X-ray crystallography of tetracycline, doxycycline and sancycline

Article abstract of DOI:10.1007/s10870-013-0407-0

The molecular and crystal structures of α-6-deoxy-β-5- oxytetracycline (doxycycline) hydrochloride, of tetracycline hydrochloride and tetracycline hexahydrate were re-determined. The crystal structures of 6-deoxy-6-dimethyl-tetracycline (sancycline) hydrochloride were, for the first time, determined by single crystal X-ray diffraction technique. All crystals studied exhibit the orthorhombic space group P2₁2₁2 ₁ with 4 molecules per unit cell. The starting material tetracycline (TC) hydrochloride crystallizes from aqueous solution, independently of the pH of the mother liquor, as hexahydrate complex of the zwitterion with the same molecular structure (a = 9.585(3) A, b = 12.112(3) A, c = 21.671(6) A). From methanolic solution, tetracycline hydrochloride crystallizes as hydrochloride (a = 11.001(3) A, b = 12.852(4) A, c = 15.795(3) A). Doxycycline hydrochloride crystallizes from acidic aqueous solution as dihydrate complex (a = 11.115(4) A, b = 12.768(4) A, c = 16.921(5) A). In both hydrochloride crystals, the amide group is protonated and oriented such that an intramolecular hydrogen bond is formed between the amide oxygen and O3. In the tetracycline-hexahydrate crystal, the amide group of the zwitterion is rotated by about 180 (vs the cation) with a hydrogen bond being made by one hydrogen of its amino group and O3^-. Sancycline hydrochloride crystallized from an acidified solvent mixture (water and methanol) adopts a geometry close to that of the tetracycline cation, e.g., with a hydrogen bond between the protonated amide oxygen and O3 (a = 6.8944(4) A, b = 16.815(2) A, c = 18.190(2) A). But the dimethyl ammonium group of sancycline hydrochloride is disordered with respect to its orientation. In the majority fraction (65.9 %), the proton at N4 is directed towards O3, which has a water molecule next to it. In the minority fraction (34.1 %), the proton at N4 points away from O3 and has a methanol molecule in its neighborhood. The bond length variations in ring A of all compounds studied do not reflect the location of single and double bonds in any of the classical mesomeric structures. By comparison with calculated Wiberg-bond orders we show that this apparent discrepancy is not due to the existence of different tautomers in the crystals as discussed previously but merely a consequence of the π-conjugation in one tautomer. Graphical Abstract: Although the packing of the three title compounds in the respective crystals is different, the geometries of the molecular framework are nearly the same as the one shown for the sancycline cation. A comparison of experimentally determined bond lengths with calculated Wiberg-Bond orders provides evidence that only one tautomer is present in the crystal.[Figure not available: see fulltext.].

Full text of DOI:10.1007/s10870-013-0407-0

J Chem Crystallogr (2013) 43:213–222
DOI 10.1007/s10870-013-0407-0
ORIGINAL PAPER
X-Ray Crystallography of Tetracycline, Doxycycline
and Sancycline
•
Frank W. Heinemann Clemens F. Leypold
•
•
Cyprian R. Roman Matthias O. Schmitt
•
Siegfried Schneider
Received: 14 April 2011 / Accepted: 25 February 2013 / Published online: 9 March 2013
Ó Springer Science+Business Media New York 2013
Abstract The molecular and crystal structures of a-6-
deoxy-b-5-oxytetracycline (doxycycline) hydrochloride, of
tetracycline hydrochloride and tetracycline hexahydrate
were re-determined. The crystal structures of 6-deoxy-6-
dimethyl-tetracycline (sancycline) hydrochloride were, for
the first time, determined by single crystal X-ray diffraction
technique. All crystals studied exhibit the orthorhombic
space group P212121 with 4 molecules per unit cell. The
starting material tetracycline (TC) hydrochloride crystallizes
from aqueous solution, independently of the pH of the
mother liquor, as hexahydrate complex of the zwitterion
hydrogen of its amino group and O3-. Sancycline hydro-
chloride crystallized from an acidified solvent mixture
(water and methanol) adopts a geometry close to that of the
tetracycline cation, e.g., with a hydrogen bond between the
˚
protonated amide oxygen and O3 (a = 6.8944(4) A,
˚
˚
b = 16.815(2) A, c = 18.190(2) A). But the dimethyl
ammonium group of sancycline hydrochloride is disordered
with respect to its orientation. In the majority fraction
(65.9 %), the proton at N4 is directed towards O3, which has
a water molecule next to it. In the minority fraction (34.1 %),
the proton at N4 points away from O3 and has a methanol
molecule in its neighborhood. The bond length variations in
ring A of all compounds studied do not reflect the location of
single and double bonds in any of the classical mesomeric
structures. By comparison with calculated Wiberg-bond
orders we show that this apparent discrepancy is not due to
the existence of different tautomers in the crystals as dis-
cussed previously but merely a consequence of the p-con-
jugation in one tautomer.
