1-Aminocyclobutanecarboxylic acid derivatives as novel structural elements in bioactive peptides: Application to tuftsin analogs

Article abstract of DOI:10.1021/jm960390t

Four novel 2,4-methano amino acids (MAAs, 1-aminocyclobutane-1- carboxylic acids) were synthesized. These include the basic MAA analogs of lysine (16), ornithine (5), and arginine (6) and the neutral methanovaline (22), related to proline. The above MAAs,

Full text of DOI:10.1021/jm960390t

J . Med. Chem. 1996, 39, 4833-4843
4833
1-Am in ocyclobu ta n eca r boxylic Acid Der iva tives a s Novel Str u ctu r a l Elem en ts
in Bioa ctive P ep tid es: Ap p lica tion to Tu ftsin An a logs
Eytan Gershonov,† Ruth Granoth,† Esther Tzehoval,‡ Yehiel Gaoni,† and Mati Fridkin*,†
Departments of Organic Chemistry and Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel
Received J une 3, 1996X
Four novel 2,4-methano amino acids (MAAs, 1-aminocyclobutane-1-carboxylic acids) were
synthesized. These include the basic MAA analogs of lysine (16), ornithine (5), and arginine
(6) and the neutral methanovaline (22), related to proline. The above MAAs, as well as the
MAA analog of homothreonine (7), were incorporated into the peptide chain of the immuno-
modulatory peptide tuftsin, Thr-Lys-Pro-Arg, known to enhance several biological activities
mediated by phagocytic cells. The synthetic methano tuftsin analogs were assayed for their
ability to stimulate interleukin-6 (IL-6) secretion by mouse peritoneal macrophages and for
their stability in human serum toward enzymatic degradation. It was found that, at 2 × 10-7
M, [MThr1]tuftsin (24) and an isomer of [MVal3]tuftsin (27a ) were considerably more active
than the parent peptide in augmentation of cytokine release. [MOrn2]Tuftsin (25) was equally
potent. The analogs [MThr1]tuftsin (24) and [MOrn2]tuftsin (25), both pertaining to the
proteolytically sensitive Thr-Lys bond of tuftsin, exhibited high resistance to enzymatic
hydrolysis as compared to tuftsin. Using specific rabbit anti-tuftsin antibodies in a competitive
enzyme-linked immunosorbent assay (ELISA) revealed that none of the MAA analogs can cross-
react with tuftsin. It may indicate that the peptides assume global structures different than
that of tuftsin.
In tr od u ction
model. Toward this goal we have synthesized four novel
MAA derivatives.
The use of peptides in medically-oriented research,
basic as well as applied, is constantly increasing. The
major clinical potential of this family of compounds has,
as yet, only slightly been realized. Hence, there is a
growing demand for diversification of the arsenal of
suitable building blocks whose incorporation into pep-
tide chains may lead to augmentation of corresponding
biopotency, selectivity and bioavailability. Efforts have
been directed toward these goals through, notable
among others, improving resistance to proteolysis1,2 and
stabilization of putative bioactive structures.1,3 Meth-
odologies such as peptide-bond modifications,4 rational
cyclization,5-9 and introduction of bulky, motility-
constraining elements into the peptide chain7,10,11 have
been investigated. 1-Aminocyclobutane-1-carboxylic acid
(2,4-methano amino acid; MAA) derivatives belong to
the latter category. Several of these amino acid analogs,
bearing carboxyl or phosphoryl side chain moieties, have
been studied in relation to excitatory receptors in the
mammalian central nervous system.12-14 The applica-
tion of simple MAA derivatives, i.e. without side-chain
functions, along with other CR,R-symmetrically disub-
stituted cycloaliphatic glycine, has been limited, so far,
to the synthesis of compounds related to L-aspartyl
dipeptide sweeteners15 and to basic conformational
considerations.16 Both of these latter studies have
clearly pointed at the potentiality of MAA derivatives
as beneficial structural components in bioactive peptide
research. Aiming to test the feasibility of incorporating
MAA, bearing “side-chain” moieties, into peptide chains
and to evaluate its consequences we choose tuftsin as a
Tuftsin, Thr-Lys-Pro-Arg, is a serum basic peptide,
originating from immunoglobulin G through specific
proteolysis, which has been found to exhibit a wide
spectrum of biological activities associated with the
reticuloendothelial and immune system function.17 It
is a potent immunomodulator whose capacity is mani-
fested through and following binding to specific recep-
tors present on virtually all phagocytic cells.18,19 Tuftsin
has a marked therapeutic potential, e.g. as an antimi-
crobial and anticancer agent,17,20,21 and thus the devel-
opment of potent and metabolically stable analogs is of
major significance. The steric hindrance and confor-
mational constrain induced on the tuftsin molecule by
the cyclobutane ring of MAA may lead to realization of
the above goals. Furthermore, all four amino acid
residues in the tuftsin molecule are important, with
certain allowed alterations, for manifestation of optimal
biopotency.17,22 Systematic substitution of amino acids
by corresponding MAA derivatives may shed additional
light on structure-function relationships and mode of
action of tuftsin.17 It may also lead to novel tuftsin-
related drugs.
Ch em istr y
Four novel trans-2,4-methano amino acids derivatives
were prepared in the present study and utilized in
peptides synthesis. These compounds are analogs of
three basic amino acids, i.e. ornithine (5; MOrn), argi-
nine (6; MArg), and lysine (16; MLys), and of valine (22;
MVal), used here as a substitute for proline. A previ-
ously reported23,24 analog of homothreonine (7; MThr)
was also synthesized (Scheme 1). The synthetic ma-
nipulations which led to compounds 5-7 and 16 are
summarized in Schemes 1 and 2. They refer to and are
* Author to whom correspondence should be addressed. Tel: 972-
8-9342505. Fax: 972-8-9344142.
† Department of Organic Chemistry.
‡ Department of Immunology.
X Abstract published in Advance ACS Abstracts, November 1, 1996.
S0022-2623(96)00390-1 CCC: $12.00 © 1996 American Chemical Society

