Amino acid derivatives of β-cyclodextrin

Article abstract of DOI:10.1021/jo951396d

The syntheses of the heptaamino acid-substituted β-cyclodextrins per-6-[(phenylalanyl)amino]-β-cyclodextrin (6), per-6-cysteinyl-β-cyclodextrin (7), as well as the per-2,3-dimethyl-per-6-cysteinyl-β-cyclodextrin (12) are described. The amino acids were coupled to the primary face of the β-cyclodextrin torus using the backbone carboxylic acid functionality of phenylalanine and the side chain thiol group of cysteine. In the case of the heptacysteinyl derivatives, polyzwitterionic compounds were obtained and shown to be highly water soluble.

Full text of DOI:10.1021/jo951396d

J . Org. Chem. 1996, 61, 903-908
903
Am in o Acid Der iva tives of â-Cyclod extr in
Peter R. Ashton, Rainer Ko¨niger, and J . Fraser Stoddart*
School of Chemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.
David Alker and Valerie D. Harding
Central Research, Pfizer Limited, Sandwich, Kent CT13 9NJ , U.K.
Received J uly 28, 1995X
The syntheses of the heptaamino acid-substituted â-cyclodextrins per-6-[(phenylalanyl)amino]-â-
cyclodextrin (6), per-6-cysteinyl-â-cyclodextrin (7), as well as the per-2,3-dimethyl-per-6-cysteinyl-
â-cyclodextrin (12) are described. The amino acids were coupled to the primary face of the
â-cyclodextrin torus using the backbone carboxylic acid functionality of phenylalanine and the side
chain thiol group of cysteine. In the case of the heptacysteinyl derivatives, polyzwitterionic
compounds were obtained and shown to be highly water soluble.
In tr od u ction
The synthesis of cyclodextrins that are singly substi-
tuted with amino acids has been reported previously.9-15
However, only one report of cyclodextrins that are sym-
metrically substituted with amino acids has appeared
recently in the literature.16 Despite their relative ease
of characterization, highly-substituted, symmetrical cy-
clodextrins present a synthetic challenge. Nonetheless,
the challenge is worthwhile since a higher degree of
substitution should confer upon cyclodextrin derivatives
characteristics which are expected to deviate more strongly
from those of the native cyclodextrin. As a consequence
of these considerations, amino acids have been identified
as residues to attach to all the primary hydroxyl groups
on â-cyclodextrin. Here, we report the efficient syntheses
(Schemes 1-3) of â-cyclodextrin derivatives which are
fully substituted on their primary faces with the amino
acids L-phenylalanine and L-cysteine.
Composed of six, seven, or eight R-1,4-linked D-(+)-
glucopyranose units, the cyclodextrins (R, â, and γ,
respectively) are cyclic oligosaccharides1 which have an
overall shape reminiscent of a lampshade or truncated
cone. The most significant characteristic of the cyclo-
dextrins is their ability to form2 inclusion complexes in
aqueous solutions with a wide variety of substrates.
Since compounds that are complexed by an appropriate
cyclodextrin often display much improved water solubili-
ties, the cyclodextrins may act as enhancers of the
aqueous solubilities of small organic molecules.3-5 In this
context, cyclodextrins have found applications6 in the
solubilization of organic compounds when organic sol-
vents cannot be employed because of their toxicity,
flammability, or expense.
As a consequence of its good binding ability toward
aromatic units, as well as its ready availability, â-cyclo-
dextrin has emerged at present as the naturally-occur-
ring cyclodextrin with the highest commercial potential.
It is, therefore, unfortunate that its aqueous solubility
is so low.7 As a result, the extensive investigations into
the chemical modifications of cyclodextrins8 have been
concerned primarily with influencing their solubilities as
well as with modifying their binding behaviors.
Resu lts a n d Discu ssion
The opportunity for the symmetric derivatization of
â-cyclodextrin was found in an important reaction re-
ported recently by Defaye and Gadelle.17 This reaction
allows the selective replacement of all the primary
hydroxyl groups of a cyclodextrin by iodine atoms. Thus,
treatment of â-cyclodextrin (1) with I2 and Ph3P in DMF
at 70 °C afforded (Scheme 1) the heptaiodide 2. The
reaction was performed as described in the literature.17
However, the workup procedure, which was recom-
X Abstract published in Advance ACS Abstracts, J anuary 15, 1996.
(1) Wenz, G. Angew. Chem., Int. Ed. Engl. 1994, 33, 803-822.
Strattan, C. E. Biopharm. 1991, Nov/Dec, 44-51. Stoddart, J . F., Ed.
Carbohydr. Res. 1989, 192. Szejtli, J . Cyclodextrin Technology; Kluwer
Academic: Dordrecht, 1988. Ducheˆne, D., Ed. Cyclodextrins and their
Industrial Uses; Editions de la Sante´: Paris, 1987. Szejtli, J . Cyclo-
dextrins and their Inclusion Complexes; Akade´miai Kiado: Budapest,
1982. Bender, M. L.; Komiyama, M. Cyclodextrin Chemistry;
Springer-Verlag: Berlin, 1978. Bergeron, R. J . J . Chem. Educ. 1977,
54, 204-207.
(9) Parrot-Lopez, H.; Galons, H.; Coleman, A. W.; Djeda¨ıni, F.;
Keller, N.; Perly, B. Tetrahedron Asymmetry 1990, 1, 367-370.
(10) Parrot-Lopez, H.; Djeda¨ıni, F.; Perly, B.; Coleman, A. W.;
Galons, H.; Miocque, M. Tetrahedron Lett. 1990, 31, 1999-2002.
(11) Parrot-Lopez, H.; Galons, H.; Dupas, S.; Miocque, M.; Tsoucaris,
G. Bull. Soc. Chim. Fr. 1990, 127, 568-571.
(2) Pagington, J . S. Chem. Brit. 1987, 455-458. Saenger,W. In
Inclusion Compounds; Atwood, J . L., Davies, J . E. D., MacNicol, D.
D., Eds.; Academic Press: London, 1984; Vol. 2, pp 231-259.
