of Chemical Crystallography, Vol. 29, No. 4, 1999
The structure and conformation of the tryptophanyl
diketopiperazines cyclo(Trp–Trp)ؒC2H6SO
and cyclo(Trp–Pro)
G.D. Grant,(1) A.L. Hunt,(1) P.J. Milne,(1)* H.M. Roos,(2) and J.A. Joubert(3)
Received August 6, 1998
The structure and conformation of the cyclic dipeptides [cyclo(L-Trp–L-Trp)иC2H6SO] and
cyclo(L-Trp–L-Pro) have been investigated with X-ray crystallographic and spectroscopic
methods. Cyclo(L-tryptophanyl-L-tryptophanyl)иDMSO solvate crystallized in the space
˚
group P212121 with cell dimensions a ϭ 6.193(2), b ϭ 11.545(3), c ϭ 31.117(4) A. The crystal
structure is stabilized by four hydrogen bonds (three intermolecular hydrogen bonds and
one intramolecular bond). The first intermolecular bond is between the oxygen of DMSO
and the nitrogen of indole ring 2, in contrast to the second intramolecular hydrogen bond
between the nitrogen of indole ring 1 and the oxygen of DMSO. The two remaining intermo-
lecular hydrogen bonds are between the nitrogens of the DKP ring and the carbonyl oxygens
1A
1A
of the DKP ring. The values of ␹ (Ϫ45.764) and ␹ (67.437) indicate an extended side
1
2
chain conformation for Trp residue 1 (EN) and a folded conformation for Trp residue 2. The
DKP ring is more planar than in other cyclic dipeptide compounds (␸ ϭ 11.414, ⌿1 ϭ
1
Ϫ7.516, ␸ ϭ 12.471, and ⌿2 ϭ Ϫ8.256). In cyclo(L-Trp–L-Trp) the Cͱ resonance of
2
L-tryptophan (29.88 ppm) is shifted upfield 0.82 ppm when compared with the same resonance
in cyclo(L-Trp–L-Gly) (30.7 ppm) and cyclo(L-Leu–L-Trp) (30.7 ppm). Two conformations
of cyclo(Trp–Pro) crystallized in the space group P1 with cell dimensions a ϭ 5.422(1),
˚
b ϭ 9.902(1), c ϭ 13.443(2) A, Ͱ ϭ 80.42(1), ͱ ϭ 78.61(1), and Ͳ ϭ 89.13(1)Њ. The conformation
of the backbone and the orientation of the aromatic side chains for these conformers are
very similar. The DKP rings for both conformers adopt a typical boat conformation in
contrast to the flattened chair conformation observed for cyclo(Tyr–Pro) and cyclo(Phe–F-
Pro). The tryptophan side chains of these conformers are folded towards the diketopiperazine
(DKP) ring. The pyrrolidine ring for conformer 1 can be described as an envelope (Cs–Cͱ-
endo) conformation in contrast to the pyrrolidine ring symmetry for conformer 2 which is
an intermediate between Cs and C2 rather than pure Cs for the proline ring with Cͱ-endo
and CͲ-exo with respect to CЈ. The two prolyl rings are puckered at the ͱ-carbon atoms
which deviate from the best planes defined by the four remaining atoms. The crystal structures
are stabilized by four intermolecular hydrogens bonds. An intermolecular bond between the
nitrogen of the indole ring (conformer 1) and the carbonyl oxygen of the DKP ring (conformer
2) was observed. The second hydrogen bond is between the nitrogen of the indole ring
(conformer 2) and the carbonyl oxygen of the DKP ring (conformer 1). The last two hydrogens
involve the carbonyl oxygens of the DKP rings and the nitrogens of the DKP rings [carbonyl
oxygen of DKP ring (conformer 1)––––nitrogen of DKP ring (conformer 2); nitrogen of
DKP ring (conformer 1)––––––carbonyl oxygen of DKP ring (conformer 2)].
KEY WORDS: Cyclic dipeptides; Tryptophan; Proline; x-rays, NMR.
(1) Department of Pharmacy, University of Port Elizabeth, Box
1600, Port Elizabeth, 6000, Republic of South Africa.
(2) Department of Chemistry, University of Pretoria, Pretoria, 0001,
Republic of South Africa.
(3) Department of Chemistry, Potchefstroom University of C.H.E.,
Potchefstroom, 2520, Republic of South Africa.
* To whom correspondence should be addressed.
435
1074-1542/99/0400-0435$16.00/0  1999 Plenum Publishing Corporation

436
Grant, Hunt, Milne, Roos, and Joubert
Introduction
Proline is an important imino acid of many pro-
teins and neuropeptides and imposes certain confor-
mational restraints on these biomolecules.10,11 In addi-
tion, the conformational aspects of the pyrrolidine
ring system are of particular interest as they reveal
different modes of puckering of the five-membered
ring system.12 Second, proline is the only residue
which leads to an N-alkylamide bond when incorpo-
rated into a peptide via natural biochemical path-
ways. Numerous peptides with important biological
activity (e.g., didemnin and cyclosporin) contain
N-methyl amino acids.13 Thirdly, the cis–trans iso-
merism of the N-alkylamide bond involving the
amino group of proline has been implicated in the
biological activity of peptides. Brandl and Deber14
have proposed that cis–trans isomerism of proline
residues might play a role in transduction of trans-
membrane proteins.
Cyclic dipeptides, both in the solid state and
in solution, are extensively used as suitable models
to gain information on more complex peptides
and proteins. Due to their relative simplicity 2,5-
diketopiperazines (DKP’s) are excellent models for
theoretical studies and are of primary interest for
comparison with experimental findings obtained
through X-ray experiments and NMR studies. The
properties of DKP’s differ from those of ordi-
nary peptides:
* They do not exist as zwitterions and are often neu-
tral compounds.1
* The simpler members of this group are water
soluble.1
* They provoke the destruction of the secondary
globular protein structure.2
In continuation of our interest in conformation-
ally restricted small peptides containing the prolyl
residue and/or the aromatic amino acid residue, and
as part of our studies on the biological activity of
selected cyclic dipeptides, we report here the synthe-
sis, conformational and spectroscopic properties of
* They are sensitive to oxidation, especially when
imino acid residues are the building blocks.3
* They may act as powerful hydrolytic catalysts.4
* They are of interest in studies on the thermody-
namic behavior of non-ionic compounds in aque-
ous medium because they have the ability of form-
ing hydrogen bonds with the solvent (via the two
cis-amide groups in the ring) and of giving rise
to hydrophobic interactions (determined by the R
substituents).5
cyclo(L-Trp–L-Trp)
(Scheme 1).
and
cyclo(L-Trp–L-Pro)
Experimental
* Many derivatives show antiviral properties while
others are powerful antibiotics, and anti-tumor
agents.6,7,8
All reagents and solvents were of reagent grade
and used without further purification.