4834 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24
Gershonov et al.
Sch em e 1a
Sch em e 3a
a
(a) MeSO2Cl, NEt3, CH2Cl2; (b) TMGA, NMP; (c) H2, 10% Pd/
C, EtOH; (d) 6 N HCl; (e) DMPCN, NEt3, DMF.
Sch em e 2a
a
(a) Basic CuCO3, H2O; (b) PhCH2CO2Cl, MgO, H2O; (c) Na2S,
0.9 N HCl; (d) ((CH3)3COCO)2O, NEt3, DMF/H2O.
5-7 and 16 from 8 were 11%, 10%, 22%, and 7%,
respectively. Racemic trans-1-amino-2-methylcyclobu-
tane-1-carboxylic acid (MVal; 22) was synthesized by
the same procedures described for the preparation of
intermediate 8 using crotyl bromide instead of allyl
chloride.29 Protection of the side-chain amino group and
of the R-amino group of the MAA is described in Scheme
3. The purity of MAA, as well as that of en-route
intermediates, was ascertained by NMR, by mass
spectrometry, and by homogeneity in amino acid analy-
sis. This latter technique was further employed for the
quantitative determination of MAA content in the
corresponding peptides.
Four analogs of tuftsin, [MThr1]tuftsin (24), [MOrn2]-
tuftsin (25), [MLys2]tuftsin (26), and [MVal3]tuftsin (27)
(Figure 1), were synthesized following solid phase
methodology,30 on a chloromethylated polystyrene car-
rier, utilizing NR-t-Boc strategy. An equimolar combi-
nation of N,N′-dicyclohexylcarbodiimide (DCC) and
1-hydroxybenzotriazole (HOBT) was employed as cou-
pling agent. The various stages of peptide chain exten-
sions were monitored by qualitative ninhydrin test. As
a rule, double couplings were performed throughout
syntheses. Respective incorporation of MAA derivatives
did not create difficulties related to incomplete cou-
plings, as might perhaps been anticipated due to steric
considerations. The methanoarginine analog of tuftsin,
[MArg4]tuftsin (28) (Figure 4), was prepared in solution
by coupling of unprotected MArg (6) with the tripeptide
active ester Boc-Thr-Lys(Boc)-Pro-OSu17 following a
corresponding deprotection. All crude tuftsin analogs
were purified to homogeneity by semipreparative HPLC
on silica RP-18 column and their correct structures
ascertained by amino acid analysis and mass spectrom-
etry.
a
(a) BuLi, THF; (b) oxirane; (c) DHP, PPTS, CH2Cl2; (d) Na/
Hg 6%, MeOH:THF, 3:1; (e) unsuccessful isomers separation; (f)
EtOH, PPTS; (g) MeSO2Cl, NEt3, CH2Cl2; (h) TMGA, NMP; (i) H2,
10% Pd/C, EtOH; (j) 6 N HCl. Tetrahydropyranyl ether.
b
based on the general scheme for the preparation of
substituted cyclobutanes from 1-(phenylsulfonyl)bicyclo-
[1.1.0]butane (8).12,25 Thus, the high strain of the
bicyclobutane ring, coupled with the olefinic π-bond
nature of the central C-C bond and with the enhanced
acidity of the bridgehead proton, make this system
particularly prone to a large variety of chemical modi-
fications.26,27 As previously shown, the amino functions,
at both R and side-chain positions of MAA derivatives,
were introduced via reduction of corresponding azido
groups of 3 and 14. The guanidino side chain of the
MAA analog of arginine (6) was introduced by reacting
the free amino group of intermediate 4 with 3,5-
dimethylpyrazole-1-carboxamidine nitrate (DMPCN).28
The overall yields in the reactions leading to the MAA