(3) Szejtli, J . Pharm. Technol. Int. 1991, Feb, 15-22. Szejtli, J .
Pharm. Technol. Int. 1991, March, 16-21. Szejtli, J . J . Incl. Phenom.
1992, 14, 25-36.
(12) Takahashi, K.; Ohtsuka, Y.; Nakada, S.; Hattori, K. J . Incl.
Phenom. 1991, 10, 63-68.
(13) Cucinotta, V.; D’Alessandro, F.; Impellizzeri, G.; Pappalardo,
G.; Rizzarelli, E.; Vecchio, G. J . Chem. Soc., Chem. Commun. 1991,
293-294.
(14) Di Blasio, B.; Pavone, V.; Nastri, F.; Isernia, C.; Saviano, M.;
Pedone, C.; Cucinotta, V.; Impellizzeri, G.; Rizzarelli, E.; Vecchio, G.
Proc. Natl. Acad. Sci. USA 1992, 89, 7218-7221.
(4) Viernstein, H.; Reiter, S.; Wolschann, P. Monatsh. Chem. 1994,
125, 681-689.
(5) Hedges, A. R. In Starch Hydrolysis Products; Schenk, F. W.,
Hebeda, R. E., Eds.; VCH Publishers: New York, 1992; pp 319-333.
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Saenger, W. Angew. Chem., Int. Ed. Engl. 1980, 19, 344-362.
(7) The native R- and γ-cyclodextrins exhibit high water solubilities
(145 and 232 g L-1, respectively) compared with â-cyclodextrin, which
has a low (18.5 g L-1) water solubility. French, D.; Levine, M. L.; Pazur,
J . H.; Norberg, E. J . Am. Chem. Soc. 1949, 71, 353-356.
(8) Bartsch, R. A.; Croft, A. P. Tetrahedron 1983, 39, 1417-1474.
(15) Djeda¨ıni, F. Etude par Re´sonance Magne´tique Nucle´aire des
phe´nome`nes d’inclusion et d’adaption mole´culaire dans les cyclodex-
trines naturelles et des de´rive´s synthe´tiques; Universite´ de Paris Sud
Centre d’Orsay, 1991.
(16) Symmetrically-substituted â-cyclodextrin as a scaffold for su-
pramolecular peptide assemblies. See: A° kerfeldt, K. S.; DeGrado, W.
F. Tetrahedron Lett. 1994, 35, 4489-4492.
(17) Gadelle, A.; Defaye, J . Angew. Chem., Int. Ed. Engl. 1991, 30,
78-80.
0022-3263/96/1961-0903$12.00/0 © 1996 American Chemical Society

904 J . Org. Chem., Vol. 61, No. 3, 1996
Ashton et al.
Sch em e 1
Sch em e 2
Geiger and Ko¨nig.21 N-Boc-phenylalanine (Boc ) tert-
butyloxycarbonyl) was treated with dicyclohexylcarbodi-
imide (DCC) in DMF in the presence of N-hydroxyben-
zotriazole (HBT) and N-methylmorpholine in order to
couple it exhaustively with per-6-amino-â-cyclodextrin
(4). The Boc-protected per-6-[(phenylalanyl)amino]-â-
cyclodextrin derivative 5 was characterized only by fast
atom bombardment mass spectrometry (FABMS) since
hindered rotation associated with the Boc carbamate
1
bonds gives rise to substantial line broadening in the H
NMR spectrum at 300 MHz. Removal of the Boc groups
from 5 was achieved in neat trifluoroacetic acid (TFA),
yielding the per-6-[(phenylalanyl)amino]-â-cyclodextrin
1
(6) after a basic workup. Both the FABMS and the H
NMR spectra confirmed the fully substituted nature of
the compound which is soluble in H2O under neutral or
acidic conditions. However, in alkaline aqueous media,
as the free amine, per-6-[(phenylalanyl)amino]-â-cyclo-
dextrin (6), is insoluble.
mended by Defaye and Gadelle,17 proved to be unsatis-
factory in our hands. And so, a simple and efficient
method for purifying the desired product by selective
precipitation, followed by Soxhlet extraction, was devel-
oped. We believe that this method is superior even to a
revised workup procedure which was reported subse-
quently.18 Next, the heptaiodide 2 was treated with
NaN3 in DMF to give in 96% yield the heptaazide 319
which was found to be a stable, easy-to-handle compound.
Treating the heptaazide 3 with a Pd/C catalyst under 1
atm of H2 did not prove to be successful as a means of
reducing the azide functional groups to the corresponding
amino functions.20 However, a different reduction pro-
cedure, employing Ph3P, was successful. This reduction
of 3 proceeded very efficiently, and subsequent treatment
of the intermediate with aqueous NH3 solution allowed
the isolation of the heptaamine 4, which surprisingly,
proved to be extremely insoluble in H2O as well as in
organic solvents. However, as the pH of an aqueous
suspension of 4 was lowered to neutrality or slightly
acidic conditions, the compound dissolved rapidly. In-
deed, for NMR spectroscopic analysis, 4 had to be
converted into its HCl salt, which is highly soluble in
D2O.
In order to address this problem, an amino acid
derivative of a cyclodextrin was targeted that would
retain the zwitterionic character of the pendant amino
acids. This goal could only be reached if the amino acids
were attached by their side chain functionalities22 to the
cyclodextrin torus. A synthetic approach, using cysteine
and the per-6-iodo-â-cyclodextrin (2), proved to be suc-
cessful (Scheme 2). The thiol group in the cysteine side
chain can be converted easily under basic conditions into
the thiolate anion, which can be used to displace23 all the
iodine atoms as anions from 2. The best experimental
conditions for generating the thiolate anion and inducing
it to react with the heptaiodide 2 were found to involve
the dissolution of cysteine or cystine in liquid NH3 at -33
°C followed by the addition of 2 equiv of Na metal. Any
(21) Ko¨nig, W.; Geiger, R. Chem. Ber. 1970, 103, 788-798.