The inclusion/incorporation of essential aro-
matic amino acids offers a model system of limited
complexity for studying the influence of solvents and
solvent mixtures on intramolecular interactions dur-
ing the excited lifetime of the chromophore.9 Trypto-
phan contains an indole ring (a benzene ring fused
to a pyrrole ring) which is found in many pharmaco-
logically active compounds, including hallucinogens
and other drugs which have mental or emotional ef-
fects, e.g., LSD (Lysergic acid diethylamide), Psilocy-
bin, DMT (Dimethyltryptamine), Harmaline, and
Strychnine.
Synthesis of cyclo(Trp–Trp)
Triethylamine (0.96 ml, 6.89 mmol) and diethyl-
phosphoryl cyanide (0.52 ml, 3.61 mmol) were added
The therapeutic uses of drugs containing an
indole ring that are available on the market today
range from anti-emetics, and anti-inflammatories
to the treatment of hypertension, migraine, and
Parkinson’s disease (e.g., Bromocriptine, Ergota-
mine, Indapamide, Indomethacin, Sumatriptan, and
Ondansetron).
Scheme 1.

Structure of cyclo(Trp–Trp)ؒC2H6SO and cyclo(Trp–Pro)
437
Table 1. Crystal Data and Summary of Intensity Data Collection and Structure Refinement of
cyclo(Trp–Trp) and cyclo(Trp–Pro)
Compound
CCDC deposit no.
Color/shape
C22H20O2N4·C2H6SO
CCDC-1003/5497
Colorless/parallelepiped
450.55
P212121
18
C16H17O2N3
CCDC-1003/5498
Colorless/parallelepiped
283.33
P1
Formula weight
Space group
Temp., ЊC
18
Cell constantsa
˚
a, A
6.193(2)
11.545(3)
31.117(4)
5.422(1)
9.902(1)
13.443(2)
78.61(1)
697.5
˚
b, A
˚
c, A
ͱ, deg
Cell volume, A
3
˚
2224.9
Formula units/unit cell
4
1.34
1.38
2
1.35
0.53
Dcalc, g cmϪ3
Ȑcalc, cmϪ1
Max. crystal dim., mm
Reflections measured
2␪ range, deg
Range of h, k, l
Reflections observed
[Ͼ2␴(1)]b
0.40 ϫ 0.08 ϫ 0.13
2819
3 Յ ␪ Յ 27
Ϫ7, Ϫ14, Ϫ39
0.57 ϫ 0.15 ϫ 0.30
4444
3 Յ ␪ Յ 30
0.7, Ϫ13.13, Ϫ18.18
1268
2786
Diffractometer/scan
Radiation, graphite
monochromator
Computer programsc
Structure solution
No. of parameters varied
Weights
Enraf-Nonius CAD-4/Ͷ-2␪
Enraf-Nonius CAD-4/Ͷ-2␪
MoKͰ(␭ ϭ 0.71073)
SHELX76
MoKͰ(␭ ϭ 0.71073)
SHELX76
SHELX86, SHELX76
224
SHELX86, SHELX76
377
␴
Ϫ2(Fobs
)
␴ )
Ϫ2(Fobs
R ϭ ⌺͉͉Fo͉Ϫ ͉Fc͉͉⌺͉Fo͉
Rw
0.1199
0.0544
0.0739
0.0386
Largest feature
final diff. map
Ϫ3
Ϫ3
1.11eϪ
A
0.410eϪ
˚
˚
A
Ϫ1.14eϪ
Ϫ0.383eϪ
Ϫ3
Ϫ3
˚
A
˚
A
a Least-squares refinement of [(sin ␪)/␭]2 values for 25 reflections ␪ Ͼ 20 deg.
b Corrections: Lorentz-polarization.
c Neutral scattering factors and anomalous dispersion corrections.
* Ellipsoids were at a 50% level.
to a stirred solution of N-t-Boc–L-Trp (1 g, 3.28
mmol) and L-Trp–OMe (0.84 g, 3.28 mmol) in 1,2-
dimethoxyethane (40 ml) at 0ЊC. After 1 h at 0ЊC
and 4 h at room temperature the reaction mixture
was diluted with chloroform (250 ml) and washed
successively with 5% hydrochloric acid (50 ml), aque-
ous sodium hydrogen carbonate (50 ml), and satu-
rated brine (50 ml). Removal of the solvent in vacuo
furnished the protected product as a colorless
syrup [Rf ϭ 0.66, chloroform–methanol–acetic acid
(14 : 2 : 1)].
The N–t-Boc dipeptide ester was dissolved in
formic acid (20 ml, 98%) containing anisole (0.2 ml)
and stirred for 3 h at room temperature. After re-
moval of the excess formic acid in vacuo, the residue
containing the crude dipeptide ester formate was re-
fluxed in 40 ml of sec-butanol and toluene (4 : 1) for
3 h at 120ЊC. After concentrating the solution to 8
ml and cooling to 0ЊC, the product was filtered off
and recrystallized from a suitable solvent. [yield
70%; Rf ϭ 0.55, chloroform–methanol–acetic acid
(14 : 2 : 1)].
Synthesis of cyclo(Trp–Pro)
Triethylamine (0.96 ml, 6.89 mmol) and diethyl-
phosphoryl cyanide (0.55 ml, 3.61 mmol) were added
to a stirred solution of N–t-Boc–L-Trp (1 g, 3.28
mmol) and L–Pro–NH2 (0.37 g, 3.28 mmol) in 1,2-
dimethoxyethane (40 ml) at 0ЊC. After 1 h at 0ЊC
and 4 h at room temperature, the reaction mixture

438
Grant, Hunt, Milne, Roos, and Joubert
Table 2. Fractional Atomic Coordinates (ϫ104) and Equivalent
crude dipeptide formate salt and the mixture was
stirred for 3 days at low temperature (5–8ЊC). The
cyclic dipeptide cyclo(Trp–Pro) was extracted with
chloroform (4 ϫ 30 ml) and crystals were grown from
2
3
˚
Thermal Factors (A ϫ 10 ) for cyclo (Trp–Trp)·DMSO
a
Atom
x/a
y/b
3(8)
794(11)
272(12)
z/c
Ueq
chloroform-n-hexane [Rf
ϭ 0.58, chloroform–
N(1)
C(1)
C(2)
O(1)
N(2)
C(3)
C(4)
O(2)
C(5)
C(6)
C(7)
5415(17)
7224(22)
9409(23)
10953(17)
9436(20)
7726(21)
5468(23)
3925(15)
7393(23)
5361(25)
4213(25)
2352(22)
2480(26)
1173(25)
1692(27)
3521(27)
4802(24)
4341(24)
8241(21)
8253(26)
9854(24)
9318(26)
7363(32)
6274(27)
4320(29)
3602(31)
4652(25)
6727(25)
826(19)
Ϫ2150(3)
Ϫ2134(4)
Ϫ2245(5)
Ϫ2272(4)
Ϫ2295(3)
Ϫ2232(5)
Ϫ2184(5)
Ϫ2187(3)
Ϫ1677(4)
Ϫ1491(5)
Ϫ1212(5)
Ϫ1078(4)
Ϫ1269(5)
Ϫ1233(5)
Ϫ1480(4)
Ϫ1742(5)
Ϫ1767(4)
Ϫ1520(4)
Ϫ1851(4)
Ϫ1430(5)
Ϫ1273(5)
Ϫ853(4)
Ϫ738(6)
Ϫ364(5)
Ϫ336(5)
Ϫ664(5)
Ϫ1047(5)
Ϫ1106(5)
Ϫ249(3)
172(2)
28(3)
26(4)b
40(4)
60(3)
31(3)b
31(3)
29(3)
41(3)
42(4)
41(4)
44(4)
43(4)b
38(4)b
48(4)b
57(5)b
58(5)b
39(4)b
31(4)b
35(3)
38(4)b
46(4)
68(4)
55(5)b
58(5)b
60(5)b
70(6)b
47(5)b
42(4)
65(3)
132(3)
83(5)
110(6)
methanol–acetic acid (14 : 2 : 1); Rf ϭ 0.19, isopropyl
ether–chloroform–acetic acid (6 : 3 : 1)].
