(4R)-4-Hydroxy-1-nitroso-l-proline: synthesis, X-ray structure, ab initio and conformational calculations

Article abstract of DOI:10.1016/j.tet.2006.06.097

4-Hydroxy-l-proline, an amino acid, an important component of collagen, was transformed into its N-nitroso-derivative, (4R)-4-hydroxy-1-nitroso-l-proline, 1 by butylnitrite in the acidic medium. The structure is a cyclic hydroxy-N-nitrosoacid with the carboxyl and hydroxyl groups trans to each other. The carboxyl group is in the syn-conformation. In the structure, the neutral molecules are connected via classical intermolecular O-H?O hydrogen bonds involving the hydroxyl and carboxyl groups [O?O=2.6251(14) ?], and form chains along the a-axis direction. The chains are linked into sheets via O-H?O hydrogen bond, [O?O=2.6813(15) ?] with participation of oxygen atom of nitroso group. Ab initio calculations based on density functional theory at the B3LYP/6-311++G(d, p) level of theory were performed to analyze the influence of 4-hydroxy-l-proline (Hyp) nitrosation on the conformation of the synthesized N-nitroso-compound. The geometry optimization of 1 and initial 4-hydroxy-l-proline was carried out in the gas phase and in solution using the polarizable continuum model. The single-point calculation was performed for the crystal structure of 1. The most stable conformer of 1 is observed in an aqueous solution. In this state, the pyrrolidine ring adopts an envelope conformation, which is also maintained in the gas phase. The twisted conformation of the pyrrolidine ring is present in all states of Hyp and in the crystal structure of 1. In 1 the interchange of five-membered ring conformation in solution and in the gas phase in comparison with the crystal is accompanied by an increase of the dipole moment of the molecule, which is maximal in solution.

Full text of DOI:10.1016/j.tet.2006.06.097

Tetrahedron 62 (2006) 8779–8786
(4R)-4-Hydroxy-1-nitroso-L-proline: synthesis, X-ray structure,
ab initio and conformational calculations
a
b
c
Marina S. Fonari,a, Yurii M. Chumakov, Gheorghe G. Duca, Eduard V. Ganin,
*
Arkadii A. Yavolovskii,d Tadeusz Lise and Yurii A. Simonova
aInstitute of Applied Physics, Academy of Sciences of R. Moldova, Academy Str. 5, MD2028 Chisinau, Republic of Moldova
bAcademy of Sciences of R. Moldova, bul St. cel Mare 1, Chisinau, Republic of Moldova
cOdessa State Environmental University, Ministry of Education and Science of Ukraine, ul. Lvovskaya 15, 65016 Odessa, Ukraine
dA.V. Bogatsky Physico-Chemical Institute, National Academy of Sciences of Ukraine, Lustdofskaya doroga 86, Odessa, Ukraine
eFaculty of Chemistry, University of Wroclaw, 50-383 Joliot-Curie 14, Wroclaw, Poland
Received 30 March 2006; revised 12 June 2006; accepted 29 June 2006
Available online 21 July 2006
Abstract—4-Hydroxy-L-proline, an amino acid, an important component of collagen, was transformed into its N-nitroso-derivative, (4R)-
4-hydroxy-1-nitroso-L-proline, 1 by butylnitrite in the acidic medium. The structure is a cyclic hydroxy-N-nitrosoacid with the carboxyl
and hydroxyl groups trans to each other. The carboxyl group is in the syn-conformation. In the structure, the neutral molecules are connected
˚
via classical intermolecular O–H/O hydrogen bonds involving the hydroxyl and carboxyl groups [O/O¼2.6251(14) A], and form chains
˚
along the a-axis direction. The chains are linked into sheets via O–H/O hydrogen bond, [O/O¼2.6813(15) A] with participation of oxygen
atom of nitroso group. Ab initio calculations based on density functional theory at the B3LYP/6-311++G(d, p) level of theory were performed to
analyze the influence of 4-hydroxy-L-proline (Hyp) nitrosation on the conformation of the synthesized N-nitroso-compound. The geometry
optimization of 1 and initial 4-hydroxy-L-proline was carried out in the gas phase and in solution using the polarizable continuum model.
The single-point calculation was performed for the crystal structure of 1. The most stable conformer of 1 is observed in an aqueous solution.