(22) Coupling of the side chain of a protected glutamic acid to per-
6-amino-â-cyclodextrin (4) in an analogous synthesis was attempted
and afforded a crude product, from which it was not possible to separate
and characterize the expected derivative. The coupling reaction
between 4 and N-Boc-glutamic acid R-benzyl ester is believed not to
have gone to completion on account of the steric bulk of the protecting
groups preventing exhaustive amide coupling at the 6-position of the
cyclodextrin.
In the first instance, phenylalanine was chosen as the
amino acid residue because of its inert side chain.
Moreover, we argued that a method that would allow the
exhaustive coupling of phenylalanine to the â-cyclodex-
trin torus would be applicable to any other amino acid
which bears a less sterically demanding, inert side chain.
The peptide coupling procedure employed in the case of
phenylalanine was analogous to that introduced by
(23) Further studies showed that nucleophilic displacement of iodide
anions from per-6-iodo-â-cyclodextrin, employing poorer nucleophiles
or elevated temperatures, favors the intramolecular substitution
reaction, resulting in the formation of 3,6-anhydro-D-glucopyranose
residues within the structure of per-6-iodo-â-cyclodextrin. See: Fujita,
K.; Tahara, T.; Egashira, Y.; Yamamura, H.; Imoto, T.; Koga, T.;
Fujioka, T.; Mihashi, K. Chem. Lett. 1988, 705-708. Fujita, K.;
Yamamura, H.; Imoto, T.; Fujioka, T.; Mihashi, K. J . Org. Chem. 1988,
53, 1943-1947. Fujita, K.; Egashira, Y.; Tahara, T.; Imoto, T.; Koga,
T. Tetrahedron Lett. 1989, 30, 1285-1288. Fujita, K.; Tahara, T.; Koga,
T. Chem. Lett. 1989, 821-824. Fujita, K.; Tahara, T.; Yamamura, H.;
Imoto, T.; Koga, T.; Fujioka, T.; Mihashi, K. J . Org. Chem. 1990, 55,
877-880. Yamamura, H.; Fujita, K. Chem. Pharm. Bull. 1991, 39,
2505-2508. Gadelle, A.; Defaye, J . Angew. Chem., Int. Ed. Engl. 1991,
30, 78-80. Ashton, P. R.; Ellwood, P.; Staton, I.; Stoddart, J . F. Angew.
Chem., Int. Ed. Engl. 1991, 30, 80-81. Ashton, P. R.; Ellwood, P.;
Staton, I.; Stoddart, J . F. J . Org. Chem. 1991, 56, 7274-7280.
Yamamura, H.; Ezaka, T.; Kawase, Y.; Kawai, M.; Butsugan, Y.; Fujita,
K. J . Chem. Soc., Chem. Commun. 1993, 636-637.
(18) Baer, H. H.; Berenguel, A. V.; Shu, Y. Y.; Defaye, J .; Gadelle,
A.; Gonza´lez, F. S. Carbohydr. Res. 1992, 228, 307-314.
(19) Parrot-Lopez, H.; Ling, C.-C.; Zhang, P.; Baszkin, A.; Albrecht,
G.; de Rango, C.; Coleman, A. W. J . Am. Chem. Soc. 1992, 114, 5479-
5480.
(20) Boger, J .; Corcoran, J .; Lehn, J .-M. Helv. Chim. Acta 1978, 61,
2190-2218.

Amino Acid Derivatives of â-Cyclodextrin
J . Org. Chem., Vol. 61, No. 3, 1996 905
Sch em e 3
F igu r e 1. Space-filling representation of the solid state
structure of per-6-bromo-per-2,3-O-dimethyl-â-cyclodextrin (10)
viewed from the primary face of the molecule.
excess of Na had to be destroyed by the addition of NH4-
Cl before the per-6-iodo-â-cyclodextrin (2) was added to
the reaction mixture, which was maintained at the
temperature of refluxing NH3 for 1 h before the NH3 was
allowed to evaporate. After the NH3 had evaporated
completely, the residue was dissolved in a small amount
of H2O and neutralized to pH 7 by the addition of 2 N
aqueous HCl. The heptazwitterionic compound 7 was
then precipitated using an excess of EtOH to give a white
hygroscopic powder. The solubility of 7 in H2O was found
to be similar to that of amino acids: at high and low pH,
7 displays very high solubility, whereas at neutral pH,
procedure.24,25 The per-2,3-O-dimethyl derivative 9 was
accessible by exhaustive methylation of 8, using a large
excess of MeI and oil-free NaH in THF at room temper-
ature. Desilylation and bromination were accomplished
in one step by adding 9 to a suspension of Ph3PBr2 in
CH2Cl2. As the reaction proceeded, the suspension
cleared slowly. The product was recrystallized from Me2-
CO and characterized as per-6-bromo-per-2,3-O-dimethyl-
â-cyclodextrin (10). Analytically pure 10 could be ob-
tained by flash column chromatography on silica gel
using 70% t-BuOMe in hexane as the eluant. Even at
this stage, the solubility of 10 in low boiling organic
solvents was noted to be much enhanced compared with
that of the unmethylated analog, per-6-bromo-â-cyclo-
dextrin,17 which could be prepared by a procedure
analogous to that employed in the synthesis of per-6-iodo-
â-cyclodextrin (2). Single crystals of the per-6-bromo-
per-2,3-O-dimethyl-â-cyclodextrin (10), which proved to
be of suitable quality for X-ray crystallographic analy-
sis,28 were grown by slow evaporation from an Me2O
solution. The X-ray crystal structure reveals (Figure 1)
that the conformation of the molecule departs signifi-
cantly from C7 symmetry. The R-1,4-linked D-glucopy-
ranose rings adopt different orientations with respect to
each other, resulting in four of the bromomethyl groups
being directed outward and three inward, partially
blocking the free passage through the core of the mol-
ecule. The loss of symmetry within 10 is undoubtedly a
consequence of the loss of the chain of circumferal
intramolecular hydrogen bonds between the secondary
hydroxyl groups of adjacent D-glucopyranose units in
â-cyclodextrin.29 The increased flexibility of 10 becomes
apparent when its crystal structure is compared to that
1
concentrated solutions are turbid. Similarly, in the H-
NMR spectrum of 7, the chemical shifts of all the signals
are dependent on the pH, with the largest change in
chemical shift being exhibited by the R protons of the
cysteine residues. The hepta-Na salt of 7 is highly
deliquescent and H2O soluble, so much so that precipita-
tion cannot be achieved at any concentration. A viscous
mass and finally a glass forms as an aqueous solution is
concentrated.