925(9)
Ϫ881(9)
Ϫ1703(11)
Ϫ1160(12)
Ϫ1799(8)
1338(12)
1755(12)
1163(12)
1777(11)
2847(11)
3806(12)
4763(12)
4819(13)
3895(12)
2884(11)
Ϫ2513(11)
Ϫ1934(12)
Ϫ1229(11)
Ϫ849(11)
Ϫ1287(14)
Ϫ1174(13)
Ϫ1763(12)
Ϫ2438(13)
Ϫ2549(12)
Ϫ1962(12)
899(9)
X-ray analysis
All diffraction measurements were obtained at
room temperature and the data was collected with
an Enraf Nonius CAD4 diffractometer with MoKͰ
N(3)
C(8)
C(9)
˚
radiation (Graphite monochromator, ␭ ϭ 0.7107 A).
Accurate unit cell parameters were obtained by least-
squares methods from the position of 25 centered
reflections for each crystal. There was no significant
crystal decay and intensities were corrected for ab-
sorbtion, as well as Lorentz and polarization effects.
An empirical method for absorption correction was
applied.15 Standard intensity checks and orientation
control were carried out. The structures were solved
by Patterson and direct methods.16,17 Refinement was
by full matrix least-squares methods, using ␴Ϫ2 (Fobs)
weights.17 All the nonhydrogen atoms for cyclo(Trp–
Trp) and cyclo(Trp–Pro) were refined anisotropi-
cally. Atomic scattering factors were taken from
the literature.18
Diffraction quality crystals of cyclo(Trp–Trp)
were obtained by slow cooling of a hot DMSO solu-
tion. The crystals crystallized from DMSO in the
space group P212121 and are listed with other relevant
crystal data in Table 1. Due to the low ratio of number
of reflections: number of parameters, a selection of
atoms were refined anisotropically. All hydrogen
atoms, except the experimentally located and refined
HN3, were placed in calculated positions and were
included in the refinement with common isotropic
thermal parameters. Fractional coordinates and
equivalent thermal factors, and the relevant torsion
angles for cyclo(Trp–Trp) are listed in Table 2 and
Table 3, respectively.
C(10)
C(11)
C(12)
C(13)
C(14)
C(15)
C(16)
N(4)
C(17)
C(18)
C(19)
C(20)
C(21)
C(22)
O(3)
S(1)
1888(15)
1463(30)
971(33)
1014(5)
2314(12)
66(12)
C(23)
C(24)
408(5)
543(5)
a
* *
Ueq ϭ 1/3 ⌺i ⌺j Uijai aj (ai · aj).
b Isotropic temperature factor.
was diluted with ethylacetate (250 ml) and washed
successively with 5% hydrochloric acid (50 ml), aque-
ous sodium hydrogen carbonate (50 ml), and satu-
rated brine (50 ml). Removal of the solvent in vacuo
furnished the protected product as a colorless
syrup [Rf ϭ 0.68, chloroform–methanol–acetic acid
(14 : 2 : 1)].
Diffraction quality crystals of cyclo(Trp–Pro)
crystallized from chloroform-n-hexane in the space
group P1 and are listed with other relevant data in
Table 1. All hydrogen atoms were placed in calcu-
lated positions and were included in the refinement
with common isotropic thermal parameters.
Perspective views of the molecules, prepared
with ORTEP,19 are represented in Figs. 1 and 2, illus-
trating the crystallographic numbering schemes used.
Colorless crystals of the protected dipeptide
crystallized from ethylacetate-n-hexane
The N–t-Boc–dipeptide (0.2 g, 1.24 mmol) was
dissolved in formic acid (20 ml, 98%) containing ani-
sole (0.2 ml) and stirred for 3 h at room temperature.
After removal of the excess formic acid in vacuo,
saturated NaHCO3 (50 ml) was added to the above

Structure of cyclo(Trp–Trp)ؒC2H6SO and cyclo(Trp–Pro)
439
Table 3. The Relevant Torsion Angles of cyclo(Trp–Trp)·DMSO
Torsion angle
C4
C2
N1
N2
C1
C1
N1
N2
C1
C3
C2
N1
C1
C3
C2
C4
N2
C4
C2
C4
N2
N1
C3
C3
␸
␸
11.414(1.881)
12.471(1.931)
Ϫ7.516(1.869)
Ϫ8.256(1.806)
Ϫ4.259(2.278)
Ϫ3.107(1.911)
1
2
⌿
⌿
1
2
Ͷ1
Ͷ2
Indole residue 1 or
(Trp side chain 1)
Indole residue 2 or
(Trp side chain 2)
N1(N2)a
C2(C4)
C1(C3)
C1(C3)
C1(C3)
C5(C14)
C5(C14)
C6(C15)
C6(C15)
C7(C16)
C7(C16)
C13(C22)
C13(C22)
N3(N4)
C13(C22)
C13(C22)
C13(C22)
C8(C17)
C9(C18)
C12(C21)
C10(C19)
C11(C20)
C6(C15)
C6(C15)
C13(C22)
C7(C16)
N3(N4)
Ϫ45.764(1.702)
Ϫ172.464(1.275)
Ϫ89.748(1.926)
99.018(1.763)
178.669(1.337)
5.628(1.891)
Ϫ177.138(1.412)
Ϫ4.708(1.752)
Ϫ4.468(1.732)
2.056(1.724)
67.437(1.612)
Ϫ60.795(1.649)
104.126(1.865)
Ϫ75.895(1.999)
Ϫ178.897(1.391)
1.086(1.908)
179.144(1.542)
Ϫ0.839(2.075)
Ϫ0.992(1.947)
0.258(2.132)
0.963(2.411)
Ϫ2.018(2.195)
0.430(1.849)
Ϫ0.855(2.582)
3.248(2.568)
1.823(2.588)
C5(C14)
C5(C14)
C6(C15)
C6(C15)
C6(C15)
C6(C15)
C7(C16)
C8(C17)
C8(C17)
C12(C21)
N3(N4)
C1(C3)
C5(C14)
C13(C22)
C5(C14)
C7(C16)
C6(C15)
N3(N4)
C9(C18)
C11(C20)
C7(C16)
C13(C22)
C10(C19)
C8(C17)
C9(C18)
N3(N4)
C8(C17)
C8(C17)
C8(C17)
C6(C15)
C12(C21)
C8(C17)
C13(C22)
C10(C19)
C13(C22)
C11(C20)
C12(C21)
Ϫ4.682(2.201)
3.163(2.185)
1.194(1.737)
C8(C17)
C11(C20)
C9(C18)
C10(C19)
4.761(2.205)
Ϫ1.820(2.343)
Ϫ3.502(2.430)
2.068(2.392)
Ϫ3.174(2.766)
a Atom designation of Trp residue 1 with Trp residue 2 in brackets.