˚
with the same molecular structure (a = 9.585(3) A, b =
˚
12.112(3) A, c = 21.671(6) A). From methanolic solution,
˚
tetracycline hydrochloride crystallizes as hydrochloride
˚
˚
˚
(a = 11.001(3) A, b = 12.852(4) A, c = 15.795(3) A).
Doxycycline hydrochloride crystallizes from acidic aqueous
˚
solution as dihydrate complex (a = 11.115(4) A, b =
˚
˚
12.768(4) A, c = 16.921(5) A). In both hydrochloride
crystals, the amide group is protonated and oriented such that
an intramolecular hydrogen bond is formed between the
amide oxygen and O3. In the tetracycline–hexahydrate
crystal, the amide group of the zwitterion is rotated by about
180° (vs the cation) with a hydrogen bond being made by one
Keywords Molecular and crystal structures Á
Tetracycline Á Doxycycline Á Sancycline Á Wiberg-bond
order
In memoriam: Prof. Dr. Dieter Sellmann.
Introduction
F. W. Heinemann
Lehrstuhl fu¨r Anorganische und Allgemeine Chemie,
Since the discovery of the antibiotic action of tetracyclines
[1] (for chemical structure and generally adopted num-
bering of atoms see Fig. 1), much work has been devoted
to developing an understanding of the protonation–depro-
tonation equilibria (Scheme 1), the tautomerism of the
neutral and mono-anionic form and the geometry adopted in
dependence on the state of protonation and kind of envi-
ronment (solvent). Despite the large effort and the great
¨
Friedrich-Alexander-Universitat Erlangen-Nurnberg,
Egerlandstr.1, 91058 Erlangen, Germany
¨
C. F. Leypold Á C. R. Roman Á M. O. Schmitt Á
S. Schneider (&)
Lehrstuhl fu¨r Physikalische Chemie I,
¨
Friedrich-Alexander-Universitat Erlangen-Nurnberg,
Egerlandstr.3, 91058 Erlangen, Germany
¨
e-mail: Siegfried.Schneider@chemie.uni-erlangen.de
123

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J Chem Crystallogr (2013) 43:213–222
dissolved directly in aqueos buffer at pH 2 or pH 11,
respectively [7, 11, 13–15]. During the course of a titration
experiment, the conformation changes with the effect that
the mixture of conformers (tautomers) depends on titration
history (Remember, drugs taken orally experience during
their passage through the intestines an environment of
largely different pH-values!).
In order to contribute towards an answer of unresolved
questions we present here the molecular structures of
tetracycline (zwitterion), tetracycline hydrochloride (cat-
ion), doxycycline hydrochloride (cation) and sancycline
hydrochloride (cation) obtained by conventional X-ray
crystallography from samples crystallized under differ-
ent experimental conditions. ZDO Atomic Charges and
Wiberg-bond orders are calculated for various tautomers
of sancycline to rationalize the observed variations of
bond lengths.
Fig. 1 Molecular structure of tetracyclines investigated and conven-
tional numbering of atoms and rings (only one of the possible
isomeric/tautomeric forms is shown)
Experimental Section
variety of spectroscopic techniques applied many of the
addressed questions are still not satisfactorily answered. The
elucidation of these properties is, however, considered
essential to estimate the pharmacokinetic properties, as, e.g.,
the bio-availability or the permeability through membranes.
With the advent of new applications in various medical
fields (non-antibiotic actions) a complete knowledge of the
structural properties is more desirable than ever before. (For
a review of all topics related to tetracyclines reference [2] is
recommended.)
Preparation of (Single) Crystals
Tetracycline hydrochloride was bought from Aldrich
(Germany), doxycycline hydrochloride from Fluka
(Germany) and sancycline from Clariant (Origgio, Italy).
For the preparation of aqueous solutions of tetracycline,
the poly-crystalline material was dissolved in bi-distilled
water. For adjustment of the desired pH, either HCl or
ammonia water was added [11]. Three different types of
tetracycline crystals and one each of doxycycline and
sancycline were grown by the following procedures:
Most crystallographic work on tetracycline and deriva-
tives was done in the late 1970s and early 1980s and free
refinement of H atoms was not possible in all cases [3–9].
Consequently, very similar geometries of the heavier atoms
have been interpreted in terms of different tautomers [8, 9]
with severe consequences on the assignment of experimen-
tally determined pKa-values. Only recently it could be
unambiguously shown that tetracycline hydrochloride exists
in the crystalline state as that tautomer in which the proton is
located at the amide oxygen rather than at O3 [10, 11].
Tetracycline and oxytetracycline (b-5-oxytetracycline)
crystallize in the absence of water in the neutral form with a
conformation termed B by Prewo and Stezowski [7], in
which both the dimethylamino group and the tricarbonyl
system are above the plane spanned by rings B, C and D.