Protection of a more permanent kind for the secondary
hydroxyl groups on â-cyclodextrin (1) was investigated
next as a means of improving the organic solubility of
per-6-cysteinyl-â-cyclodextrin (7), which is only soluble
in H2O. Methylation emerged as the method of choice
for the modification of the secondary hydroxyl groups of
â-cyclodextrin 1. It was hoped that the analogue of 7 in
which the secondary hydroxyl groups were methylated
would exhibit better solubility characteristics in organic
solvents. Direct methylation of 7 or the precursor hep-
taiodide 2 was not contemplated seriously because of the
anticipated large number of side reactions. Conse-
quently, a completely new synthesis of the methylated
derivative of 7 was proposed. The selectivity with which
all the primary hydroxyl groups of a cyclodextrin can be
converted into TBDMS ether groups24-26 and the pos-
sibility of direct replacement27 of these TBDMS ether
functions with bromine atoms suggested to us an efficient
route (Scheme 3) to the desired compound 12 starting
from â-cyclodextrin 1. Per-6-(tert-butyldimethylsilyl)-â-
cyclodextrin (8) was prepared from 1 using a literature
(24) Fu¨gedi, P. Carbohydr. Res. 1989, 192, 366-369.
(25) Takeo, K.; Mitoh, H.; Uemura, K. Carbohydr. Res. 1989, 187,
203-211.
(26) Mulligan, D. C. MPhil Thesis, The University of Sheffield, 1986.
(27) Aizpurua, J . M.; Cossio, F. P.; Palomo, C. J . Org. Chem. 1986,
51, 4941-4943.
(28) Alker, D.; Ashton, P. R.; Harding, V. D.; Ko¨niger, R.; Stoddart,
J . F.; White, A. J . P.; Williams, D. J . Tetrahedron Lett. 1994, 35, 9091-
9094.

906 J . Org. Chem., Vol. 61, No. 3, 1996
Ashton et al.
of its unmethylated analog.30 Attempts were made to
react the heptabromide 10 with an N-protected cysteine
derivative. For example, per-6-bromo-per-2,3-O-di-
methyl-â-cyclodextrin (10) was reacted with N-Boc-cys-
teine31 under basic conditions. A variety of different
conditions32,33 were tried from which one employing
powdered KOH in DMSO34 emerged as the most effective
method. These reaction conditions resulted in a very
short reaction time of only a few minutes at room
temperature to give a product which was found to move
filtered, (ii) pyridine (C5H5N) was dried over CaH2 and
distilled, and (iii) THF was distilled under N2 from a solution
containing the Na/Ph2CO ketal. Thin layer chromatography
(TLC) was performed on aluminum sheets coated with silica
gel. Where appropriate, the TLC plates were scrutinized by
ultraviolet light, before being developed with 5% H2SO4 in
EtOH, followed by heating in an oven or, preferably, by using
a heat gun. Column chromatography was performed using
silica gel (particle size 0.040-0.063 mm). Fast atom bombard-
ment mass spectra (FABMS) were obtained with a spectrom-
eter on which the FAB unit was operated at 8 keV by use of a
krypton primary atom beam with a 3-nitrobenzyl alcohol
(NOBA) matrix. Matrix-assisted laser desorption ionization
time of flight (MALDI-TOF) mass spectrometry was carried
out using a gentisic acid (2,5-dihydroxybenzoic acid) matrix.
Liquid secondary ion mass spectrometry (LSIMS) was per-
formed using a cesium ion beam. 1H nuclear magnetic
resonance (NMR) spectra were recorded at either 270, 300, or
400 MHz using the solvent peaks as internal references. 13C
NMR spectra were recorded at either 75 or 100 MHz.
1
as one component by TLC. The H NMR spectrum of 11
shows only extremely broad signals. Also, it could not
be characterized by a variety of mass spectrometric
techniques. As in the case of the Boc-protected per-6-
[(phenylalanyl)amino]-â-cyclodextrin (5), this compound
exhibits slow interconversions between the Z and E
conformations for the carbamoyl functions associated
with each of the seven cysteine residues on the per-(2,3-
O-dimethyl)-â-cyclodextrin torus. Deprotection of 11 in
neat TFA gave compound 12, which displayed sharp
P er -6-iod o-â-cyclod extr in (2). The reaction conditions for
the preparation of this compound are analogous to those
described by Defaye and Gadelle;14 i.e., Ph3P (40.1 g, 153 mmol)
was dissolved with stirring in dry DMF (160 mL). To this
solution was carefully added I2 (40.5 g, 160 mmol) over 10 min
with the evolution of heat: the solution reaches approximately
50 °C. Dry â-cyclodextrin (1) (11.6 g, 10.2 mmol) was then
added to this dark brown solution, and the temperature was
raised to 70 °C. At this temperature, the solution was stirred
under an atmosphere of N2 for 18 h. Heating was then
discontinued and the solution concentrated under reduced
pressure by the removal of DMF (approximately 100 mL).
NaOMe in MeOH (3 M, 60 mL) was then prepared by adding
Na (4.2 g) to MeOH (60 mL) under an inert atmosphere with
efficient cooling. This NaOMe solution was then added to the
reaction vessel with cooling, and the reaction mixture was
stirred for 30 min. The following reaction workup deviates
from the literature procedure. Instead of precipitating the
product with ice-water, the reaction mixture was poured into
MeOH (800 mL) to form a precipitate, which was washed with
MeOH, superficially dried, and Soxhlet extracted with MeOH
for 20 h or until no more discoloration of the solvent could be
detected. The product was removed from the Soxhlet extractor
and allowed to air dry before being dried under high vacuum.