In this state, the pyrrolidine ring adopts an envelope conformation, which is also maintained in the gas phase. The twisted conformation of the
pyrrolidine ring is present in all states of Hyp and in the crystal structure of 1. In 1 the interchange of five-membered ring conformation in
solution and in the gas phase in comparison with the crystal is accompanied by an increase of the dipole moment of the molecule, which is
maximal in solution.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
Hyp, respectively. The occurrence of Pro and Hyp in collagen
restricts the orientational freedom of the chain in relation to
the fiber axis and permits only left-handed helices.2
The functional specificity of proteins and polypeptides
depends on the conformational behavior of the initial amino
acids, which are extensively studied by theoretical and
experimental methods.1 L-Proline (Pro) and 4(R)-hydroxy-
L-proline (Hyp) are abundant amino acids in collagen and
exceptional among amino acids, as they are the only ones
that have the amino group fixed within a pyrrolidine ring,
making it rigid and directional in biological systems despite
its conformational flexibility. Due to its crucial biological
role (it is the most abundant protein in vertebrates) and
very peculiar structure, collagen has been deeply investi-
gated, together with several related polypeptides. Collagen
is composed of approximately 300 repeats of the sequence
XaaYaaGly, where Xaa and Yaa are in most cases Pro and
The importance of Pro and Hyp explains the extensive theo-
retical and structural studies of these amino acids and their
derivatives including several di- and polypeptides. Aside
from recent electron-density studies and quantum chemical
investigations, the study of amino acid clusters in the gas
phase has attracted considerable attention. The stereoelec-
tronic effects crucial for the collagen stability have been
studied, as well as quantum mechanical calculations for
Pro, Hyp, and fluorinated dipeptide analogues in aqueous
solution and for the Pro gaseous isomers have been fulfilled.3
Moreover, in a recent paper, Conticello et al. discuss in detail
the stereoelectronic effects in elastin-mimetic polypeptides
due to Pro substitution.4
Keywords: Oxyproline; Nitrosation; X-ray structure; Theoretical calcula-
tions.
* Corresponding author. Tel.: +373 22 73 81 54; fax: +373 22 72 58 87;
e-mail: fonari.xray@phys.asm.md
The structural studies of vitally important amino acids are
fundamentally important. With respect to the metal free Pro
and Hyp, so far the crystal structures of L-Pro and DL-Pro,5,6
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.06.097

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M. S. Fonari et al. / Tetrahedron 62 (2006) 8779–8786
the monohydrates of L-Pro and DL-Pro7,8 and numerous other
solvates and salts of Pro have been determined. For Hyp
and its derivatives, structural data are available for the
zwitterion of 4-hydroxy-L-proline itself defined by X-ray
analysis9,10 and precision neutron diffraction,11 4-hydroxy-
L-proline hydrate,12 4-hydroxy-N-methylproline,13 and
N,N0-dimethylated 4-hydroxy-D- and 4-hydroxy-L-proline
chloride.14 The acyclic peptides, ALB*-ALB-PRO* (three
residues) and PRO*-PRO* were also described.15,16
groups are in the trans configuration relative to the ring, as
found earlier for the zwitterions of L-Hyp,9–11 4-hydroxy-
N-methylproline13 and its hydrochloride salt14 and differs
from cis-zwitterion of Hyp in its dihydrate.12 Molecular
bond distances and bond angles as well as torsion angles,
labeled according to the IUPAC conventions,21 are given in
Tables 1 and 2, respectively. The average C–C length of
˚
˚
1.530(2) A is in agreement with 1.533(8) A in Hyp dihydrate.
The carboxylic group is in a syn-orientation and practically
planar with the asymmetrical C(1)–O(2) 1.204(2) and
˚
As the only amino acids with the imino group, Pro and Hyp
were extensively studied to elucidate the conditions respon-
sible for the generation of carcinogenic N-nitroso-deriva-
tives. A dose–response study on the endogenous formation
of N-nitrosoproline was performed in rats, dogs, and in
smokers and non-smokers. Solutions containing nitrite,
proline, and in some cases ascorbic acid (ASC) and/or
SCNꢀ were infused into the stomach and samples taken to
determine gastric [nitrite], [NPro], [ASC], [SCNꢀ], and pH
as functions of time. The ability of ASC to inhibit nitrosation
(by reaction with nitrite) was shown to be highly dependent
on initial [ASC] and on the rate of entry of O2 into the sto-
mach from blood. The rate of N-nitroso-Pro formation
in the absence of ASC and SCNꢀ, the inhibitory effects on
nitrosation of ASC, and the catalytic effects of SCNꢀ were
all accurately predicted by the mathematical model.17
C(1)–O(1) 1.328(2) A distances which are consistent with
the double and single C–O bonds. C(4)–O(3) length of
˚
˚
1.433(2) A is consistent with 1.435(7) A obtained by Sha-
mala et al.12 The N-nitroso-group has an ordinal geometry19
with the equalized O(4)–N(2) 1.266(1) and N(1)–N(2)
˚
1.289(2) A distances and N–N–O bond angle O(4)–N(2)–
N(1) 113.3(1)ꢁ. The dihedral angles formed by the carboxyl
[O(1)/C(1)/O(2)] and nitroso [N(1)/N(2)/O(4)] groups
with the pyrrolidine ring are equal 65.4(1)ꢁ and 11.4(1)ꢁ,
respectively.