Fractional coordinates and equivalent thermal fac-
tors, and the relevant torsion angles for cyclo(Trp–
Pro) are listed in Table 4 and Table 5, respectively.
Results and discussion
X-ray analysis
Figures 1 and 2 show the ORTEP drawing of
cyclo(Trp–Trp)иDMSO and cyclo(Trp–Pro) com-
plete with their respective numbering schemes. The
X-ray data appears in Table 1 and the torsion angles
of the backbone and the relevant side chains appear
in Tables 3 and 5.
The X-ray data (Table 1) indicated the crystalli-
zation of only one conformation for cyclo(Trp–Trp)
in comparison with the two conformations for
cyclo(Trp–Pro). The DKP ring is appreciably more
planar than in other cyclic dipeptide compounds due
Spectroscopic analyses
1H Proton (300 MHz) and 13C carbon (75 MHz)
spectra were recorded on a Bruker AM-300 spec-
trometer, with DMSO-d6, and CDCl3 as solvents, and
TMS as internal standard. Hetcor and Cosy spectra
were recorded to assist with the H and 13C assign-
1
ments.
to the conformational angles being reduced to ␸ ϭ
Infrared spectra were recorded on a Perkin-
Elmer 1600 FTIR spectrophotometer as KBr disks.
Fast atom bombardment (FAB) mass spectra of both
cyclo(Trp–Trp) and cyclo(Trp–Pro) dissolved in
DMSO with 3-nitrobenzyl alcohol as matrix, were
obtained on a VG-7070E spectrometer.
1
11.414, ⌿1 ϭ Ϫ7.516, ␸2 ϭ 12.471, and ⌿2 ϭ Ϫ8.256.
The values of ␹1A (Ϫ45.764) and ␹1A (67.437) indicate
1
2
an extended side chain conformation for Trp residue
1 (EN) and a folded conformation for Trp residue 2.
The extended side chain conformation for indole ring

440
Grant, Hunt, Milne, Roos, and Joubert
Fig. 1. ORTEP view of cyclo(Trp–Trp) и DMSO.
1 is in agreement with Morris et al.,20 who observed
an extended conformation of the tryptophan side
chain in crystals of cyclo(Trp–Gly). However, a
folded conformation is characteristic of some cyclic
dipeptides containing an aromatic side chain.21–25 The
amide bonds Ͷ1 and Ͷ2 are Ϫ4.259 and Ϫ3.107, re-
spectively (Table 3), and deviate only slightly from
planarity. Such angles have been found to be as large
as 8Њ in cyclic dipeptides.26 The internal angles at CͰ
in cyclo(Trp–Trp) are 114.5 and 115.6Њ whereas in
unsubstituted diketopiperazines the corresponding
angles are 115.1Њ.27 The ␸ (11.414, 12.471) and ⌿
(Ϫ7.516, Ϫ8.256) values are smaller and of opposite
sign to the corresponding angles in cyclo(L-Trp–L-
Pro): conformer 1 [(␸ ϭ Ϫ43.739, Ϫ38.954) (⌿ ϭ
39.005, 34.682)]; conformer 2 [(␸ ϭ Ϫ43.272,
Ϫ38.056) (⌿ ϭ 40.431, 35.415)].
trogens of the DKP ring and the carbonyl oxygens
of the DKP ring (Table 6). Molecular modeling of
cyclo(Trp-Trp) is given in Fig. 3.
The X-ray data (Table 1) indicated the crystalli-
zation of two conformations for cyclo(Trp–Pro) as
shown in Fig. 2. The two conformers show general
similarity with respect to conformational orientation
for the backbone around the following torsion angles
(conformer 1: ␸ ϭ Ϫ43.739, ␸ ϭ Ϫ38.954, ⌿1 ϭ
1
2
39.005, ⌿2 ϭ 34.682; conformer 2: ␸ ϭ Ϫ43.272,
1
␸ ϭ Ϫ38.056, ⌿1 ϭ 40.431, ⌿2 ϭ 35.415) and the
2
orientation of the tryptophan side chains (conformer
1A
2A
1A
1
1: ␹ ϭ 62.027, ␹ ϭ 167.964; conformer 2: ␹
ϭ
1
1
2A
60.934, ␹ ϭ 171.214). The DKP ring of cyclo(Trp–
1
Pro) for both conformers can be considered a typical
boat conformation with the CͰUCͱ bond of Pro
orientated equatorially and is in agreement with the
observation that the majority of DKP’s prefer a deep
boat-like conformation for the DKP ring with the
CͰUCͱ bond of Pro orientated equatorially.28,29 This
is in contrast to the conformation of the DKP ring
of cyclo(Tyr–Pro)30 and cyclo(Phe–F-Pro)31, that can
be considered a flattened chair with the CͰUCͱ bond
The crystal packing is stabilized by four hydro-
gen bonds: N4UHN4UO3, inter; N3UHN3UO3
intra; N1UHN1UO1, inter; N2UHN2UO2, inter.