Compound 2 (17.9 g, 92%) was recovered as a white powder:
FABMS m/ z 1927 for [M + Na]+, calcd for C42H63O28I7 M 1904;
δH (300 MHz, CD3SOCD3) 3.28 (t, J ) 9 Hz, 7H), 3.34-3.48
(m, 14H), 3.54-3.68 (m, 14H), 3.80 (bd, J ) 9 Hz, 7H), 4.99
(d, J ) 3 Hz, 7H), 5.94 (d, J ) 2 Hz, 7H), 6.05 (d, J ) 6.5 Hz,
7H); δC (75 MHz, CD3SOCD3) 9.5, 71.0, 72.0, 72.3, 86.0, 102.2.
P er -6-a zid o-â-cyclod extr in (3). Per-6-iodo-â-cyclodextrin
(2) (2.99 g, 1.57 mmol) was dissolved in DMF (50 mL), and
NaN3 (1.00 g, 15.4 mmol) was added. The resulting suspension
was stirred at 60 °C under an atmosphere of N2 for 20 h. The
suspension was then concentrated under reduced pressure to
a few milliliters before a large excess of H2O was added. A
fine white precipitate was formed and was filtered off carefully.
The precipitate was washed with H2O and dried under high
vacuum to yield a stable white powder. The product never
showed any sign of decomposition, even when the dry powder
was heated to 80 °C. Compound 3 (2.01 g, 98%) was recov-
ered: IR (Nujol suspension on NaCl plates) 3355 cm-1 (OH),
2106 cm-1 (N3); LSIMS m/ z 1332 for [M - H + Na]+, 1354 for
[M - 2H + 2Na]+, 1376 for [M - 3H + 3Na]+; calcd for
C42H63N21O28 M 1310; δH (300 MHz, CD3SOCD3) 3.30-3.42 (m,
14H), 3.53-3.65 (m, 14H), 3.68-3.82 (m, 14H), 4.91 (d, J ) 3
Hz, 7H), 5.77 (d, J ) 2 Hz, 7H), 5.92 (d, J ) 7 Hz, 7H); δC
(75MHz, CD3SOCD3) 51.4 , 70.4, 72.1, 72.7, 83.3, 102.1. Anal.
Calcd for C42H63N21O28: C, 38.5; H, 4.81; N, 22.4. Found: C,
38.5; H, 5.05; N, 21.9.
1
signals in its H NMR spectrum. As expected, compound
12 showed improved solubilities in organic solvents. For
example, it dissolves readily in DMF, DMSO, and C5H5N,
and as its hepta-TFA salt, it dissolves in Me2CO and
THF.
Con clu sion
The synthetic methodology which is described in this
paper affords gram quantities of the â-cyclodextrin
derivatives 6, 7, and 12, which are symmetrically sub-
stituted with either phenylalanine or cysteine. It is
hoped that these chemically modified cyclodextrins will
show higher and more selective complexation and solu-
bilization of guests than the naturally occurring cyclo-
dextrins. Furthermore, these amino acid derivatives of
â-cyclodextrin may be used to assemble peptides on
â-cyclodextrins.16,35
Exp er im en ta l Section
Gen er a l. Unless otherwise stated, chemicals were used as
received. When dry solvents are referred to, they were dried
by the following procedures: (i) DMF was dried over CaH2 and
(29) Le Bas, G.; Rysanek, N. in Cyclodextrins and their Industrial
Uses; Ducheˆne, D., Ed.; Editions de la Sante´: Paris, 1987; pp 105-
130. Retzel, C.; Saenger, W.; Hingertz, B. E.; Brown, G. M. J . Am.
Chem. Soc. 1984, 106, 7545-7557. Zahel, V.; Saenger, W.; Mason, S.
A. J . Am. Chem. Soc. 1986, 108, 3664-3673.
(30) Nicolis, I.; Coleman, A. W.; Charpin, P.; Villain, F.; Zhang, P.;
Ling, C.-C.; de Rango, C. J . Am. Chem. Soc. 1993, 115, 11596-11597.
(31) The N-Boc-cysteine was obtained in good yields by reduction
of the corresponding cystine with sodium in liquid ammonia and was
isolated as a white solid as its ammonium salt. See: Zahn, H.;
Hammerstro¨m, K. Chem. Ber. 1969, 102, 1048-1052.
(32) Gundermann, K.-D.; Hu¨mke, K. In Methoden der Organischen
Chemie; Klamann, D., Ed.; Georg Thieme Verlag: Stuttgart, 1985; Vol.
E11, pp 33-35.
(33) Buter, J .; Kellogg, R. M. J . Org. Chem. 1981, 46, 4481-4485.
Khurana, J . M.; Sahoo, P. K. Synth. Commun. 1992, 22, 1691-1702.
Meier, H.; Dai, Y. Tetrahedron Lett. 1993, 34, 5277-5280.
(34) J ohnstone, R. A. W.; Rose, M. E. Tetrahedron Lett. 1979, 35,
2169-2173. Ciucanu, I.; Kerek, F. Carbohydr. Res. 1984, 131, 209-
217.
(35) Lear, J . D.; Wasserman, Z. R.; DeGrado, W. F. Science 1988,
240, 1177-1181; Science 1989, 243, 622-628. Sasaki, T.; Kaiser, E.
T. J . Am. Chem. Soc. 1989, 111, 380-381. Mutter, M.; Vuilleumier, S.
Angew. Chem., Int. Ed. Engl. 1989, 28, 535-554. Mutter, M.; Tuch-
scherer, G. G.; Miller, C.; Altmann, K.-H.; Carey, R. I.; Wyss, D. F.;
Labhardt, A. M.; Rivier, J . E. J . Am. Chem. Soc. 1992, 114, 1463-
1470. Hahn, K. W.; Klis W. A.; Stewart, J . M. Science 1990, 248, 1544-
1547. Ghadiri, M. R.; Soares, C.; Choi, C. J . Am. Chem. Soc. 1992,
114, 825-831. Ghadiri, M. R.; Soares, C.; Choi, C. J . Am. Chem. Soc.