Novel 2,4-Methano Amino Acids
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24 4835
a
F igu r e 1. Structures of methano tuftsin analogs. Tuftsin analog 26 was obtained as a mixture of both cis and trans isomers of
methanolysine and was separated by HPLC. They will be referred to as 26a and 26b. bTuftsin analog 27 was obtained as a
mixture of two diastereomers (due to racemic MVal) and was separated by HPLC. They will be referred to as 27a and 27b.
Cir cu la r Dich r oism (CD) Mea su r em en ts
ity of its biological action as well as its binding to specific
receptors are therefore indicative of the possibility that
tuftsin assumes an “active conformation” only upon
adhering to its target cells.
The possibility that tuftsin may assume a preferred
conformation in solution was investigated by many
groups, using a variety of physical techniques. How-
ever, rather conflicting conclusions have been drawn.
Considerations based on X-ray, NMR, and IR data31 as
well as on IR studies32 suggested that tuftsin has a
certain tendency to form a 4 f 1 hydrogen-bonded
â-turn of type 3. It has also been suggested that this
structure is further stabilized by ionic interaction
between the carboxyl terminal of Arg4 and the R-amino
terminal of Thr1. This was further supported by CD
spectra of tuftsin and its analogs.33 Conformational
calculations based on energy minimization34 revealed
that the peptide may assume numerous low-energy
conformations which share a general characteristic
described as a “hairpin with two split ends”, where each
of the splits is composed of one terminus of the backbone
and a Lys or Arg side chain. Other conformational
studies based on calculations35 and CD spectra36 pro-
posed that the tuftsin chain is folded to form a quasi-
cyclic structure, stabilized by ionic interaction between
the carboxylic group of Arg4 and the ꢀ-amino group of
Lys2. Further 1H NMR and 13C NMR studies37 sug-
gested that tuftsin in aqueous solution exists in a
random conformation, while in DMSO a slightly ordered
structure is obtained. More recently it was postulated,
on the basis of results of high-temperature quenched
molecular dynamics calculations of tuftsin in solution,
that a type 4 â-turn characterize the Lys2-Pro3 posi-
tion.38 In view of the above it is evident that there is
no consensus as to the structure of tuftsin in aqueous
media. It can perhaps be assumed that tuftsin has a
rather random structure in aqueous solution. This
notion is further supported by extensive structure-
function studies on tuftsin analogs.22 The high specific-
Our present CD studies of tuftsin in water (pH 5)
reveal, in agreement with previous studies,36 a maximal
negative ellipticity at 198 nm characteristic of an
ensemble of random conformations.39 The positive
maximum observed at 221 nm36 appears, however, now
as a positive shoulder at 226 nm (Figure 2A). The CD
spectrum of tuftsin in trifluoroethanol (TFE) is similar
in shape to that obtained in water but with a marked
decrease in intensity of the negative maximum at 198
nm. This effect was considered,36 on the ground of pH
dependence of CD spectra, to be indicative of an interac-
tion of the carboxylic Arg4 group with the amino group
Thr1 contributing to the stabilization of the tuftsin’s
conformation. However, this change was also inter-
preted as the result of an increasing population of
â-turns conformers in solution. This suggestion is
further supported by the appearance of the small
negative maximum at 225-226 nm.
The CD curves of [MArg4]tuftsin 28 in water is similar
to that of tuftsin but with a slight decrease of maximum
negative intensity in the region 198-200 nm. No
changes were observed between the CD spectra of 28
in water and in TFE, suggesting that this analog had
no apparent secondary structure in solution (not shown).
The CD curves of [MVal3]tuftsin (27a ) (Figure 2A),
[MOrn2]tuftsin (25) (Figure 2B), and [MLys2]tuftsin
(26b) (not shown) are completely different from the
spectrum of tuftsin. There is a marked decrease in
intensity of ellipticity and a red concomitant shift of the
negative maximum, which becomes broader. The CD
spectra obtained for [MVal3]tuftsin (27a ) in TFE and
for [MOrn2]tuftsin (25) in water display a negative