Two of the hydrogen bonds involve the nitrogens of
the two indole rings and the oxygen of DMSO. The
two remaining hydrogen bonds are between the ni-

Structure of cyclo(Trp–Trp)ؒC2H6SO and cyclo(Trp–Pro)
441
Fig. 2. ORTEP view of cyclo(Trp–Pro).
of Pro and the CͰUCͱ bond of Tyr orientated equa-
torially and axially, respectively. The orientations of
the aromatic ring for both the conformers are folded
towards the DKP ring. These side chain conforma-
CͰ follow an expected trend [110.4Њ, 108.6Њ (con-
former 1) and 110.4Њ, 108.8Њ (conformer 2)], and are
smaller than the corresponding angles (115.1Њ) in the
unsubstituted diketopiperazine cyclo(Gly–Gly).27
The crystal packing is stabilized by four inter-
molecular hydrogen bonds: N3AUHN3AUO2B,
N3BUHN3BUO2A, N1AUHN1AUO1B, and
N1BUHN1BUO1A. The first hydrogen bond is be-
tween the nitrogen of the indole ring (conformer 1)
and the carbonyl oxygen of the DKP ring (conformer
2). The second hydrogen bond is between the nitro-
gen of the indole ring (conformer 2) and the carbonyl
oxygen of the DKP ring (conformer 1). The two re-
maining hydrogens involve the carbonyl oxygens of
the DKP rings and the nitrogens of the DKP rings
[carbonyl oxygen of DKP ring (conformer 1)––––
nitrogen of DKP ring (conformer 2), nitrogen of DKP
ring (conformer 1)–––––carbonyl oxygen of DKP
ring (conformer 2)] (Table 7).
tions are very similar, although not identical to the
1
folded arrangement of cyclo(Tyr-Pro) [␹ ϭ 64.1
1
(conformer 1); ␹ ϭ 64.2, (conformer 2)]30 and
1
1
cyclo(PheUF-Pro) (␹ ϭ 65.7)31 and in contrast to
1
1
the extended N, NE for the Phe side chain of
cyclo(PheUPro) (␹ ϭ Ϫ79.7).32
1
1
4
The small values for the conformational angle ␹
2
[CͲUCͳ-NUCͰ], Ϫ1.086 (conformer 1) and Ϫ3.009
(conformer 2) indicate that the two prolyl rings are
puckered at the ͱ-carbon atoms which deviate from
the planes defined by the four remaining atoms.33 The
conformation of the pyrrolidine ring of cyclo(Trp–
Pro) conformer 1 can be described as an envelope
(CsUCͱ-endo) conformation34 and the puckering
mode for conformer 2 as an intermediate34 between
Cs and C2 with Cͱ-endo and CͲ-exo with respect
to CЈ.
Spectroscopic analyses
The amide bonds Ͷ1 and Ͷ2 are 0.230Њ; 5.649Њ
(conformer 1) and Ϫ1.017Њ; 4.816Њ (conformer 2), re-
spectively. In cyclo(Trp–Pro), the internal angles at
The mass spectra of cyclo(Trp–Trp) and
cyclo(Trp–Pro) show a parent ion peak at m/z 373

442
Grant, Hunt, Milne, Roos, and Joubert
Table 4. Fractional Atomic Coordinates (ϫ104) and Equivalent
The cis-amide bond nature of cyclo(Trp–Trp)
is revealed through the specific values of ␯(NUH)
͕3214.7 cmϪ1͖ and ␯(CuO) ͕1661.8 cmϪ1͖. The cis-
amide bond also shows a further (amide III) absorp-
tion at 1333.2 cmϪ1 not shown by trans-amide bonds.37
For cyclo(TrpUPro), the cis CONH group ex-
hibits the amide I band at 1676 cmϪ1. The amide II
band (NH-in plane vibration) occurs at 1423.4 cmϪ1.
In addition, the cis-amide band shows a further (am-
ide III) absorption at 1300.6–1313.7 cmϪ1. The NH
bending and CN stretching vibrations are observed
at 1457.6 cmϪ1 and 1342 cmϪ1, respectively. Although
the presence of the amide band II (NH in-plane vibra-
tion) at 1550 cmϪ1 is characteristic of the trans-amide,
its nonexistence in a spectrum does not imply the
absence of all trans-amide bands.37
In cyclo(Trp–Trp) the Cͱ resonance of L-trypto-
phan (29.88 ppm), is shifted upfield 0.82 ppm when
compared to the same resonance in cyclo(L-Trp–L-
Gly) (30.7 ppm) and cyclo(L-Leu–L-Trp)(30.7
ppm).38 (Table 8). The four ͱ and two Ͱ protons of
cyclo(Trp–Trp) give rise to a single ABX pattern
with the chemical shifts indicated in Fig. 4. The two
kinds of ͱ-protons differ by 0.51 ppm; the more
shielded one has an apparent coupling to the Ͱ proton
of 6.7 Hz, the less shielded, one of 4.2 Hz. If a folded
conformation was important and persisted long
enough to prevent averaging of this nonequivalence,
there would be separated resonance patterns for the
two kinds of methylene. Therefore, it seems that a
preferred conformation is likely to be one in which
each tryptophan residue shares the space over the
DKP ring in such a fashion that the two ͱ-methylenes
have identical environments.
These findings are in agreement with the studies
done on other aromatic cyclic dipeptides where it
was revealed that diketopiperazines assume a flat-
tened 2,5-piperazinedione ring conformation with the
aromatic rings sharing the space over the pipera-
zinedione nucleus, each aromatic residue being in a
‘‘face to face’’ position.24 This type of conformation
would support the energy transfer seen by Edelhoch
et al.9 A planar diketopiperazine, above which the
indole rings of each tryptophan residue face each
other, would allow ȏ–ȏ interactions between the in-
dole rings.