1992, 114, 4000-4002. Ghadiri, M. R.; Case, M. A. Angew. Chem., Int.
Ed. Engl. 1993, 32, 1594-1597.
P er -6-a m in o-â-cyclod extr in (4). The heptaazide 3 (2.01
g, 1.53 mmol) was dissolved in DMF (40 mL), and Ph3P (6.36
g, 24.2 mmol) was added. The evolution of N2 can be observed
by the formation of bubbles in the reaction vessel. After 1 h,
during which time the evolution of N2 ceased, concentrated
aqueous NH3 (6 mL, approximately 35%) was added dropwise

Amino Acid Derivatives of â-Cyclodextrin
J . Org. Chem., Vol. 61, No. 3, 1996 907
to the solution. Shortly after the addition of the NH3 solution
was complete, the reaction mixture turned into an off-white
suspension. It was stirred at rt for 18 h before the resulting
suspension was concentrated under reduced pressure to ap-
proximately 10 mL. The product was then precipitated by the
addition of EtOH (100 mL). The precipitate was washed with
EtOH and dried under high vacuum to yield a white solid.
Compound 4 (1.69 g, 98%) was recovered. To allow charac-
terization by NMR spectroscopy, the HCl salt of 4 was formed
by suspending compound 4 in a small volume of H2O followed
by the addition of a dilute solution of HCl until the pH had
reached 6. At this pH, a clear solution formed which gave a
yellow glass when evaporated under reduced pressure: [R]D
+112° (c ) 1 in H2O at 25 °C); FABMS m/ z 1128 for [M]+,
calcd for C42H77O28N7 M 1128; δH (300 MHz, D2O) 3.26 (dd, J
) 7, 13 Hz, 7H), 3.44 (dd, J ) 3, 13 Hz, 7H), 3.57 (t, J ) 9 Hz,
7H), 3.66 (dd, J ) 3.5, 9.5 Hz, 7H), 3.98 (dd, J ) 9, 9.5 Hz,
7H), 4.15-4.25 (ddd, J ) 3, 7, 9 Hz, 7H), 5.15 (d, J ) 3.5 Hz,
7H); δC (75MHz, D2O) 42.9, 70.5, 74.3, 74.8, 84.8, 104.1.
P er -6-[(N-Boc-p h en yla la n yl)a m in o]-â-cyclod extr in (5).
N-Boc-phenylalanine21 (0.850 g, 3.20 mmol) was dissolved in
dry DMF (40 mL). HBT (0.460 g, 3.40 mmol) was added, and
the solution was cooled to 0 °C in an ice bath. DCC (0.670 g,
3.25 mmol) was then added, and the temperature was main-
tained at 0 °C for a further 60 min. The reaction mixture was
then allowed to warm to rt, during which time dicyclohexy-
lurea precipitated out. It was stirred for a further 60 min at
ambient temperature, a suspension of per-6-amino-â-cyclo-
dextrin (4) (0.510 g, 0.452 mmol) and ethylmorpholine (0.34
mL) in DMF (20 mL) was added to the reaction vessel, and
the reaction mixture was stirred at rt for 20 h. The precipi-
tated dicyclohexylurea was filtered off, and the filtrate was
concentrated under reduced pressure at 50 °C to obtain an
oil. Then, saturated aqueous NaHCO3 (200 mL) was added
to this oil to give a suspension which was stirred for 1 h and
then filtered. The precipitate was washed with H2O and dried
under high vacuum. Compound 5 (1.13 g, 89%) was recovered.
As the 1H NMR spectrum showed very broad signals, this
compound was only identified by FABMS: m/ z 2880 for [M +
Na]+, calcd for C140H196N14O49 M 2857.
P er -6-[(p h en yla la n yl)a m in o]-â-cyclod extr in (6). Per-
6-[(N-Boc-phenylalanyl)amino]-â-cyclodextrin (5) (1.13 g, 0.395
mmol) was added to neat TFA (2 mL). The solution was
agitated for 1 h before the TFA was evaporated off under
reduced pressure at rt. The resulting oil was added to Et2O
(50 mL) and sonicated to give a fine suspension which was
carefully filtered. The precipitate was washed with Et2O and
dried under high vacuum to give a yellow/orange hepta-TFA
acid salt. This solid was dissolved in a small amount of H2O
and precipitated by the addition of concentrated aqueous NH3
solution. The precipitate was filtered off and dried under high
vacuum. Compound 6 (0.295 g, 35%) was recovered: [R]D +78°
(c ) 1 in DMF at 25 °C); FABMS m/ z 2158 for [M]+, calcd for
C105H140N14O35 M ) 2158; δH (300 MHz, CD3SOCD3) 1.71 (bs,
14H), 2.56 (dd, J ) 9, 14 Hz, 7H), 2.90 (dd, J ) 4, 14 Hz, 7H),
3.15-3.65 (m, 42H), 3.85 (bd, J ) 9 Hz, 7H), 4.77 (bd, J ) 3
Hz, 7H), 5.78 (bs, 7H), 5.87 (d, J ) 6.5 Hz, 7H), 7.13-7.25 (m,
35H), 8.08 (bs, 7H); δC (75MHz, CD3SOCD3) ) 39.2, 41.2, 55.7,
70.2, 72.3, 72.7, 83.2, 102.0, 126.1, 128.0, 129.4, 138.9, 175.1.
Anal. Calcd for C119H147F21N14O49 (the hepta-TFA salt): C,
48.9; H, 5.04; N, 6.72. Found: C, 48.8; H, 5.52; N, 7.06.