Two approaches could be applied to describe the puckering
of the pyrrolidine ring. Generalization by Cremer and
Pople22 requires specification of an appropriate mean plane
and uses the displacements of each atom from that plane to
define puckering coordinates (q is a puckering amplitude and
j is a phase angle). Geise et al.23 use endocyclic torsion
angles and thus avoid the need to define a mean plane. We
use the advantages of both of these approaches and say that
2. Results and discussion
the pyrrolidine ring is in a twisted form with Cg [C(4)] atom
11
˚
˚
In continuation of our previous studies devoted to the condi-
tions of mutagenesis of the biologically important molecules
to their carcinogenic N-nitroso-derivatives18 and the stereo-
chemistry of N-nitroso-compounds,19 the nitrosation of 4-hy-
droxy-L-proline (Hyp) by butylnitrite20 in the acidic medium
has been fulfilled and the crystal structure of N-nitroso-deriv-
ative, 4-hydroxy-1-nitrosopyrrolidine-2-carboxylicacid1,was
studied by X-ray crystallography (Fig. 1). The aim of this
article is to study the product of Hyp nitrosation analyzing
the structural peculiarities of N-nitrosooxyproline in the
crystalline, liquid, and gaseous phases.
lying 0.608(2) A [0.516 A in Hyp ] below the plane through
N, Ca, Cb, and Cd, and with the latter four atoms coplanar to
˚
˚
within 0.0367 A [0.037(3) A in Hyp]. The dihedral angle
between the planes N–Ca–Cb–Cd and Ca–Cb–Cg–Cd is
39.6(1)ꢁ [33.5ꢁ in Hyp]. The pyrrolidine ring conformation
corresponds to the Cg-endo or down puckering. The Cremer
and Pople puckering ring parameters for 1 are gathered in
Table 3 in comparison with those for Hyp and Pro deriva-
tives. The closeness of the conformations for 1, Hyp, and
Pro monohydrates is evident.
To compare the puckering of the pyrrolidine ring in 1 and
some derivatives of Hyp we use the data available in the
Cambridge Structural Database.24 Compound 1 and some
structurally related molecules were fitted by the same atoms
in the pyrrolidine ring skeleton. Figure 2 reveals the overlap
X-ray structural analysis was carried out at 100 K that per-
mits the unambiguous localization of all the hydrogen atoms
and to state that the molecule exists in the crystal in its neutral
form. In the neutral molecule 1, the hydroxyl and carboxyl
(a)
(b)
Figure 1. ORTEP (a) top and (b) side view of 1. Thermal ellipsoids are drawn with the 50% probability level.

M. S. Fonari et al. / Tetrahedron 62 (2006) 8779–8786
8781
˚
Table 1. Bond lengths (A) and angles (degree) for 1 in crystal, in the gas
phase (3¼1), and in solution (3¼78.4)
links the molecules into chains parallel to the [100] direction
that can be described by the first level graph set, C(7).26
Along the [010] direction the hydroxyl group acts as H-
donor, being involved in an O–H/O hydrogen bond with
the nitroso group oxygen via again C(7) graph set and the
second level graph set, R44(23), thus generating the H-bonded
sheet. Between the layers only one type of contact C(3)–
Crystal
3¼1
3¼78.4
Bond distances
O(1)–C(1)
O(1)–H(1)
O(2)–C(1)
O(3)–C(4)
O(3)–H(3O)
O(4)–N(2)
N(1)–N(2)
N(1)–C(2)
N(1)–C(5)
C(1)–C(2)
C(2)–C(3)
C(2)–H(2)
C(3)–C(4)
C(3)–H(31)
C(3)–H(32)
C(4)–C(5)
C(4)–H(4)
C(5)–H(51)
C(5)–H(52)
1.328(2)
0.83(3)
1.204(2)
1.433(2)
0.90(2)
1.266(1)
1.289(2)
1.473(2)
1.477(2)
1.529(2)
1.536(2)
0.97(2)
1.532(2)
0.98(2)
0.96(2)
1.522(2)
0.97(2)
0.91(2)
0.96(2)
1.349
0.97
1.203
1.426
0.96
1.226
1.319
1.466
1.465
1.524
1.547
1.09
1.541
1.09
1.09
1.334
0.99
1.211
1.427
0.98
1.242
1.299
1.472
1.472
1.523
1.548
1.1
1.541
1.09
1.09
˚
H(31)/O(4) 3.152(2) A deserves to be mentioned.
The rather close crystal packing is observed in Hyp itself,9–11
where the molecules are also combined in the chains via
the same C(7) O–H/ꢀOOC synthon, and the chains being
further combined in the layer via NH+2(ammo-
nium)/ꢀOOC(carboxyl) interactions analogous to those
with nitroso group involvement in 1.
In the analysis of the conformational and intermolecular
characteristics of N-nitroso-compounds, it is of basic impor-
tance to consider environmental effects, so the ab initio cal-
culations at the density functional theory (DFT) level have
been thus fulfilled both in gas phase (3¼1) and in an aqueous
solution (3¼78.4). To obtain a more definitive estimate of the
conformational flexibility and relative stabilities of 1 in dif-
ferent states, geometry optimization was carried out in the
solution using the polarizable continuum model (PCM)
and in the gas phase. The single-point calculation was
performed for the crystal structure. The computed SCF
energies and energy differences for 1 in different states are
summarized in Table 5.