The X-ray structure published by Koziol et al. verifies this
conclusion [12]. (because there exists a confusing termi-
nology in the literature describing various conformations of
tetracyclines, a survey is given in the ‘‘Appendix’’ section ).
More recent studies provide strong evidence that in
aqueous solution tetracycline hydrochloride adopts the
same conformation as in the crystal if the solid material is
(Crystal I) Tetracycline hexahydrate (acidic):
Aqueous solutions of TcÁHCl (69 mM) were adjusted to
pH 1.83 and stored in the refrigerator for several days
until crystals formed.
(Crystal II) Tetracycline hexahydrate (alkaline):
Aqueous solutions of TcÁHCl were adjusted to pH 9.0
and crystallized in the refrigerator.
(Crystal III) Tetracycline hydrochloride (Á0.25H2O):
Crystallization at room temperature from methanolic
solution by slow evaporation of the solvent.
(Crystal IV) Doxycycline hydrochloride dihydrate:
The polycrystalline material was dissolved in water
(69 mM) and the pH adjusted to about 1 by addition of
concentrated HCl. Afterwards, the pH was raised to 1.84
by addition of ammonia water and the solution stored for
24 h. Several of the separated small crystals were added
back to the decanted mother liquor. After several days of
storage in the refrigerator, single crystals large enough
for X-ray diffractometry could be isolated.
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J Chem Crystallogr (2013) 43:213–222
215
Scheme 1 .
(Crystal V) Sancycline hydrochloride (Áhydrate/methanolate):
3.6 g sancycline was given into a beaker containing
100 ml water and 10 ml of 10 M HCl (equivalent to
69 mM). Then, methanol was added slowly until a
saturated solution of sancycline hydrochloride was
formed. Aliquots of 20 ml of such solutions were
made subject to different procedures, which are usually
applied for crystallization [16]. Only that sample which
rested for several weeks at room temperature in an
open beaker produced small crystals suitable for
crystallography.
corresponding CCDC reference numbers are given in
Table 1: CCDC-243335-38 and CCD-C864575.
Results and Discussion
All samples investigated crystallized in the orthorhombic
space group P212121 (no. 19) with four molecules per unit
cell. Details of the structure refinement and selected crys-
tallographic data are summarized in Table 1 to allow an
easy comparison. Stereo views of the Ortep plots of tetra-
cycline zwitterion and doxycycline cation provide a good
impression of the extensively discussed conformation of
these compounds.
X-Ray Crystal Structure Determinations
Suitable single crystals were sealed in a glass capillary
under N2. Intensity data were either collected on a Nicolet
R3m/V (crystals I–IV) or on a Bruker–Nonius KappaCCD
(crystal V) diffractometer (using Mo-Ka radiation
Tetracycline (Cation and Zwitterion)
Crystallographic R-values as well as estimated standard
deviations for the sample TcÁ6H2O (alkaline) are worse
than those for the sample TcÁ6H2O (acidic). This reflects
the fact that the crystal produced from the alkaline solution
was smaller and obviously less good than the one produced
from the acidic solution. Nevertheless, one can conclude
that the crystal structure of TcÁ6H2O is unique and inde-
pendent of the pH of the mother liquor. In two earlier
studies, tetracycline was crystallized from a water–acetone
mixture [4] and water [5], respectively. The crystallo-
graphic data are in accord with those presented in Table 1.
Neighboring tetracycline molecules are arranged mostly
face-to-edge. Intermolecular hydrogen bonds are found
between O2AM and the hydroxyl groups attached to C6
and C12A, respectively, of different neighbors. They
should have no significant effect on the molecular struc-
ture. Figure 2 illustrates the existence of the above
described A conformation. Like in all tetracycline deriva-
tives strong intramolecular hydrogen bonds between O11
and O10 and O11 and O12 stabilize the geometry of rings
B, C and D.
˚
(k = 0.71073 A, graphite monochromator). Data were
corrected for Lorentz and polarization effects while an
absorption correction has not been applied except for
crystals II and V. Atomic scattering factors used and
corrections for anomalous dispersion were taken from
Reference [17]. Where appropriate (sufficient anomalous
dispersion present, crystals III–V), the absolute structure
parameters were determined according to Flack [18]. All
structures were solved by direct methods and refined using
full-matrix least-squares procedures on F2 values (SHEL-
XTL NT 6.12) [19]. All non-hydrogen atoms were refined
with anisotropic displacement parameters. The positions of
the hydrogen atoms of crystals I, III, and IV and those of
the solvent water molecules of II were taken from a dif-
ference Fourier synthesis and their positional parameters
were either kept fixed (I and solvent water hydrogens of II)
or refined (III (except solvent water) and IV). The
remaining hydrogen atoms of II and all of V were placed in
positions of optimized geometry. The isotropic displace-
ment parameters of all hydrogen atoms were tied to those
of their corresponding carrier atoms by a factor of either
1.2 or 1.5.