P er -6-cystein e-â-cyclod extr in (7). Cysteine (0.993 g,
8.19 mmol) was weighed into a three-necked round-bottom
flask. The flask was cooled in an i-PrOH/dry ice bath, and
NH3 (100 mL) was condensed into the vessel using a dry ice
condenser. Na (0.38 g, 16.5 mmol) was then added to the
solution, and the reaction mixture was allowed to warm to NH3
reflux temperature (-33 °C). After the dark blue color had
persisted for 20 min, the excess of Na was destroyed by the
addition of NH4Cl. Per-6-iodo-â-cyclodextrin (2) (2.208 g, 1.15
mmol) was then added to the solution, and the reaction
mixture was stirred for 30 min. The NH3 was allowed to
evaporate over 2 h. The solid residue was then taken up in a
small volume of H2O to give a white suspension, which was
filtered through a pad of Celite. The hepta-Na salt could then
be precipitated from this filtrate by the addition of an excess
of EtOH. The precipitate was washed with EtOH and dried
under high vacuum at 80 °C to give a deliquescent white solid
(2.19, 94%). To obtain the zwitterionic compound 7, the
aqueous solution was neutralized with dilute HCl before
precipitation of the product was effected with EtOH, [R]D +73°
(c ) 1 in 1 M NaOHaq at 25 °C). This compound did not give
a FABMS under a variety of different conditions: MALDI-
TOFMS m/ z 1859 for [M + H]+, 1881 for [M + Na]+, 1900 for
[M - H + 2Na]+, 1921 for [M - 2H + 3Na]+, calcd for
C63H105N7O42S7 M 1857; δH (300 MHz, 0.1 M NaOD in D2O)
2.82 (dd, J ) 8, 14 Hz, 7H), 2.93 (dd, J ) 7.5, 14 Hz, 7H), 3.05
(dd, J ) 5, 14 Hz, 7H), 3.13 (bd, J ) 14 Hz, 7H), 3.47 (dd, J )
5, 8 Hz, 7H), 3.56 (t, J ) 9 Hz, 7H), 3.58 (dd, J ) 4, 9 Hz, 7H),
3.87 (t, J ) 9 Hz, 7H), 3.95 (bdd, J ) 7.5, 9 Hz, 7H), 5.08 (d,
J ) 4 Hz, 7H); δC (75 MHz, 0.1 M NaOD in D2O) 36.2, 41.1,
58.1, 74.5, 74.7, 75.5, 86.1, 104.0, 182.2. Elemental analysis
was performed on the hepta-Na salt. Anal. Calcd for
C63H98O42N7S7Na7‚7H2O: C, 35.4; H, 5.24; N, 4.58. Found: C,
35.3; H, 5.33; N, 4.47.
P er -6-(ter t-bu tyld im eth ylsilyl)-â-cyclod extr in (8). Dry
â-cyclodextrin (1) (9.04 g, 7.96 mmol) was dissolved under
vigorous stirring in dry C5H5N (100 mL). The solution was
cooled on an ice bath, producing a thick gel. A solution of
TBDMSCl (14.5 g, 96.2 mmol) in dry C5H5N (150 mL) was then
added dropwise to the cooled reaction vessel over 3.5 h. During
this time, the gel liquified. Cooling was continued for a further
3 h before the solution was allowed to warm to rt. After a
further 18 h at rt, the solvent was removed under reduced
pressure to give a white solid, which was taken up in CH2Cl2
(300 mL). The CH2Cl2 layer was washed with KHSO4 (200
mL, 1 M) to remove any residual C5H5N, followed by saturated
aqueous NaCl solution. The CH2Cl2 layer was recovered and
evaporated to dryness. Compound 8 (14.64, 95%) was recov-
ered. In order to remove any last traces of TBDMSCl and to
obtain high purity samples for analytical purposes, the product
can be subjected to column chromatography on silica gel using
EtOAc/hexane as eluant: LSIMS m/ z 1958 for [M + Na]+,
calcd for C84H168O35Si7 M 1935; δH (300 MHz, CDCl3) 0.03 (s,
21H), 0.04 (s, 21H) 0.86 (s, 63H), 3.55 (dd, J ) 9.5, 9.5 Hz,
7H), 3.59 (bs, 7H), 3.65 (dd, J ) 3, 9.5 Hz, 7H), 3.70 (bd, J )
11 Hz, 7H), 3.89 (dd, J ) 2, 11 Hz, 7H), 4.03 (dd, J ) 9.5, 9.5
Hz, 7H), 4.88 (d, J ) 3 Hz, 7H), 5.26 (s, 7H), 6.72 (s, 7H); δC
(75 MHz, CDCl3) -5.2, -5.1, 18.3, 25.9, 61.7, 72.6, 73.4, 73.6,
81.8, 102.0.
P er -6-(ter t-bu tyld im eth ylsilyl)p er -2,3-m eth yl-â-cyclo-
d extr in (9). NaH 60% (11.6 g, 289 mmol) was weighed into
the reaction vessel. Hexane (100 mL) was added, and the
mixture was stirred for 1 min before the suspension was
allowed to settle and the hexane was decanted off. This
hexane washing procedure was repeated two more times to
obtain oil-free NaH. Per-6-(tert-butyldimethylsilyl)-â-cyclo-
dextrin (8) (16.80 g, 8.68 mmol) was dissolved in dry THF (100
mL) and added very carefully to the NaH with cooling. Once
the evolution of H2 had subsided, MeI (20 mL, 321 mmol) was
added and stirring was commenced. The reaction mixture was
protected from light and placed under an atmosphere of N2.
Cooling was maintained for the first hour of the reaction, after
which time the suspension was allowed to warm to rt. After
16 h, the reaction mixture was cooled on an ice bath and MeOH
was added dropwise to destroy the excess of NaH and MeI.
Once all the NaH had been destroyed, the THF was removed
under reduced pressure and the residue was suspended in CH2-
Cl2. The CH2Cl2 layer was washed with H2O, followed by
saturated aqueous NaCl solution. The CH2Cl2 layer was
recovered and evaporated to dryness to yield an off-white solid.
Compound 9 (18.30 g, 99%) was recovered: [R]D +95° (c ) 1
in CHCl3 at 25 °C); FABMS m/ z 2153 for [M + Na]+, calcd for
C98H196O35Si7 M 2130; δH (300 MHz, CDCl3) 0.02 (s, 21H), 0.03
(s, 21H), 0.86 (s, 63H), 3.04 (dd, J ) 3.5, 10 Hz, 7H), 3.47-
3.75 (m, 28H), 3.49 (s, 21H), 3.64 (s, 21H), 4.10 (dd, J ) 2, 12
Hz, 7H), 5.18 (d, J ) 3.5 Hz, 7H); δC (75 MHz, CDCl3) -5.2,
-4.8, 18.3, 25.9, 58.6 and 61.5, 62.3, 72.2, 78.7, 82.0, 82.2, 98.1.