1.528
1.1
1.09
1.526
1.1
1.09
1.09
1.09
Bond angles
C(1)–O(1)–H(1)
C(4)–O(3)–H(3O)
N(2)–N(1)–C(2)
N(2)–N(1)–C(5)
C(2)–N(1)–C(5)
O(4)–N(2)–N(1)
O(2)–C(1)–O(1)
O(2)–C(1)–C(2)
O(1)–C(1)–C(2)
N(1)–C(2)–C(1)
N(1)–C(2)–C(3)
C(1)–C(2)–C(3)
N(1)–C(2)–H(2)
C(1)–C(2)–H(2)
C(3)–C(2)–H(2)
C(4)–C(3)–C(2)
C(4)–C(3)–H(31)
C(2)–C(3)–H(31)
C(4)–C(3)–H(32)
C(2)–C(3)–H(32)
H(31)–C(3)–H(32)
O(3)–C(4)–C(5)
O(3)–C(4)–C(3)
C(5)–C(4)–C(3)
O(3)–C(4)–H(4)
C(5)–C(4)–H(4)
C(3)–C(4)–H(4)
N(1)–C(5)–C(4)
N(1)–C(5)–H(51)
C(4)–C(5)–H(51)
N(1)–C(5)–H(52)
C(4)–C(5)–H(52)
H(51)–C(5)–H(52)
109(2)
109(2)
107
109
109
109
124.4(1)
121.7(1)
113.5(1)
113.3(1)
125.6(1)
124.5(1)
109.8(1)
111.9(1)
101.7(1)
112.4(1)
107(1)
111(1)
113(1)
103.7(1)
111(1)
109(1)
113(1)
110(1)
110(2)
109.0(1)
110.5(1)
102.8(1)
111(1)
112(1)
112(1)
101.6(1)
108(1)
116(1)
123.4
121.6
114.2
114.4
123.9
124.8
111.2
111.6
102.2
112.0
109
109
113
104.5
109
113
110
113
108
107.1
112.4
103.1
111
112
113
124.2
121.6
113.6
115.2
124.5
124.5
111.0
111.9
102.4
112.4
109
109
112
104.4
109
110
112
112
109
107.5
112.1
102.9
111
112
111
The criteria of Cremer and Pople22 have been used to ana-
lyze the non-planar character of the five-membered ring.
Thus, a ‘pure envelope’ conformation with apex at 1 would
be such that
z2 ¼ z5; z3 ¼ z4; j ¼ k ꢂ 36;
ð1Þ
and a ‘pure twist’ with axis through 1 would have
z1 ¼ z2 þ z5 ¼ z3 þ z4; j ¼ k ꢂ 36 þ 18;
ð2Þ
where the values of zi are the displacements of ith atom
perpendicular to the least-square plane.
102.5
111
110
112
111
102.4
110
113
110
111
However, it should be noted that for a ring with unequal
lengths and angles, the conditions in Eq. 1 do not necessarily
imply coplanarity of atoms 2–5. The list of zi values for 1 in
different states is summarized in Table 6.
107(1)
111(1)
113(2)
110
110
The most stable conformer of 1 is observed in an aqueous
solution. In this state according to the criteria of Cremer
and Pople (Tables 3 and 6) the pyrrolidine ring adopts an
envelope conformation, which is also maintained in the gas
phase. The deviations of the carbon atom C(4) from the
best-fit planes [N(1)/C(2)/C(3)/C(5)] in the solution and
of two molecules. It is evident that the best fitting was found
for the zwitterion of Hyp, where the atoms of the pyrrolidine
˚
rings of the two molecules fit within 0.06 A. Figure 2 also
reveals an essential difference in an arrangement of the
carboxylic and hydroxyl groups that is evident from the
corresponding torsion angles. Torsion angles for Hyp in
Table 2 are taken from Ref. 11.
˚
gas phase are equal to ꢀ0.57 and ꢀ0.56 A, respectively. In
the crystal structure of 1 the pyrrolidine ring adopts a confor-
mation, which is twisted on C(3)–C(4) bond. Therefore for 1
we performed the conformational search (CS) implemented
in Hyperchem 6.03 using the MM+ force field varying the di-
hedral angles inside the pyrrolidine ring. The envelope con-
formation of this ring is maintained in the lowest energy
A view of the molecular packing and hydrogen-bonding
scheme is shown in Figure 3. The hydrogen-bonding geo-
metry is summarized in Table 4. A classical hydrogen bond
between the carboxyl hydrogen and the hydroxyl oxygen

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M. S. Fonari et al. / Tetrahedron 62 (2006) 8779–8786
Table 2. Torsion angles (degree) for 1 and L-Hyp11
IUPAC designation21
Atoms involved
1
L-Hyp11
j1
O1–C–Ca–N1
O2–C–Ca–N
O(1)–C(1)–C(2)–N(1)
O(2)–C(1)–C(2)–N(1)
N(1)–C(2)–C(3)–C(4)
C(2)–C(3)–C(4)–O(3)
C(2)–C(3)–C(4)–C(5)
H(3O)–O(3)–C(4)–C(3)
H(3O)–O(3)–C(4)–C(5)
O(3)–C(4)–C(5)–N(1)
C(3)–C(4)–C(5)–N(1)
C(4)–C(5)–N(1)–C(2)
C(5)–N(1)–C(2)–C(3)
O(4)–N(2)–N(1)–C(2)
O(4)–N(2)–N(1)–C(5)
151.2(1)
ꢀ31.6(2)
ꢀ30.0(1)
ꢀ73.6(1)
41.4(1)
ꢀ30.7(2)
ꢀ142.4(2)
81.7(1)
ꢀ3.2(2)
178.7(1)
ꢀ18.3(2)
ꢀ87.0(2)
32.0(2)