For the sake of completeness, it should be mentioned
that tetracycline was also crystallized from an acidified
D2O solution (CCDC 243339). The crystal structure was
found to be identical to that of crystal I produced from a
H2O solution.
Selected crystallographic data and further details of the
data collection and refinement of crystals I–V are sum-
marized in Table 1. Crystallographic data for the structures
reported in this paper have been deposited with the
Cambridge Crystallographic Data Centre (CCDC). The
The dimensions of the unit cell of crystal III (TcÁ
HClÁ0.25H2O) and the molecular parameters are very close
to those reported previously by Clegg and Teat [10]
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J Chem Crystallogr (2013) 43:213–222
Table 1 Selected crystallographic data and structure refinement details
TcÁ6H2O (acidic)
Crystal I
TcÁ6H2O (alcaline) TcÁHClÁ0.25H2O
DoxyÁHClÁ2H2O
SancyclineÁH2O/
LeOHd
Crystal V
Crystal II
Crystal III
Crystal IV
CCDC reference number
Formula
243335
243336
243337
243338
864575
C21.34H25.68ClN2O8d
C22H36N2O14
C22H36N2O14
552.53
C22H25.5ClN2O8.25
485.39
C22H29ClN2O10
516.92
Mr (g/mol)
552.53
473.65
Crystal size (mm)
Temperature (K)
F(000)
0.60 9 0.50 9 0.40 0.36 9 0.30 9 0.22 0.60 9 0.50 9 0.40 0.50 9 0.42 9 0.36 0.24 9 0.07 9 0.05
293
298
295
295
100
1176
1176
1016
1088
995
Crystal system
Space group
Orthorhombic
P212121
9.585(3)
12.112(3)
21.671(6)
2516(2)
4
Orthorhombic
P212121
9.603(4)
12.108(7)
21.658(10)
2518(2)
4
Orthorhombic
P212121
11.001(3)
12.852(4)
15.795(3)
2233(1)
4
Orthorhombic
P212121
11.115(4)
12.768(4)
16.921(5)
2401(2)
4
Orthorhombic
P212121
6.8944(4)
16.815(2)
18.190(2)
2108.8(4)
4
˚
a (A)
˚
b (A)
˚
c (A)
3
V (A )
˚
Z
d
calcd. (g/cm3)
1.459
1.457
1.444
1.43
1.492
Absorption coeff. l (mm-1
Scan technique
2h range (°)
Meas. reflections
Indep. reflections
Rint
Obs. Reflectionsa
Parameters refined
Ra1; wRb2
)
0.122
0.122
0.225
0.219
0.235
x scans
3.7–54.0
4879
x scans
3.7–52.0
4231
x scans
4.0–56.0
4771
x scans
4.0–56.0
5073
x scans
3.57–27.1
27723
4323
3765
4156
4432
4636
0.0188
3526
0.0933
1319
0.0255
3010
0.0256
2411
0.0734
3478
343
350
382
403
332
0.0388, 0.0950
0.963
0.0665, 0.1962
0.783
0.0339, 0.0723
0.866
0.0427, 0.0840
0.85
0.0476, 0.1052
1.038
GooFb
Absolute structure
parameter18
n.d.c
n.d.c
0.00(6)
0.08(8)
0.01(8)
qfin max/min) (e/nm3)
0.187/-0.196
0.318/-0.318
0.195/-0.146
0.170/-0.162
0.294/-0.283
a
b
c
For I [ 2.0r(I); for all data, not determined
The data given refer to our crystal (H2O/MeOH = 0.7/0.3); other crystals can have a different ratio of solvent molecules
d
employing synchrotron radiation to a commercial micro-
crystalline sample. The tetracycline cations again adopt
kind of an edge-to-face arrangement, but with no intermo-
lecular hydrogen bonds between them. Instead, each cation
forms four hydrogen bonds to different chloride anions [10].
The conformation of the cation is very similar to that of
the zwitterion as can be concluded from similar values of the
dihedral angles characterizing the tilt of ring A versus the
plane spanned by rings B, C and D (see Table 2). The most
apparent difference between the two structures discussed is
the orientation of the amide group. In the tetracycline–
hexahydrate crystal the amide group of the zwitterion is
oriented such that a hydrogen bond is made by one hydrogen
of its amino group, HN2AM, and O3. In the tetracycline
cation the amide group is rotated by about 160° versus the
zwitterion (compare angles C1–C2–C2AM–O2AM) and a
hydrogen bond made between HO2AM and O3.
In the cation, the fragment of ring A that exhibits a
p-electronic system can formally be described by a meso-
meric structure with three double bonds: C1–O1, C3–O3
˚
and C2–C2AM. The fairly short bonds (1.238 and 1.259 A,
respectively) found for the two C–O bonds are in accor-
˚
dance with such a mesomeric structure. But the 1.434 A
˚
determined for C2–C2AM together with the value 1.398 A
for the length of bond C2–C3 were taken as evidence that
the p-electronic system of ring A can not be described
properly by such a single mesomeric structure.