Assuming an energetically preferred staggered
conformation of the CͰUCͱ bond and approximate
validity of Pachler’s values39 of JͰͱ for J180 and J60
(13.6 Hz and 2.6 Hz, respectively) indicated that the
preferred conformation for cyclo(Trp–Trp) is a
folded one (49%) with the aromatic rings of trypto-
2
3
˚
Thermal Factors (A ϫ 10 ) for cyclo(Trp–Pro)
a
Atom
x/a
y/b
z/c
Ueq
N(1A)
C(1A)
C(2A)
O(1A)
N(2A)
C(3A)
C(4A)
C(5A)
C(6A)
C(7A)
O(2A)
C(8A)
C(9A)
C(10A)
N(3A)
C(11A)
C(12A)
C(13A)
C(14A)
C(15A)
C(16A)
N(1B)
C(1B)
C(2B)
O(1B)
N(2B)
C(3B)
C(4B)
C(5B)
C(6B)
C(7B)
O(2B)
C(8B)
C(9B)
C(10B)
N(3B)
C(11B)
C(12B)
C(13B)
C(14B)
C(15B)
C(16B)
7242
Ϫ4255
7867
41(1)
41(2)
37(2)
51(1)
45(1)
57(2)
75(3)
66(2)
43(2)
44(2)
60(1)
40(2)
39(2)
40(2)
44(1)
38(2)
50(2)
58(2)
60(2)
47(2)
36(1)
37(1)
32(1)
39(2)
47(1)
38(1)
56(2)
101(3)
68(2)
43(2)
40(2)
57(1)
40(2)
35(1)
36(1)
47(2)
43(2)
56(2)
68(2)
66(2)
52(2)
37(2)
4629(13)
4263(15)
2961(13)
5366(14)
5040(18)
6425(19)
8276(17)
6806(15)
8337(16)
10502(13)
3980(15)
5492(15)
7788(15)
8561(14)
6784(16)
6729(17)
4690(19)
2716(17)
2846(16)
4864(15)
5262(13)
7819(14)
8081(16)
9424(13)
6965(14)
7190(17)
5679(21)
4038(17)
5543(15)
4113(16)
1939(14)
8569(15)
7003(14)
4781(15)
3936(15)
5708(17)
5679(17)
7610(20)
9671(18)
9618(17)
7671(15)
Ϫ3868(7)
Ϫ2359(7)
Ϫ1602(6)
Ϫ2019(6)
Ϫ693(8)
Ϫ834(9)
Ϫ2020(8)
Ϫ2972(8)
Ϫ3909(8)
Ϫ4262(6)
Ϫ4184(7)
Ϫ3459(8)
Ϫ2749(7)
Ϫ2290(6)
Ϫ2678(7)
Ϫ2416(7)
Ϫ2949(8)
Ϫ3699(8)
Ϫ3913(8)
Ϫ3433(7)
392(6)
948(7)
2239(7)
3224(6)
2121(7)
3138(8)
2492(10)
1354(9)
888(8)
292(7)
8285(7)
7838(6)
8343(5)
6862(6)
6246(7)
5174(7)
5340(7)
6245(6)
6884(6)
6506(5)
9449(6)
10021(7)
9646(7)
10457(6)
11336(7)
12326(7)
13096(7)
12873(8)
11869(7)
11099(7)
9421(6)
9056(6)
9544(7)
9056(6)
10508(6)
11166(7)
12195(9)
12026(7)
11085(6)
10415(7)
10761(6)
7912(6)
7313(6)
7649(6)
6851(6)
5969(7)
4981(7)
4232(8)
4463(7)
5464(7)
6229(6)
Ϫ217(6)
1154(7)
2145(8)
2726(7)
3511(7)
3456(8)
4081(8)
3863(9)
3002(9)
2394(8)
2596(7)
a
* *
Ueq ϭ 1/3 ⌺i ⌺j Uijai aj (ai · aj).
and m/z 283, respectively, i.e., the expected cyclic
dipeptide form. The characteristic tryptophan side
chain cleavage yielding the fragmentation m/z 130 is
one of the highest observed fragment ions in the mass
spectra. The ion at m/z 154 corresponds to the DKP-
pyrrolidine fragment.
IR spectroscopy is a spectral method permitting
reliable discrimination between cis- and trans-sec-
ondary amide bonds.35–37

Structure of cyclo(Trp–Trp)ؒC2H6SO and cyclo(Trp–Pro)
443
Table 5. Relevant Torsion Angles of Conformers 1 and 2 of cyclo(Trp–Pro)
Cyclo(Trp–Pro)
Torsion anglesa
1
2
C7A(7B)UN1A(1B)UC1A(1B)UC2A(2B)
C2A(2B)UN2A(2B)UC6A(6B)UC7A(7B)
N1A(1B)UC1A(1B)UC2A(2B)UN2A(2B)
N2A(2B)UC6A(6B)UC7A(7B)UN1A(1B)
C1A(1B)UC2A(2B)UN2A(2B)UC6A(6B)
C1A(1B)UN1A(1B)UC7A(7B)UC6A(6B)
N1A(1B)UC1A(1B)UC8A(8B)UC9A(9B)
C1A(1B)UC8A(8B)UC9A(9B)UC16A(16B)
C8A(8B)UC9A(9B)UC10A(10B)UN3A(3B)
C16A(16B)UC9A(9B)UC10A(10B)UN3A(3B)
C8A(8B)UC9A(9B)UC16A(16B)UC11A(11B)
C9A(9B)UC10A(10B)UN3A(3B)UC11A(11B)
N3A(3B)UC11A(11B)UC16A(16B)UC9A(9B)
C10A(10B)UC9A(9B)UC16A(16B)UC11A(11B)
C10A(10B)UN3A(3B)UC11A(11B)UC16A(16B)
C12A(12B)UC11A(11B)UC16A(16B)UC15A(15B)
C14A(14B)UC15A(15B)UC16A(16B)UC11A(11B)
C16A(16B)UC11A(11B)UC12A(12B)UC13A(13B)
C13A(13B)UC14A(14B)UC15A(15B)UC16A(16B)
C11A(11B)UC12A(12B)UC13A(13B)UC14A(14B)
C12A(12B)UC13A(13B)UC14A(14B)UC15A(15B)
C4A(4B)UC5A(5B)UC6A(6B)UN2A(2B)
C3A(3B)UC4A(4B)UC5A(5B)UC6A(6B)
N2A(2B)UC3A(3B)UC4A(4B)UC5A(5B)
C6A(6B)UN2A(2B)UC3A(3B)UC4A(4B)
C3A(3B)UN2A(2B)UC6A(6B)UC5A(5B)
C2A(2B)UC1A(1B)UC8A(8B)UC9A(9B)
C1A(1B)UC8A(8B)UC9A(9B)UC10A(10B)
␸
␸
Ϫ43.739
Ϫ38.954
39.005
34.682
0.230
Ϫ43.272
Ϫ38.056
40.431
35.415
Ϫ1.017
4.816
60.934
171.214
Ϫ177.043
0.983
177.865
Ϫ1.270
Ϫ0.449
Ϫ0.312
1.035
1
2
⌿
⌿
1
2
Ͷ1
Ͷ2
5.649
1A
␹
62.027
167.964
Ϫ177.379
Ϫ0.294
178.497
Ϫ0.836
Ϫ1.810
1.274
1.654
0.858
Ϫ1.839
0.837
1.092
1
2A
␹
1
0.492
Ϫ0.363
0.013
Ϫ0.221
Ϫ0.624
0.744
Ϫ29.485
29.047
Ϫ16.363
Ϫ3.009
21.086
Ϫ63.982
Ϫ11.044
Ϫ1.602
0.680
1
2
␹
␹
␹
␹
␪
Ϫ34.820
35.372
Ϫ21.142
Ϫ1.086
23.343
Ϫ61.969
Ϫ15.424
2
2
3
2
4
2
a Atom designation of conformer 1 with conformer 2 in brackets.
phan located above the DKP ring. The values for
the two unfolded conformations were 37 and 15%,
respectively. (Considering the Ͱ–ͱ coupling values
for cyclic peptides, equal couplings near 3 Hz may
be expected for the folded conformation, whereas
one large (14 Hz) and one small (3 Hz) coupling
would present a single, unfolded conformation. If the
two unfolded conformations are equally populated
and the folded form is negligibly so, the two couplings
Table 6. Hydrogen Bonding Scheme for cyclo(Trp–Trp)
DUH–––Aa
DUA
H–––A Angle DUH–––A
˚
˚
˚
˚
˚
˚
˚
N4UHN4UO3
N3UHN3UO3
N1UHN1UO1
N2UHN2UO2
2.913 A 2.345 A
110.0Њ
137.3Њ
163.3Њ
143.2Њ
2.928 A 2.155 A
2.986 A 1.935 A
˚
2.994 A 2.059 A
Fig. 3. Molecular modeling of cyclo(Trp–
Trp)иC2H6SO.
a D-donor, H-hydrogen, A-acceptor.