ꢀ158.8(2)
87.7(2)
84.0(1)
ꢀ33.5(2)
23.1(1)
ꢀ3.1(1)
—
j2
c1
N1–Ca–Cb–Cg
Ca–Cb–Cg–Od
Ca–Cb–Cg–Cd
c2,1
c2,2
c3,1,1
c3,1,2
c3,2,1
c3,2,2
c4
Hd1–Od–Cg–Cb
Hd1–Od–Cg–Cd
Od–Cg–Cd–N
Cb–Cg–Cd–N1
Cg–Cd–N1–Ca
Cd–N1–Ca–Cb
O4–N2–N1–Ca
O4–N2–N1–Cd
ꢀ35.2(1)
17.3(1)
8.0(1)
c5
4.6(2)
176.6(1)
—
Table 3. Computed ring puckering parameters for 1, L-Hyp,a and Prob
Compound
Crystal
Solution (3¼78.4)
q/A
Gas phase (3¼1)
q/A
Conformers 1a, 1H
j/ ꢁ
˚
j/ꢁ
j/ꢁ
j/ꢁ
q/A
˚
q/A
˚
˚
1
0.403(1)
0.236
276.1(2)
309.6
0.373
0.376
0.363
0.371
279.3
270.3
281.9
314.1
0.330
0.384
280.2
336.4
L-Hyp
DL-Pro
L-Pro
0.403(2)
0.404
57.7(2)
89.1
DL-Pro monohydrate
L-Pro monohydrate
0.4033(5)
0.395(4)
308.63(7)
309.9(6)
a
Atomic coordinates for the computing calculations are taken from Cambridge Structural Database (refcode HOPROL).
All cited puckering parameters for Pro are taken from Ref. 6.
b
conformer 1a according to the criteria of Cremer and Pople
(Tables 3 and 6). In this molecule the deviation of the Cg
[C(4)] carbon atom from the best-fit plane [N(1)/C(2)/C(3)/
3. Conclusion
4-Hydroxy-L-proline, an amino acid, an important com-
ponent of collagen, was transformed into its potentially
carcinogenic N-nitroso-derivative, 4-hydroxy-1-nitroso-L-
pyrrolidine-2-carboxylic acid by butylnitrite in the acidic
medium. The X-ray structural analysis and ab initio calcula-
tions based on density functional theory at the B3LYP/
6-311++G(d, p) level of theory were performed to analyze
the influence of the N-nitroso-group on conformation of syn-
thesized N-nitroso-compound. The most stable conformer of
1 was observed in an aqueous solution. In this state the pyr-
rolidine ring adopts an envelope conformation, which is
maintained in the gas phase. In the crystal structure of 1
this ring adopts a twisted conformation. The change of the
five-membered ring conformation in solution and gas phase
in comparison with the crystal is accompanied by an
increase of the dipole moment of the molecule, which is
maximal in solution.
˚
C(5)] is equal to ꢀ0.51 A and the values of O(2)–C(1)–C(2)–
N(1) (ꢀ52.0ꢁ) and O(3)–N(2)–N(1)–C(2) (11.1ꢁ) torsion
angles are maximal in comparison with those in other states.
For the crystal, gas, and solvent states these values are equal
to ꢀ31.6(2)ꢁ, 4.6(2)ꢁ; ꢀ41.18ꢁ, 6.55ꢁ; and ꢀ33.67ꢁ, 5.86ꢁ,
respectively. The change of pyrrolidine ring conformation
in solution and in gas phase in comparison with the crystal
is accompanied by an increase in the dipole moment of the
molecule, which is maximal in solution (Table 5).
In order to study the influence of nitrosation on the conforma-
tion of pyrrolidine ring, the DFT-based geometry optimiza-
tions as well as the CS implemented in Hyperchem 6.03
were carried out for hydroxy-L-proline (Hyp) in solution,
gas, and crystal phases. The atomic numbering scheme for
hydroxy-L-proline (Hyp) is the same as that of compound
1. As in the case of 1, the most stable conformer is observed
in solution (Table 5). In the crystal and in solution, the pyrro-
lidine ring adopts the conformation twisted on the C(4)–C(5)
bond. In the gas phase and in the conformer 1H the pyrroli-
dine rings are twisted on C(3)–C(4) and N(1)–C(5) bonds, re-
spectively. Thus, the twisted conformation of the pyrrolidine
ring is present in all conformers of Hyp and in the crystal
structure of 1. However, due to nitrosation of Hyp, this ring
in the crystalline state is twisted on the C(3)–C(4) bond,
while in the same state of Hyp the ring is twisted along the
C(4)–C(5) bond. In solution and in the gas phase after nitro-
sation the pyrrolidine ring in 1 adopts an envelope conforma-
tion. The optimized geometries of 1 and Hyp in solution and
in gas phase as well as bond distances and bond angles in their
crystal structures are listed in Table 1.