The data obtained for the zwitterion show significant
differences for several of the bonds discussed above. The
bonds C2–C3, C2–C2AM and C2AM–N2AM are rela-
tively longer than in the cation, the bonds C3–O3 and
especially C2AM–O2AM significantly shorter (Table 3).
The described changes taken together suggest a dominant
mesomeric (zwitterionic) structure N4?HÁÁÁO1-, again in
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J Chem Crystallogr (2013) 43:213–222
217
Fig. 2 Stereo drawing of the
molecular structure of the
Tetracycline zwitterion (crystal
I), crystallized from acidic
aqueous solution (thermal
ellipsoids are drawn at the 50 %
probability level, dashed lines
indicate intramolecular
hydrogen bonds, solvate
molecules omitted for clarity)
Table 2 Dihedral angles (°) characterizing the two fragments with tricarbonyl systems (e.s.d.’s in parentheses)
Tetracycline
hexahydrate
(zwitterion)
(crystal I)
Tetracycline
hydrochloride
(cation)
Doxycycline
hydrochloride
(cation)
Sancycline
hydrochloride
(cation)
(crystal V)
(crystal III)
(crystal IV)
O10–C10–C10A–C11
C10–C10A–C11–O11
O11–C11–C11A–C12
C11–C11A–C12–O12
O12–C12–C12A–C1
O1–C1–C2–C2AM
C1–C2–C2AM–N2AM
C1–C2–C2AM–O2AM
C1–C2–C3–O3
-1.4(4)
-12.7(3)
-1.8(3)
-0.1(3)
-78.1(2)
5.0(4)
1.8(4)
-14.7(3)
-3.2(3)
1.6(3)
1.9(6)
-16.1(5)
-5.2(5)
0.9(5)
0.4(4)
-11.1(4)
-3.3(4)
5.8(4)
-77.8(2)
9.8(4)
-80.3(3)
12.4(6)
-81.4(3)
0.9(4)
-169.4(2)
9.4(3)
-9.6(4)
170.5(2)
-168.9(2)
21.0(3)
-7.7(6)
173.7(4)
-167.5(3)
20.0(5)
6.3(4)
-174.6(3)
174.5(3)
-2.0(4)
173.2(2)
-68.5(3)
-156.6(2)
32.9(3)
C1–C2–C3–C4
C12–C12A–C4A–C4
C5–C4A–C12A–C1
170.4 (2)
-72.5(2)
172.1(2)
-73.8(2)
174.1(3)
-71.0(3)
contrast to the tautomeric form generally assumed in the
past (N4?HÁÁÁO3-). A further discussion of this topic will
be given below.
Stezowski crystallized doxycyclineÁHCl from an ethanol–
water mixture [6]. In his sample, the unit cell (space group
P21) contained two doxycycline molecules associated with
either one water or one ethanol molecule, respectively.
Stezowski interpreted his X-ray diffraction data to the effect
that in his crystal there were two symmetry-independent
molecular cations of doxycycline hydrochloride present
which differ in the orientation of the amide group, one having
a dihedral angle C1–C2–C2AM–O2AM of 171.3°, the other
one of 3.0°. As a consequence the dimensions of the unit cell
Doxycycline
In contrast to tetracyclineÁHCl, which crystallizes from
aqueous solution as zwitterion, doxycyclineÁHCl crystal-
lizes under essentially the same conditions as dihydrate of
the hydrochloride: doxycyclineÁHClÁ2H2O. In our crystal
(space group P212121) the similar intermolecular hydrogen
bonding pattern of the four doxycycline molecules per unit
cell leads to a similar edge-to-face arrangement within the
unit cell and correspondingly to similar cell parameters a, b
and c as for the tetracycline cation (Table 1).
˚
differ significantly from our values (a = 18.203 A,
˚
˚
b = 16.045 A, c = 8.004 A, b = 94.11°).
