1H NMR analysis of isocyclosporin A prepared in organic solvent and in aqueous solution

Article abstract of DOI:10.1006/bioo.1997.1058

Isocyclosporins are isomers of cyclosporins deriving from an N,O-acyl transfer occurring under acidic conditions. For the identification by ¹H NMR of isocyclosporins prepared in organic solvents the signal at 2.3 ppm assigned to three hydrogens of the NHCH₃ group is considered. Results reported here show that the ¹H NMR spectrum of isocyclosporin A prepared in aqueous acidic conditions lacks of the above signal and profoundly differs from that of isocyclosporin A prepared in organic solvents, despite the identity of the primary structures determined on the basis of mass spectrometric analysis.

Full text of DOI:10.1006/bioo.1997.1058

BIOORGANIC CHEMISTRY 25, 110–116 (1997)
ARTICLE NO. BH971058
1H NMR Analysis of Isocyclosporin A Prepared in Organic
Solvent and in Aqueous Solution
Lolita Arnoldi, Ada Manzocchi,* Fulvio Magni, Marina Del Puppo,* and
Marzia Galli Kienle*
Scientific Institute H. S. Raffaele, Via Olgettina 58, 20132 Milano, Italy; and *Department of Medical
Chemistry and Biochemistry, University of Milan, Via Saldini 50, 20133 Milano, Italy
Received February 10, 1977
Isocyclosporins are isomers of cyclosporins deriving from an N,O-acyl transfer occurring
under acidic conditions. For the identification by 1H NMR of isocyclosporins prepared in
organic solvents the signal at 2.3 ppm assigned to three hydrogens of the NHCH3 group is
considered. Results reported here show that the 1H NMR spectrum of isocyclosporin A
prepared in aqueous acidic conditions lacks of the above signal and profoundly differs from
that of isocyclosporin A prepared in organic solvents, despite the identity of the primary
structures determined on the basis of mass spectrometric analysis.
1997 Academic Press
INTRODUCTION
Cyclosporin A (CsA) is a neutral cyclic undecapeptide [cyclo(-MeBmt1-Abu2-
Sar3-MeLeu4-Val5-MeLeu6-Ala7- -Ala8-MeLeu9-MeLeu10-MeVal11-)] with seven
D
N-methylated amino acids well known as immunosuppressant. Under acidic condi-
tions in organic solvent CsA and other known cyclosporins undergo rearrangement
by N,O-acyl migration (1–5), with formation of isomers named isocyclosporins
(isoCs). A signal at 2.3 ppm attributable to three hydrogens of the NHCH3 group
1
is reported to characterize the H NMR spectra of isoCs B, C, D, E, G, and H
(3–5). IsoCsA was described to be formed also under aqueous acidic conditions
(6, 7). Its identification was based upon the TLC and HPLC behavior in comparison
with that of authentic isoCsA prepared with methanesulphonic acid according to
Ruegger et al. (1). Formation of isoCsA under aqueous acidic conditions was also
¨
reported by us with the identification of the compound by FAB-MS/MS mass
spectrometry (8, 9). Isomerization of CsA in aqueous media may be particularly
relevant in view of the reported in vivo isomerization of isoCsA to CsA. The aim
of this work was to compare the 1H NMR spectra of isoCsA prepared under these
conditions with that of isoCsA obtained in organic solvent according to Ruegger
¨
et al. (1).
RESULTS AND DISCUSSION
The NCH3 region of proton NMR spectra in CDCl3 of CsA and of isoCsAs (Fig.
1) prepared both in organic solvent and in water are reported in Fig. 2. As already
described (10–12) CsA exhibited one major conformation (Fig. 2a) and the chemical
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1H NMR OF ISOCYCLOSPORIN A
111
F
IG. 1. IsoCsA.
shifts (Table 1) corresponded to those described previously (11). The spectrum of
isoCsA prepared in organic solvent and purified on silica gel suggested the presence
in CDCl3 of few conformations (Fig. 2b). Nevertheless, one conformation appeared
to prevail as suggested by the set of signals marked in Fig. 2b. Moreover the signal
at 2.32 ppm (s, NCH3) described to be characteristic of isoCs (3–5) was apparent.
Fig. 2c shows the NCH3 region of the spectrum of isoCsA prepared in aqueous
HCl. Also for this compound one conformation appeared to be predominant but
chemical shifts of the main signals did not correspond to those of the standard
compound. In particular no signal at 2.3 ppm was observed. On the other hand
FAB/MS-MS spectrum of isoCsA prepared under aqueous conditions was identical
to that of standard isoCsA. Moreover, mass spectrum of isoCsA acetate prepared
as described below was also indistinguishable from that of standard isoCsA acetate
(Fig. 4). The downfield shift of the signal at 2.3 ppm in the 1H NMR, as that reported
for salts of amines (14) suggested protonation of the secondary amino group which
instead appears as a free base in standard isoCsA. Protonation was accompanied
by the shift of all other CONCH3 signals, which suggested modification of the
conformation. In order to confirm this hypothesis aliquots of the CDCl3 solution
of standard isoCsA were treated as described in Fig. 3 and chemical shifts of the
NCH3 hydrogens were considered. When standard isoCsA was either dissolved in
an acidic medium (Fig. 3A) or eluted from an HPLC column (Fig. 3B) under
conditions used for the purification of isoCsA prepared in aqueous medium, signals
1
characteristic of standard isoCsA were absent in the H NMR spectrum while
main signals corresponded well to those observed for isoCsA prepared in aqueous
medium as reported in Table 1, samples A and B. The same was shown to occur
(Table 1, sample C) when a CDCl3 solution of standard isoCsA was evaporated to
dryness (Fig. 3C) followed by dissolution in CDCl3 and registration of the NMR
spectrum. The observed modifications were not attributable to low stability of
isoCsA solutions in CDCl3 because the NMR spectrum of standard isoCsA stored
in CDCl3 at 4ЊC for 7 days (Figs. 2d and 3D) suggested partial transformation of