4. Experimental
4.1. General
The initial chemicals were used as received without further
purification. IR spectra were recorded on a Specord-80 spec-
trophotometer as KBr disks. 1H and 13C NMR spectra were
recorded with a Bruker DPX-250 instrument (300 MHz 1H;
75 MHz 13C) in dimethylsulfoxide using TMS as an internal
reference. Mass spectra were recorded on a MX-1321 de-
vice. Mass spectrometer operating at 70 eV. Perkin–Elmer
241 MC polarimeter was used for [a]2D0 measurements.
Crystallographic measurements were carried out on a PX

M. S. Fonari et al. / Tetrahedron 62 (2006) 8779–8786
8783
(a)
(b)
(c)
Figure 2. Fitting of the molecules 1 and Hyp derivatives (side and top views). Molecule of 1 is shown by solid lines. C-bound H-atoms are omitted for clarity. (a)
1 and 4-hydroxy-L-proline11 (refcode HOPROL12 in CSD). Atoms of the pyrrolidine ring fit within 0.062 A; (b) 1 and 4-hydroxy-N-methylproline13 (refcode
˚
UGUHOT in CSD). Atoms of the pyrrolidine ring fit within 0.172 A; and (c) 1 and N-acetyl-L-prolyl-l-4-hydroxyproline25 (refcode GLHPRC in CSD). Atoms of
˚
˚
the pyrrolidine ring fit within 0.168 A.
kappa-geometry diffractometer with Onyx CCD camera at
100 K. The structure was solved by direct methods and
refined by full-matrix least-squares on F2 using SHELX-97
package.27 All non-hydrogen atoms were refined anisotropi-
cally. Locations of all H-atoms were justified by difference
Fourier synthesis and refined isotropically. The absolute
configuration was not been defined.
Ab initio calculations were carried out using density func-
tional theory with the Gaussian 03 package at the B3LYP/
6-311++G(d, p) level of theory.28 Polarizable continuum
model (PCM) was included in the SCF procedure for the
description of aqueous solution. The dielectric constant
was set at 78.4. All calculations were carried out using the
restricted spin formalism (closed-shell). Crystallographic

8784
M. S. Fonari et al. / Tetrahedron 62 (2006) 8779–8786
(a)
(b)
Figure 3. (a) Top and (b) side view of the layer of molecules 1.
Table 4. Hydrogen-bonding geometry in 1
:DHA/ꢁ
Symmetry code for acceptor
˚
˚
D–H/A
d(H/A)/A
d(D/A)/A
O(1)–H(1)/O(3)
O(3)–H(3O)/O(4)
O(3)–H(3O)/N(2)
C(3)–H(31)/O(4)
1.81(3)
1.79(2)
2.47(2)
2.50(2)
2.625(1)
2.681(2)
3.254(2)
3.152(2)
167(3)
171(2)
146(2)
124(1)
x+1, y, z
ꢀx+1/2, ꢀy+1, zꢀ1/2
ꢀx+1/2, ꢀy+1, zꢀ1/2
ꢀx+1, y+1/2, ꢀz+1/2
data available for 1 and Hyp provided the initial geometries.
The search of conformers was performed using the module
conformational search implemented in Hyperchem 6.0329
for finding low energy conformations of molecular systems
by varying user-specified dihedral angles. The method
involves random variation of dihedral angles to generate
new structures and then energy minimizing of these angles
using the MM+ force field. The dihedral angles in a ring
are rotated by the ‘torsion flexing’ motion of Kolossvary
and Guida,30 which effectively leads to new ring conforma-
tions while avoiding large atomic displacements that can
decrease the efficiency of optimization. The analysis of
conformations has been carried out using the PLATON
program package.31
Table 5. Computed SCF energies, energy differences, and dipole moments for 1 and Hyp compounds in the crystal, gas phase, and in solution and conformers 1a
and 1Ha
1
Hyp
E/au
DE/au
m/D
E/au
DE/au
m/D
Crystal
ꢀ605.7354
ꢀ605.864
ꢀ605.902
ꢀ605.7952
0
6.3821
6.0627
9.1683
6.0367
ꢀ476.3587
ꢀ476.5233
ꢀ476.5532
ꢀ476.4664
0
2.1041
1.87
2.4104
5.7367
Gas phase
Solution
1a, 1H
0.1286
0.1666
0.0598
0.1646
0.1945
0.1077
a
Conformers found by the conformational search implemented in Hyperchem 6.03 for 1 (1a) and Hyp (1H).