The crystal data reported by Bordner [8] for a com-
mercial sample of the a-epimer of doxycycline hydro-
chloride (Vibramycin) match our cell parameters very well
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J Chem Crystallogr (2013) 43:213–222
˚
Table 3 Selected bond distances (A), bond angles (°) and torsion angles (°) characterizing ring A as determined by X-ray crystallography
(e.s.d.’s in parentheses)
Tetracycline
hexahydrate
(zwitterion)
(Crystal I)
Tetracycline
hydrochloride
(cation)
Tetracycline
hydrochloride
(cation)
Doxycycline
hydrochloride
(cation)
Sancycline
hydrochloride
(cation)
(Crystal III)
(from reference 10)
(Crystal IV)
(Crystal V)
C1–C2
1.430(3)
1.436(3)
1.233(3)
1.241(3)
1.255(3)
1.472(3)
1.342(3)
119.2(2)
117.4(2)
-170.4(2)
9.4(3)
1.439(3)
1.398(3)
1.238(3)
1.259(3)
1.305(3)
1.434(3)
1.311(4)
118.1(2)
122.0(3)
-11.7(3)
170.5(2)
1.427(4)
1.403(5)
1.234(4)
1.260(4)
1.314(4)
1.435(4)
1.301(5)
118.4(3)
122.0(3)
-12.1(4)
170.4(3)
1.449(4)
1.402(5)
1.229(4)
1.271(4)
1.329(4)
1.434(4)
1.297(5)
116.0(3)
125.0(3)
0.7(5)
1.435(4)
1.417(4)
1.238(3)
1.261(3)
1.302(3)
1.451(4)
1.307(4)
118.3(3)
121.8(3)
1.8(4)
C2–C3
C1–O1
C3–O3
C2AM–O2AM
C2–C2AM
C2AM–N2AM
N2AM–C2AM–O2AM
N2AM–C2AM–C2
O2AM–C2AM–C2–C3
O2AM–C2AM–C2–C1
173.7(4)
-174.6(3)
Fig. 3 Stereo drawing of the
molecular structure of the
doxycyclineÁcation (crystal IV)
crystallized from acidic aqueous
solution (thermal ellipsoids are
drawn at the 50 % probability
level, dashed lines indicate
intramolecular hydrogen bonds,
chloride ion and solvate
molecules omitted for clarity)
˚
˚
(a = 11.638 A, b = 12.177 A, c = 15.840 A) although the
˚
34.1 % (methanol), respectively. The arrangement of the
sancycline molecules within the unit cell is essentially
independent of the associated solvent molecules and so is
the geometry of the rings A, B, C and D (Fig. 4).
bond length differ somewhat (Fig. 3).
Sancycline
A pronounced difference to the three cases discussed
above is found in the arrangement of the sancycline mol-
ecules within the unit cell with the effect that one dimen-
sion of the unit cell is strongly changed versus those
determined for tetracycline hydrochloride (Table 1). The
From an acidified solvent mixture (water/methanol) san-
cycline hydrochloride crystallizes with either one water or
one methanol molecule next to the A-ring of each sancy-
cline molecule protonated at N4. It proved necessary to
treat the dimethyl ammonium group (and only it) as a
disordered group throughout the refinement process. The
respective site-occupancy factors were 65.9 % (water) and
˚
reduction of the parameter ‘‘a’’ to about 6.8944 A reflects
the side-by-side arrangement of the molecules in staples
along this axis (Fig. 5). In the plane orthogonal to the
staple axis, the dimensions of the unit cell are nearly the
123

J Chem Crystallogr (2013) 43:213–222
219
O12A, respectively, is possible only in the majority case. In
both the majority and minority fraction, the amide oxygen
is protonated and their tautomeric structure corresponds to
the one of tetracycline hydrochloride. The hydrogen bonds
made up by the amide group (HOAM–O3 and HNAM–O1)
exhibit the same parameters in the majority and minority
case. Besides the two strong intramolecular hydrogen
bonds located in the tricarbonyl moiety (HO10–O11 and
HO12–O11) a hydrogen bond HO12A–O12 helps to sta-
bilise the molecular geometry.
The dihedral angles and bond distances presented in
Tables 2 and 3, respectively, confirm that the conformation
of sancycline hydrochloride resembles closely the one of
tetracycline and doxycycline, respectively. Sancycline
lacks the hydroxy group present at C6 in tetracycline and C5
in doxycycline. Because these hydroxy groups are not
involved in any intramolecular hydrogen bonding one must
not be surprised that the geometries of rings B, C and D of
the three hydrochlorides are very similar. The differences in
chemical structure are however manifested in the geometry
of the tricarbonyl system as seen by the variation of the
dihedral angles of this fragment (Table 2) and the con-
comitant variations of the precise location of the hydrogen
atoms O10H and O12H that form the stabilizing intramo-
lecular hydrogen bonding network. In our opinion, these
differences can explain the variation in the pKa-values and
binding constants of ions like Mg2? or Ca2? [13–15].
Comparison of Experimental Bond Lengths
and Calculated Wiberg-Bond Orders
Fig. 4 ORTEP plots of the molecular structure of: top) sancy-
clineÁHClÁH2O and bottom) sancycline HClÁCH3OH (crystal V)
crystallized from an acidified mixed solvent (H20/CH3OH). Thermal
ellipsoids are drawn at the 50 % probability level
In the early days the location of hydrogen atoms was less
accurate or even impossible. Therefore attempts were made
to decide which of the possible tautomers was present in
the crystal by comparing the bond lengths between heavier
atoms of ring A with the distribution of single and double
bonds in the corresponding classical mesomeric structures
[4, 6, 8, 10]. As mentioned above the variations in bond
length can not be reconciled with any one of the classical
mesomeric structures. To disprove the suggested explana-
tion that the observed discrepancies could be due to the
presence of several tautomers in the crystal under investi-
gation and to rationalize the variations in bond length we
have calculated ZDO Atomic Charges and Wiberg-bond
orders [20] for three tautomers of sancycline: San–O2AM–
H refers to the structure determined from our crystal with
the protonated amide oxygen. San–O3–H (with proton at
O3) is the tautomer that was usually assumed in the dis-
cussion of titration experiments [21]. Finally, we consid-
ered a structure without a proton on either O2AM or O3
(zwitterion).