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ARNOLDI ET AL.
F
IG. 2. NCH3 region of proton NMR spectra of CsA and isoCsA. (a) Standard CsA; (b) standard
isoCsA; (c) isoCsA prepared in aqueous medium; (d) standard isoCsA stored in CDCl3 at 4ЊC for 7
days. ᭢ and ء correspond to signals in (a) and (b), respectively.
isoCsA to CsA but showed no detectable signals at chemical shifts of isoCsA
prepared in aqueous HCl (Table 1, sample D). Modifications may be rather justified
by protonation of isoCsA. This may be true also for sample C in Table 1 due to
the Hϩ presence of CDCl3 used as the solvent. Even if low this acidity may be
sufficient for protonation of the secondary amino group of the analyzed isoCsA.
1
Results obtained here may help to interpret previous reported H NMR data,
indicating multiplets instead of singlets in the 2.7–3.4 ppm region for the CONCH3
signals of isoCs B, C, D, G, and H (3–5). Singlets, as expected, were instead reported
1
for isoCsE (5). NHCO protons in the H NMR spectrum of standard isoCsA
(doublets in the 7.2–8 ppm region) confirmed the presence of a main conforma-
tion while signals in the spectrum of isoCsA prepared in aqueous medium were
complex.
In conclusion the reported results demonstrate that isoCsA obtained by acidic

1H NMR OF ISOCYCLOSPORIN A
113

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ARNOLDI ET AL.
FIG. 3. Preparation of various samples of isoCsA for NMR analysis.
treatment of CsA in an aqueous medium is structurally identical to standard isoCsA
1
obtained in organic solvent. Differences in the H NMR spectra, particularly the
lack of the signal at 2.3 ppm in the former compound suggest it to be in a salt form,
while the latter is the free base. Moreover, protonation of the secondary amino
group causes modification of the conformation.
EXPERIMENTAL
Materials
CsA was a generous gift of Dr. G. Corbetta (Sandoz S.p.A., Milan, Italy). CDCl3
(99.95% isotopic enrichment); silica gel for column chromatography (Kieselgel 60)
and TLC plates (Silicagel 60 F254) were from Merck (Darmstadt, Germany). 3-
Nitrobenzyl alcohol was from Aldrich Chimica (Milan, Italy). Other reagents were
of analytical grade. Solvents for HPLC were used after being degassed and filtered
using 0.22-Ȑm filters.
Preparation of isoCsA
In organic solvent. IsoCsA was prepared from CsA in methanol with methane-
sulphonic acid followed by treatment with anhydrous sodium acetate as described
(1). The compound was then purified by silica gel column chromatography. Fractions
eluted from the column with CHCl3 : CH3OH (98/2, v/v) showed a single spot by
TLC with Rf 0.1 (CHCl3 : CH3OH : CH3COOH, 90/6/4, v/v/v). Rf of CsA under

1H NMR OF ISOCYCLOSPORIN A
115
F
IG. 4. FAB-MS/MS spectra of [M ϩ H]ϩ ions of acetates of standard isoCsA (top) and isoCsA
prepared under aqueous conditions (bottom).
the same conditions was 0.4. The purified compound is referred to in the text as
standard isoCsA. Reversed phase HPLC (eluting with CH3CN : 0.06% CF3COOH
32/68, v/v (A), and CH3CN : 0.052% CF3COOH, 80/20, v/v (B)) was performed
with a linear gradient of B from 0 to 100% in 20 min (9) and gave a single peak at
24 min retention time (rt). Rt of CsA under the same conditions was 35 min. FAB-
MS and FAB-MS/MS spectra were recorded as described (9). Aliquots of standard
isoCsA were processed according to Fig. 3.
In aqueous solution. IsoCsA was prepared in aqueous HCl under the conditions
reported previously (9) by mixing 4 Ȑmol of CsA with 4 ml of 3
M
HCl. The mixture
was dried under vacuum using a Savant centrifuge with mild heating (45ЊC). Residue
was dissolved in 4 ml of CH3OH : H2O (1/1, v/v) and extracted twice with 8 ml of
diethyl ether. HPLC of the aqueous phase showed a peak at rt of standard isoCsA,
which was collected. TLC, HPLC, and FAB-MS behavior of the purified compound
was as that of standard isoCsA.
1H NMR Analysis
Proton NMR spectra were recorded at 303ЊK in CDCl3 using a Bruker AM-500
(Bruker Instruments, Silberpreiten, Germany) operating at 500 MHz. Tetrameth-
ylsilane as the chemical shift reference was set at 0 ppm.

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ARNOLDI ET AL.
Acetylation of isoCsA
Both standard isoCsA and isoCsA prepared under aqueous acidic conditions
were acetylated with acetic anhydride/pyridine (1/1, v/v) for 3 h at room tempera-
ture. Reagent and solvent were then evaporated under vacuum. The residue was
dissolved in chloroform and analyzed by MS/MS as described previously (9).
ACKNOWLEDGMENTS
This research was partially supported by CNR Target Project ‘‘Biotechnology and Bioinstrumentation’’
and by IRCCS S. Raffaele.
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