M. S. Fonari et al. / Tetrahedron 62 (2006) 8779–8786
Table 6. Displacements of pyrrolidine ring atoms from the least-square plane
8785
z1
z2
z3
z4
z5
1
Atoms of the pyrrolidine ring
Crystal
Gas phase
Solution
1a
N(1)
0.027(1)
0.037
0.0485
0.0385
C(2)
0.129(1)
0.1032
0.0963
0.0894
C(3)
ꢀ0.2324(14)
C(4)
C(5)
0.249(1)
0.2269
0.2344
0.2069
ꢀ0.1707(14)
ꢀ0.1631
ꢀ0.1748
ꢀ0.1516
ꢀ0.204
ꢀ0.2044
ꢀ0.1831
Hyp
Crystal
Gas phase
Solution
1H
0.0951
0.0014
0.1657
0.223
ꢀ0.0093
0.1369
ꢀ0.0801
ꢀ0.2229
ꢀ0.1112
ꢀ0.0237
0.1389
0.2237
0.2136
0.1613
ꢀ0.1446
ꢀ0.1391
ꢀ0.2344
ꢀ0.2374
ꢀ0.0337
ꢀ0.123
4.1.1. 4-Hydroxy-1-nitroso-L-pyrrolidine-2-carboxylic
acid, 1. 4-Hydroxy-L-proline (0.2 g) was stirred with the so-
lution of butylnitrite (0.3 ml) and glacial acetic acid (0.2 ml)
at 25 ꢁS for 7 days. The colorless precipitate of 4-hydroxy-1-
nitrosopyrrolidine-2-carboxylic acid was filtered off and
recrystallized from a 1:1 mixture of n-butanol (5 ml) and
methanol (5 ml). The yield of 4-hydroxy-1-nitrosopyrrol-
idine-2-carboxylic acid was 82%, mp¼138–140 ꢁC (dec).
Anal. Calcd for C5H8N2O4: C, 37.50; H, 5.04; N, 17.49.
Found: C, 37.47; H, 5.09; N, 17.53. IR (cmꢀ1, peaks of strong
absorption only, Vaseline oil): 3500–3200, 3170, 3150, 3125,
3120, 3110, 3090, 3080, 3050, 3040, 3030, 3020, 3000–2800,
2780–2570, 1730, 1430, 1365, 1310, 1290, 1280, 1260, 1250,
1190, 1130, 1060. 1H NMR d (ppm) (DMSO-d6, 300 MHz):
References and notes
ˇ
ꢀ
ꢀˇ
1. Reha, D.; Valdes, H.; Vondrasek, J.; Hobza, P.; Abu-Riziq, A.;
Crews, B.; de Vries, M. S. Chem.—Eur. J. 2005, 11, 6803–
6817.
2. (a) Collagen; Nimni, M. E., Ed.; CRC: Boca Raton, FL, 1988;
Vols. 1–4; (b) Biochemistry of Collagen; Ramachandran, G. N.,
Reddi, A. H., Eds.; Plenum: New York, NY, 1976.
3. (a) Improta, R.; Benzi, C.; Barone, V. J. Am. Chem. Soc. 2001,
123, 12568–12577; (b) Benzi, C.; Improta, R.; Scalmani, G.;
Barone, V. J. Comput. Chem. 2002, 23, 341–350; (c) Czinki,
ꢀ ꢀ
E.; Csaszar, A. G. Chem.—Eur. J. 2003, 9, 1008–1019; (d)
Stepanian, S. G.; Reva, I. D.; Radchenko, E. D.; Adamowicz,
L. J. Phys. Chem. A 2001, 105, 10664–10672; (e) Myung, S.;
Julian, R. R.; Nantia, S. C.; Cooks, R. G.; Clemmer, D. E.
J. Phys. Chem. B 2004, 108, 6105–6111.
1.61 m (2H, CH2), 2.78 m (2H, CH2), 3.35 m (1H, CH). 13
C
NMR d (ppm) (DMSO-d6, 300 MHz): 34.62 (S–CH2–S),
54.15 (S–CH2–N), 56.18 (S–CH–COOH), 67.54 (S–CH2–
OH), 172.94 (SOOH). MS: m/z 160(22) (calcd for
C5H8N2O4, 160.128) 130(5), 115(46), 88(20), 86(24),
85(16), 84(22), 68(15), 56(100); [a]2D0 ꢀ1.47.
4. Kim, W.; McMillan, R. A.; Snyder, J. P.; Conticello, V. P. J. Am.
Chem. Soc. 2005, 127, 18121–18132.
5. Kayushina, R. L.; Vainshtein, B. K. Kristallografiya 1965, 10,
833–844.
6. Myung, S.; Pink, M.; Baik, M.-H.; Clemmer, D. E. Acta
Crystallogr., Sect. C 2005, 61, o506–o508.
7. Janczak, J.; Luger, P. Acta Crystallogr., Sect. C 1997, 53, 1954–
1956.
8. Padmanabhan, S.; Suresh, S.; Vijayan, M. Acta Crystallogr.,
Sect. C 1995, 51, 2098–2100.
9. Zussman, J. Acta Crystallogr. 1951, 4, 493–495.
10. Donohue, J.; Trueblood, K. N. Acta Crystallogr. 1952, 5,
414–419.