˚
same as for the other derivatives (b = 16.815 A and
˚
c = 18.190 A).
Each chloride ion establishes four hydrogen bonds. Two
involve the amino group hydrogen atoms HN2AM of two
neighboring sancyclines, and the other two are associated
with the respective hydrogen atoms HO12A of two san-
cycline molecules in different staples. In the majority case,
HN4? makes an intermolecular hydrogen bond to the water
˚
oxygen (2.11 A), in the minority case to the methanol
˚
oxygen (1.89 A). In view of the different location of the
water and methanol molecule, respectively, relative to ring
A the formation of the hydrogen bonds mentioned is pos-
sible only because of a rotation of the C4–N4 bond
(dihedral angles d(C3C4N4HN4?) are 59.1° (water) and
124.4° (methanol), respectively).
Due to the different rotation of the C4–N4 bond an
intramolecular hydrogen bond between HN4? and O3 and
The calculated ZDO Atomic Charges (Table 4) show
for all three structures a strong alternance of the net
123

220
J Chem Crystallogr (2013) 43:213–222
Fig. 5 Packing diagram of
sancyclineÁHClÁH2O/CH3OH
(crystal V) crystallised from an
acidified mixed solvent (H20/
CH3OH)
charges.The shift of the proton from 2OAM to O3 results in
the expected higher negative charge at 2OAM and a lower
value at O3. For the other atoms the changes are less
pronounced. In the zwitterion the net charges of the three
oxygen atoms (O1, 2OAM, and O3) are about the same.
This can be taken as a hint that its actual electronic
structure must be described by a superposition of three
mesomeric structures with the negative charge located on
either one of the three oxygen atoms.
Table 4 ZDO atomic charges of selected atoms in ring A
San–O2AM–H
San–O3–H
San–zwitterion
O(1)
-0.29
-0.21
-0.38
0.32
-0.24
-0.46
-0.26
0.30
-0.35
-0.45
-0.34
0.31
O(2AM)
O(3)
C(1)
C(2)
-0.51
0.38
-0.39
0.36
-0.48
0.36
C(2AM)
C(3)
The variation of the bond lengths determined for the
three compounds under study are of the same magnitude as
the variations observed for the same compound by different
authors. The conclusions drawn here for sancycline there-
fore should be valid also for the other derivatives.
0.28
0.22
0.26
C(4)
-0.06
-0.13
0.08
-0.02
-0.13
0.07
-0.09
-0.12
0.18
C(4A)
C(12A)
N(4)
-0.02
-0.23
-0.30
-0.03
-0.28
-0.30
-0.01
-0.36
-0.30
Table 5 shows that within each group of bonds the
changes of the experimentally determined bond lengths are
very well imaged by the corresponding changes of the
Wiberg-bond orders tabulated for San–2OAM–H. For the
N(2AM)
O(12A)
Calculated with AM1 Hamiltonian [20]
123

J Chem Crystallogr (2013) 43:213–222
221
only C–C bond with unique double bond character,
˚
C(11A)–C12), the small bond length (1.384 A) corresponds
et al. [22] for the zwitterion one gets as bond length dif-
˚
ferences: -0.08 A for C(2A)–O(2AM) and ?0.11 A for
˚
to a Wiberg-bond order of 1.61; in contrast, single bonds
˚
C(2A)–N(2AM).
(*1.53 A) exhibit a Wiberg-bond order of about 0.95. The
most discussed bonds, C(1)–C(2), C(2)–C(3) and C(2)–
C(2A), must therefore be categorized as conjugated bonds
both with respect to bond length and Wiberg-bond order.
Noteworthy is also the decrease of the bond order parallel
to the increase of the bond length found for the four C–O
bonds included in Table 5. In summary: For the chosen
tautomer, San–O2AM–H, the variations of experimentally
determined bond lengths are in perfect agreement with the
variations of the calculated bond orders.
Summary and Conclusion
The molecular geometries of the hydrochlorides of tetra-
cycline, doxycycline and sancycline are essentially the
same if they are crystallized from a methanolic (tetracy-
cline), from an acidified aqueous solution (doxycycline)
and from a water–methanol mixture (sancycline), respec-
tively. In all cases the amide group is oriented such that the
protonated amide oxygen can form an intramolecular
hydrogen bond with O3. From aqueous solution, tetracy-
cline crystallizes independently of the pH of the mother
liquor as a hexahydrate complex with the zwitterion.
Because the amide group is rotated, an intramolecular
hydrogen bond is formed between a hydrogen atom of the
amino group and O3.