444
Grant, Hunt, Milne, Roos, and Joubert
Table 7. Hydrogen Bonding Scheme for cyclo(Trp–Pro)
pared with the same resonances in cyclo(Phe–Pro)
(1.72 ppm) and cyclo(Tyr–Pro) (1.70 ppm).
Angle
DUH––A
For cyclo(Trp–Pro), a low field 13C shift of the
Cͱ resonance of L-tryptophan (25.74 ppm, DMSO-
d6, 26.76 ppm, CDCL3) (Table 10) was observed in
comparison with the corresponding resonances seen
in the spectra of cyclo(Trp–Trp) (29.88 ppm, DMSO-
d6) and cyclo(Trp–Leu) (30.7 ppm, DMSO-d6)38. The
CͰ resonance of tryptophan (55.20 ppm, DMSO-d6)
is shifted upfield when compared with the same reso-
nance in cyclo(L-Leu–L-Trp)(57.1 ppm, DMSO-d6)
and cyclo(L-Trp–Gly) (57.0 ppm, DMSO-d6)38. From
the 13C NMR data shown in Table 10 it is seen that
for cyclo(Trp–Pro), the value of ⌬ͳ(ͱͲ) is nearly
constant regardless of solvent (5.8 in DMSO-d6 and
5.7 in CDCl3). Assuming, however, that the ⌬ͳ(ͱͲ)
value reflects an equilibrium state between the planar
and boat forms of this compound, expressed by the
equilibrium constant K ϭ [planar form]/[boat form]
and remembering that for the planar form (␪ ϭ 60Њ)
⌬ͳ(ͱͲ) ϭ 7.33 ppm and for the boat form (␪ ϭ 30Њ)
⌬ͳ(ͱͲ) ϭ 4.90 ppm, the amounts of both conformers
can be calculated by the equation: 5.8 or 5.7 ϭ
4.90a ϩ 7.33(1-a) where a and (1-a) are the molar
fractions of boat and planar forms, respectively. Us-
ing this procedure, the values of 0.63 (boat) and 0.37
(planar form) in DMSO-d6 and 0.67 (boat) and 0.33
(planar form) in CDCl3 were obtained.40 These results
support the stability of the boat conformation for this
proline-containing cyclic dipeptide.
DUH––Aa
DUA
H––A
˚
˚
˚
˚
˚
˚
˚
N3AUHN3AUO2B
N3BUHN3BUO2A
N1AUHN1AUO1B
N1BUHN1BUO1A
2.903 A 2.257 A
116.4Њ
117.2Њ
136.1Њ
147.0Њ
2.902 A 2.244 A
3.085 A 2.215 A
˚
3.094 A 2.087 A
a D-donor, H-hydrogen, A-acceptor.
should be equal and about 8 Hz. If however, all three
conformations are equally populated, the couplings
should be equal and near 6.5 Hz.)24
For cyclo(Trp–Trp), no chemical shift change is
detected in the indole ring as a consequence of the
proximity of the two aromatic residues.
In DMSO-d6 solution (Table 9), the two ͱ-pro-
tons of the tryptophan residue of cyclo(Trp–Pro) dif-
fer by 0.18 ppm, the more shielded has an apparent
coupling to the Ͱ proton of 5.7 Hz, the less shielded,
one of 4.8 Hz. A predominance (52%) of the folded
conformation for the tryptophan side chain in
DMSO-d6 was estimated using Pachler’s39 analysis.
Judging from the 3J(Trp-Ͱ,ͱ) coupling constants, the
tryptophan side chain prefers the unfolded conforma-
tion (73%) in CDCL3. There is a shift of the signal
CͰ-H (Trp) (4.29 ppm) to lower field in comparison
with the corresponding resonance in cyclo(Trp-Trp)
(3.87 ppm). The CͲ proton resonance of proline in
DMSO-d6 (1.65 ppm) is shifted upfield when com-
In a slightly different approach to the method
of preparation described in the experimental section,
the N-protected dipeptide ester (Boc–Pro–Trp–
OMe) was treated with formic acid (20 ml) containing
0.2 ml anisole, to remove the boc-group. The resulting
unprotected dipeptide formate salt was dissolved in
a neutral medium [toluene-sec-butanol (1 : 4)] and
refluxed in an oil bath (120ЊC) for 3 h.41 After concen-
trating the solution to 8 ml and cooling to 0ЊC the
products were filtered off and recrystallized from
chloroform-n-hexane.
Table 8. The 13C NMR Data of
cyclo(Trp–Trp)a
Carbon atom
ppm
Trp-ͱ
Trp-Ͱ
29.88
55.24
Trp–Ar (C3)
Trp–Ar (C7)
Trp–Ar (C4)
Trp–Ar (C5)
Trp–Ar (C6)
Trp–Ar (C2)
Trp–Ar (C8)
Trp–Ar (C9)
CuO
108.85
111.29
118.41
118.60
120.85
124.47
127.45
136.17
166.89
Two conformations of cyclo(Pro–Trp) crystal-
lized in the space group P1 with cell dimensions a ϭ
˚
5.404(1), b ϭ 9.888(2), c ϭ 13.438(2) A, Ͱ ϭ 80.45(1),
ͱ ϭ 78.67(1), and Ͳ ϭ 89.09(2)Њ. The conformation
of the backbone, the orientation of the aromatic side
chain, the puckering modes for the pyrrolidine rings,
and the hydrogen bonding of these conformers are
to a certain extent similar, although not identically
a The assignments of the indole ring
of tryptophan are based on those
of Parker and Roberts,42 and Des-
lauriers et al.38
to that of cyclo(Trp–Pro) [(conformer 1: ␸
ϭ
1
Ϫ46.229, ␸ ϭ Ϫ36.307, ⌿1 ϭ 40.683, ⌿2 ϭ 33.171,
2
Ͷ1 ϭ Ϫ1.367, Ͷ2 ϭ 7.574, ␹1A ϭ 60.327, ␹2A ϭ 167.584,
1
1

Structure of cyclo(Trp–Trp)ؒC2H6SO and cyclo(Trp–Pro)
445
1
Fig. 4. H NMR spectrum of cyclo(Trp–Trp).