Crystal data for (1), orthorhombic, P212121, a¼7.644(2),
3
˚
˚
b¼8.849(3), c¼10.303(3) A, V¼696.9(4) A , Z¼4, Dx¼
1.526 g cmꢀ3
,
T¼100 K, l(Mo Ka)¼0.71073 A, m¼
˚
1.33 cmꢀ1, F(000)¼336, GooF¼1.097, R indices (all data)
R1¼0.0337, wR2¼0.0836 for 1369 reflections and 132
parameters and R indices R1¼0.0309, wR2¼0.0821 for
1261 reflections obeying I>2s(I) criterion of observability.
Crystallographic data for the structural analysis of com-
pound 1 have been deposited at the Cambridge Crystallo-
graphic Data Center, CCDC no. 602946. Copies of this
information may be obtained free of charge from The Direc-
tor, CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (fax:
+44 1233 336 033; e-mail: deposit@ccdc.cam.ac.uk or
http://www.ccdc.cam.ac.uk).
11. Koetzle, T. F.; Lehmann, M. S.; Hamilton, W. C. Acta
Crystallogr., Sect. B 1973, 29, 231–236.
12. Shamala, N.; Row, T. N. G.; Venkatesan, K. Acta Crystallogr.,
Sect. B 1976, 32, 3267–3270.
13. Yapp, D. T. T.; Steed, J. W.; Houghton, P. J. Acta Crystallogr.,
Sect. E 2002, 58, o988–o989.
14. Jones, G. P.; Naidu, B. P.; Paleg, L. G.; Tiekink, E. R. T. Acta
Crystallogr., Sect. C 1988, 44, 2208–2211.
€
15. Gessmann, R.; Bruckner, H.; Kokkinidis, M. Acta Crystallogr.,
Sect. E 2001, E57, o492–o494.
Acknowledgements
16. Sabesan, M. N.; Venkatesan, K. Acta Crystallogr., Sect. B 1971,
27, 1879–1883.
The authors thank Professor A. S. Dimoglo for the computa-
tional facility.
17. (a) Licht, W. R.; Fox, J. G.; Deen, W. M. Carcinogenesis 1988,
9, 373–377; (b) Satarug, S.; Haswell-Elkins, M. R.; Tsuda, M.;
Mairiang, P.; Sithithaworn, P.; Mairiang, E.; Esumi, H.;
Sukprasert, S.; Yongvanit, P.; Elkins, D. B. Cancer Res.
1983, 43, 5072–5076; (c) Ladd, K. F.; Newmark, H. L.;
Archer, M. C. J. Natl. Cancer Inst. 1984, 73, 83–87; (d)
Supplementary data
Supplementary data associated with this article can be found
in the online version, at doi:10.1016/j.tet.2006.06.097.

8786
M. S. Fonari et al. / Tetrahedron 62 (2006) 8779–8786
Satarug, S.; Haswell-Elkins, M. R.; Tsuda, M.; Mairiang, P.;
28. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.;
Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.;
Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi,
M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.;
Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.;
Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.;
Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.;
Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.;
Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski,
J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.;
Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels,
A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, D.;
Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.;
Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.;
Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin,
R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.;
Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson,
B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A.
Gaussian 03, Revision B.01; Gaussian: Pittsburgh, PA, 2003.
29. Hypercube Inc. Hyperchem 6; Gainesville, FL, 2001.
30. Kolossvary, I.; Guida, C. J. Comput. Chem. 1993, 14,
691–702.
Sithithaworn, P.; Mairiang, E.; Esumi, H.; Sukprasert, S.;
Yongvanit, P.; Elkins, D. B. Carcinogenesis 1996, 17, 1075–
1081; (e) Ohshima, H.; Bartsch, H. Cancer Res. 1981, 41,
3658–3666.
18. Simonov, Yu. A.; Fonari, M. S.; Duca, G. G.; Gonta, M. V.;
Ganin, E. V.; Yavolovskii, A. A.; Gdaniec, M.; Lipkowski, J.
Tetrahedron 2005, 61, 6596–6601.
19. Simonov, Yu. A.; Gonta, M. V.; Yavolovskii, A. A. Acta
Crystallogr., Sect. E 2005, 61, o3136–o3139.
20. Roth, E., Jr.; Rinaldi, A.; Fiori, G. M.; Filippi, G. Am. J.
Hematol. 1985, 20, 153–159.
21. IUPAC–IUB Commission on Biochemical Nomenclature.
J. Mol. Biol. 1970, 52, 1–17.
22. Cremer, D.; Pople, J. A. J. Am. Chem. Soc. 1975, 97, 1354–
1358.
23. Geise, H. J.; Altona, C.; Romers, C. Tetrahedron Lett. 1967, 8,
1383–1386.
24. Allen, F. N. Acta Crystallogr., Sect. B 2002, 58, 380–388.
25. Garbay-Jaureguiberry, C.; Arnoux, B.; Prange, T.; Wehri-
Altenburger, S.; Pascard, C.; Roques, B. P. J. Am. Chem. Soc.
1980, 102, 1827–1837.
26. Etter, M. Isr. J. Chem. 1985, 25, 312–319.
27. Sheldrick, G. M. SHELXS97 and SHELXL97; University of
31. Spek, A. L. PLATON; University of Utrecht: Utrecht, The
Netherlands, 2002.
€
Gottingen: Gottingen, Germany, 1997.
€