In accordance with the differences in the classical
mesomeric structures a transfer of the proton from 2OAM
to O3 induces a pronounced increase of the Wiberg-bond
order of bond C(2)–C(3)and C(2A)–O(2AM), but not to the
values characteristic for full double bonds; furthermore,
a strong reduction of the bond strength of C(3)–O(3) is
predicted, but again not to values characteristic for single
bonds. In summary: The tautomer San–O3–H should
exhibit a pattern of bond lengths pronouncedly different
from that of San–2OAM–H and without typical single and
double bonds.
The calculated Wiberg-bond orders provide convincing
evidence that the pattern of bond lengths can be rational-
ized by the exclusive presence of the proposed tautomers.
CCDC-243335 (for crystal I), CCDC-243336 (for crys-
tal II), CCDC-243337 (for crystal III), CCDC-243338 (for
crystal IV), and CCDC-864575 (for crystal V) contain the
supplementary crystallographic data for this paper. These
data can be obtained free of charge via www.ccdc.cam.
ac.uk/conts/retrieving.html, by e-mailing data reques-
t@ccdc.cam.ac.uk, or by contacting The Cambridge Crys-
tallographic Data Centre, 12 Union Road, Cambridge CB2
1EZ, UK; fax ?44(0)1223-336033.
According to the calculations significant changes in
bond length versus the cation are predicted for two bonds
in the zwitterion: a decrease for C(2A)–O(2AM) and an
increase for C(2A)–N(2AM). This prediction is verified by
˚
our experimental data for tetracycline: -0.05 A for C2A)–
˚
O(2AM) and ?0.03 A for C(2A)–N(2AM) (for other bonds
the trend is also predicted correctly). Taking our data for
the doxycycline cation and those presented by Legendre
Table 5 Comparison of
Exp. bond
Wiberg-bond
order
San–O2AM–H
Wiberg-bond
order
San–O3–H
Wiberg-bond
order
San–zwitterion
experimentally determined bond
lengths and Wiberg-bond orders
of selected bonds in ring A
˚
length (A)
San–O2AM–H
Bond:
C(1)–O(1)
1.237
1.260
1.303
1.413
1.308
1.517
1.435
1.417
1.451
1.530
1.536
1.533
1.556
1.384
1.86
1.71
1.22
1.00
1.41
0.87
1.03
1.17
1.13
0.90
0.96
0.94
0.87
1.61
1.90
1.33
1.49
1.00
1.21
0.89
1.00
1.48
0.98
0.94
0.96
0.94
0.86
1.62
1.78
1.76
1.61
0.99
1.19
0.87
1.12
1.19
1.00
0.84
0.97
0.94
0.83
1.57
C(3)–O(3)
C(2A)–O(2AM)
C(12A)–O(12A)
C(2AM)–N(2AM)
C(4)–N(4)
C(1)–C(2)
C(2)–C(3)
C(2)–C(2AM)
C(3)–C(4)
C(4)–C(4A)
C(4A)–C(12A)
C(12A)–C(1)
C(11A)–C(12)
Calculated with AM1
Hamiltonian [20]
123

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J Chem Crystallogr (2013) 43:213–222
Acknowledgments We thank Mrs. Julianne Roth for performing
the crystallization experiments. Financial support by Deutsche Fors-
chungsgemeinschaft (SFB 473 and SFB 583) and Fonds der Chem-
ischen Industrie is gratefully acknowledged.
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11. Leypold CF (2003) Spektroskopische Untersuchungen von Tet-
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Thesis, Universitat Erlangen-Nurnberg
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Appendix
¨
¨
sitat Erlangen-Nurnberg
In the literature one finds essentially three different termi-
nologies to describe the conformations of tetracyclines.The
conformations ‘‘A’’ and ‘‘B’’ introduced by Prewo and
Stezowski [7] correspond to conformations ‘‘B’’ and ‘‘E’’,
respectively, in the publications of Mitscher et al. [23], and
approximately to the ‘‘extended’’ and ‘‘twisted’’ confor-
mation introduced by Berthon and coworkers [24, 25].
14. Schmitt MO, Schneider S (2006) Z Phys Chemie 220:441
15. Schmitt MO, Schneider S, Nelson M (2007) Z Phys Chemie
221:235
16. Hulliger J (1994) Angew Chemie 106:151
17. Wilson AJC (ed) (1992) International tables for crystallography,
vol. C. Kluwer, Dordrecht, pp 193, 219, 500
18. Flack HD (1983) Acta Crystallogr A 39:876
19. Bruker (2002) SHELXTL NT 6.12. Bruker AXS Inc, Madison
20. ArgusLab 4.0, Mark A. Thompson, Planaria Software LLC,
Seattle WA. http://www.arguslab.com
21. Schneider S (2001) In: Nelson M, Hillen W, Greenwal RA (eds)
¨
Tetracyclines in Biology, Chemistry and Medicine. Birkhauser,
Basel, pp 65–104
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