Table 10. 13C NMR Data of cyclo(Trp–Pro)a
1
Table 9. H NMR Data of cyclo(Trp–Pro)a
CDCL3
ppm
DMSO-d6
ppm
Carbon atom
CDCL3
ppm
DMSO-d6
ppm
Proton
Pro-Ͳ
Trp-ͱ
22.48
26.76
21.75
25.74
NH (Indole)
NH
8.35
10.85
7.67
7.56
7.32
7.18
7.05
6.96
4.29
4.05
3.24
3.37
3.29
3.06
1.97
1.65 (2H)
1.39
—
Pro-ͱ
Pro-ͳ
Trp-Ͱ
Pro-Ͱ
28.19
45.33
54.60
59.18
27.55
44.52
55.20
58.38
5.72
7.57
7.37
7.21
7.12
7.06
4.35
4.04
3.73
3.59 (2H)
—
2.96
2.29
1.99
1.94
1.87
Trp–Ar
Trp–Ar
Trp–Ar
Trp–Ar
Trp–Ar
Trp-Ͱ
Pro-Ͱ
Trp-ͱ
Pro-ͳ
Pro-ͳ
Trp–Ar (C3)
Trp–Ar (C7)
Trp–Ar (C4)
Trp–Ar (C5)
Trp–Ar (C6)
Trp–Ar (C2)
Trp–Ar (C8)
Trp–Ar (C9)
Pro–CuO
Trp–CuO
109.99
111.61
118.55
120.04
122.81
123.39
126.85
136.81
165.69
169.48
109.36
111.27
118.28
118.69
120.93
124.45
127.44
136.10
165.65
169.13
Trp-ͱ
Pro-ͱ
Pro-Ͳ
Pro-ͱ
a The assignments of the indole ring of tryptophan
are based on those of Parker and Roberts, 1970,42
but resonances C5 and C6 have been interchanged
based on studies of indoles deuterated in position
5 and position 6.38
Pro-Ͳ
a
2J(Trp-ͱ) ϭ 15 Hz, 3J(Trp-Ͱ,ͱ) ϭ 5.7 Hz and 4.8
2
3
Hz)(DMSO-d6). J(Trp-ͱ) ϭ 15 Hz, J(Trp-Ͱ,ͱ) ϭ
10.7 Hz and 3.8 Hz)(CDCL3).

446
Grant, Hunt, Milne, Roos, and Joubert
as an envelope (CsUCͱ-endo) conformation34 in
contrast to the pyrrolidine ring symmetry for con-
former 2 which is an intermediate34 between Cs and
C2 with Cͱ-endo and CͲ-exo with respect to CЈ. The
two prolyl rings are puckered at the ͱ-carbon atoms
which deviate from the best planes defined by the
four remaining atoms. The crystal structures are sta-
bilized by four intermolecular hydrogens bonds (Ta-
ble 11). Very evident is the difference in the angles
of donor-hydrogen-acceptor and the lengths of the
hydrogen bonds between cyclo(Trp–Pro) and
cyclo(Pro–Trp).
Molecular modeling (Fig. 5) showed that there
are great similarities between conformer 1 of
cyclo(Trp–Pro) and conformer 1 of cyclo(Pro–Trp).
Similarities were also observed between conformer
2 of cyclo (Trp–Pro) and conformer 2 of cyclo(Pro–
Trp). The difference is mainly between conformers
1 and conformers 2 due to the puckering of the pyrrol-
idine ring (Cs compared to an intermediate between
Cs and C2).
Table 11. Hydrogen Bonding Scheme for cyclo(Pro–Trp)
Angle
DUH––A
DUH––Aa
DUA
H––A
˚
˚
N2AUHN2AUO2B
N3AUHN3AUO1B
N2BUHN2BUO2A
N3BUHN3BUO1A
3.078 A
2.138 A
130.7Њ
119.8Њ
121.5Њ
120.4Њ
˚
˚
2.877 A
2.182 A
˚
˚
3.023 A
2.092 A
˚
˚
2.260 A
2.914 A
a D-donor, H-hydrogen, A-acceptor.
␹21 ϭ Ϫ34.274, ␹22 ϭ 37.275, ␹23 ϭ Ϫ24.064, ␹24 ϭ 1.508,
␪ ϭ 20.483); (conformer 2: ␸ ϭ Ϫ44.902, ␸ ϭ
1
2
Ϫ42.014, ⌿1 ϭ 40.900, ⌿2 ϭ 38.133, Ͷ1 ϭ 1.729, Ͷ2 ϭ
1A
2A
1
4.587, ␹ ϭ 59.562, ␹ ϭ 172.769, ␹ ϭ Ϫ36.683,
1
1
2
␹22 ϭ 34.086, ␹23 ϭ Ϫ17.941, ␹24 ϭ Ϫ4.944, ␪ ϭ 26.476)].
The DKP ring of cyclo(Pro–Trp) for both con-
formers can be considered a typical boat conforma-
tion. The tryptophan side chains of these conformers
are folded toward the diketopiperazine (DKP) ring.
The pyrrolidine ring for conformer 1 can be described
Fig. 5. Molecular modeling of cyclo(Trp–Pro) and cyclo(Pro–Trp).

Structure of cyclo(Trp–Trp)ؒC2H6SO and cyclo(Trp–Pro)
Data Given in Figure 5 (A, B, C, D)
447
12. Chacko, K.K.; Swaminathan, S.; Veena, K.R. Curr. Sci. 1983,
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15. North, A.C.T.; Phillips, D.C.; Matthews, F.S. Acta Crystallogr.
1968, 24A, 351.
16. Sheldrick, G.M. SHELX86. A program for the solution of
crystal structures; University of Go¨ttingen: Germany, 1986.
17. Sheldrick, G.M. SHELX76. Program for crystal structure de-
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18. International Tables for X-ray crystallography, Vol. 4; Kynoch
Press: Birmingham, 1974; Cromer, D.T.; Liberman, D. J.
Chem. Phys. 1970, 4, 1891.
A. Molecular fit of cyclo(Trp–Pro)[conformer 1:
(gray line rendering), conformer 2: (gray stick ren-
dering)]
B. Molecular fit of cyclo(Pro–Trp)[conformer 1:
(black line rendering), conformer 2: (black stick ren-
dering)]
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(gray line rendering)] and cyclo(Pro–Trp)[conformer
1: (black line rendering)]
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[conformer 2: (black stick rendering)]
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Biological studies on cyclo(Trp–Trp), cyclo
(Trp–Pro), and two other selected aromatic diketo-
piperazines indicated that these cyclic dipeptides ex-
hibit biological activity in both prokaryotes and eu-
karyotes. Three of the cyclic dipeptides block cation
channels in ventricular myocytes, whilst all increase
the expression of alkaline phosphatase. All of the
cyclic dipeptides exhibit concentration dependent an-
tibacterial properties. (Another 25 cyclic dipeptides
are currently being tested and evaluated for biologi-
cal activity.)
21. Webb, L.E.; Lin, C.-F. J. Am. Chem Soc. 1971, 93, 3818.
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