P er -6-br om o-p er -2,3-m eth yl-â-cyclod extr in (10). Ph3-
PBr2 was freshly prepared by careful addition of Br2 (1.1 mL,
21 mmol) to Ph3P (5.59 g, 21.3 mmol) dissolved in CH2Cl2 (100
mL) maintained on an ice bath If the solution is slightly red

908 J . Org. Chem., Vol. 61, No. 3, 1996
Ashton et al.
in color, more Ph3P may be added to dispel the coloration.
When the reaction mixture was warmed to rt, a white
suspension was formed. Per-6-(tert-butyldimethylsilyl)per-2,3-
methyl-â-cyclodextrin (9) (5.99 g, 2.81 mmol) was then added
to this suspension, and the reaction mixture was stirred for
24 h at rt, during which time the suspension cleared. The CH2-
Cl2 solution was then washed with saturated aqueous NaHCO3
solution, followed by saturated aqueous NaCl solution. The
CH2Cl2 layer was recovered and concentrated under reduced
pressure to yield a thick oil. The oil was triturated with EtOH
to yield a white precipitate after sonication. The precipitate
was allowed to settle for 10 min before filtering it off.
Compound 10 (3.93 g, 79%) was recovered. Further purifica-
tion was often not necessary. However, analytically pure
samples may be prepared by submitting the product to column
chromatography on silica gel using a t-BuOMe/hexane eluant.
Crystals of compound 10 were grown by slow evaporation from
a concd Me2CO solution in only a few hours: [R]D +109° (c )
1 in CHCl3 at 25 °C); FABMS m/ z 1794 for [M + Na]+, calcd
for C56H91Br7O28 M 1771; δH (300 MHz, CDCl3) 3.22 (dd, J )
3.5, 9.5 Hz, 7H), 3.47-3.65 (m, 14H), 3.53 (s, 21H), 3.65 (s,
21H), 3.76-3.95 (m, 21H), 5.24 (d, J ) 3.5 Hz, 7H); δC (75
MHz, CDCl3) 34.3, 58.9 and 61.5, 70.8, 81.5, 81.6, 81.9, 98.3
column and eluted using 10% MeOH in CH2Cl2 to give a white
solid. Compound 11 (2.00 g, 63%) was recovered, [R]D +88° (c
) 1 in CHCl3 at 25 °C). It was not possible to obtain a mass
spectrum of this compound despite applying a range of
different mass spectrometric techniques to this sample: δH
(300 MHz, CDCl3) 1.46 (bs, 63H), 2.80-3.90 (2 bs, 81H), 4.80
(bs, 7H). Anal. Calcd for C112H189N7O56S7 + 7.CH2Cl2: C, 42.5;
H, 6.06; N, 2.93. Found: C, 42.5; H, 6.09; N, 3.05.
P er -6-cystein yl-per -2,3-m eth yl-â-cyclodextr in (12). Per-
6-[N-(tert-butyloxycarbonyl)cysteinyl]-per-2,3-methyl-â-cyclo-
dextrin (11) was dissolved in neat TFA, and the reaction
mixture was stirred at rt for 1 h. The TFA was then removed
under reduced pressure, ensuring that the temperature did
not exceed rt. The residual glass was dissolved in a minimum
of H2O and neutralized using i-Pr2EtN followed by precipita-
tion with an excess of Me2CO. The flask was allowed to stand
for 2 h before the precipitate was filtered off in near-
quantitative yield, [R]D +78° (c ) 1 in DMF at 25 °C). The
specific optical rotation for this compound was also determined
on the hepta-TFA salt: [R]D +49° (c ) 1 in H2O at 25 °C);
MALDI-TOFMS m/ z 2079 for [M
+
Na]+, calcd for
C77H133O42N7S7 M 2053; the hepta-TFA salt of 12 was analyzed
by 1H-NMR δH (300 MHz, D2O) 3.02 (bd, J ) 13 Hz, 7H), 3.10-
3.20 (m, C-6, 14H), 3.25 (dd, J ) 4, 13 Hz, 7H), 3.30 (dd, J )
3, 9 Hz, 7H), 3.43 (s, 21H), 3.54 (s, 21H), 3.57 (m, 7H), 3.70 (t,
J ) 9 Hz, 7H), 3.91-3.98 (m, 7H), 4.21 (dd, J ) 4, 8 Hz, 7H),
5.19 (d, J ) 3 Hz, 7H); δC (75 MHz, D2O) ) 36.7, 37.5, 56.8,
60.8, 62.9, 74.5, 82.5, 83.4, 100.5, 176.0.
(C-1). Anal. Calcd for C56H91Br7O28
Found: C, 37.9; H, 5.50.
: C, 38.0; H, 5.18.
P e r -6-[N -(t er t -b u t yloxyca r b on yl)cyst e in yl]-p e r -2,3-
m eth yl-â-cyclod extr in (11). Per-6-bromo-per-2,3-methyl-â-
cyclodextrin (10) (2.03 g, 1.15 mmol) was dissolved in DMSO
(35 mL). N-Boc-cysteine31 (2.43 g, 10.2 mmol) was then added
to the solution, followed by powdered KOH (3.258 g, 58 mmol).
The suspension was placed under an atmosphere of N2 and
stirred at rt for 1 h. The solution was then partitioned between
aqueous KHSO4 (150 mL, 1 M) and EtOAc (100 mL). The
EtOAC layer was washed with saturated aqueous NaCl
solution (3 × 75 mL) and then dried over MgSO4, filtered, and
evaporated to dryness. The residue was then applied to a
Ack n ow led gm en t. We thank Pfizer Limited for the
provision of a Studentship to R.K. and the Biotechnology
and Biological Sciences Research Council for grants to
purchase mass spectrometry equipment.
J O951396D