4836 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24
Gershonov et al.
tuftsin (24), both in water and TFE (Figure 2B), and
[MVal3]tuftsin (27b) (not shown) gave a spectrum which
did not correspond to any of the characteristic common
patterns of well-defined structures.
In contrast to the above, major alterations in CD
spectra were obtained with [MVal3]tuftsin (27a), [MOrn2]-
tuftsin (25), and [MLys2]tuftsin (26b), as compared to
tuftsin, indicating perhaps the presence of certain
preferred conformers, probably of the â-turn types. The
[MArg4]tuftsin (28) spectrum was, however, similar to
that of tuftsin, suggesting the existence of random
conformations. [MVal3]Tuftsin (27b ) and [MThr1]-
tuftsin (24), as already mentioned, were not indicative
of any known structure. A further conclusion that can
be drawn from our CD data is that more pronounced
effects on the peptide conformation are afforded by a
change of configuration in residues 2 or 3 of the peptide
chain. It is also very tempting to relate the information
obtained from CD to the biological activity of the
analogs, tested for their effect on augmentation of IL-6
secretion by macrophages. [MVal3]Tuftsin (27a ), which
showed a certain ordered structure, was very active as
compared to tuftsin, indicating that this structure might
be closer to the “bioactive conformation”. This sugges-
tion is further supported by the inactivity of [MVal3]-
tuftsin (27b), the optical isomer of 27a , which showed
unordered structure and diminished bioactivity.
Biologica l Eva lu a tion s
Sta bility in Ser u m . In order to apply tuftsin as a
drug, its stability toward enzymatic degradation would
be of utmost importance. It has been reported41 that
the biological activity of tuftsin is destroyed by leucine
aminopeptidase, carboxypeptidase B, pronase, or sub-
tilisin but not by trypsin, chymotrypsin, papain, or
pepsin. Leucine aminopeptidase located on the plasma
membrane of human neutrophils plays, presumably, a
role in the degradation of tuftsin.42 This enzyme
removes the N-terminal threonine to yield Lys-Pro-Arg,
an inhibitor of tuftsin activity.43 The blood obviously
contains a battery of various nonspecific peptidases. It
was not surprising to find, using an assay of phagocy-
tosis, that indeed tuftsin is degraded efficiently by blood
enzymes.44
Various approaches were carried out to develop more
stable, potent long-acting analogs of tuftsin and to avoid
generation of the Lys-Pro-Arg inhibitor. These studies
included substitution of the N-terminal threonyl resi-
due10 or its cyclization,45,46 substitution of L-amino acids
by D-amino acids,47 elongation of the tetrapeptide chain
by the addition of further amino acid residues to the
N-terminus,17 sequential coupling of two molecules of
tuftsin to create the dimer tuftsinyltuftsin,48 and intro-
duction of an isopeptide bond into the molecule, taking
advantage of lysine’s ꢀ-amino group.2 Although some
of these approaches increased metabolic stability of
tuftsin, it was usually accompanied by a marked de-
crease or even an abolishment of biological activity.
Tuftsinyltuftsin, as an exception, was found to exert
antitumor activity but did not stimulate the phagocytic
capacity of macrophages.49 Loss of activity might stem
from major receptor-related structural alterations or be
due to blocking of functional groups essential for activ-
ity, caused by the above modifications.22
F igu r e 2. CD spectra of aqueous and TFE solutions of tuftsin
and methano tuftsin analogs. Symbols correspond to the
following peptides: (A) (s) tuftsin (T) in H2O, (‚‚‚) tuftsin in
TFE, (- - -) [MVal3]T 27a in H2O, (- ‚ -) [MVal3]T 27a in TFE.
(B) (-) [MThr1]T 24 in H2O, (‚‚‚) [MThr1]T 24 in TFE, (- - -)
[MOrn2]T 25 in H2O, (- ‚ -) [MOrn2]T 25 in TFE.
maximum at 204 nm and a positive maximum at 192-
194 nm with a shoulder at about 214 nm. This curve
shape is very similar to that observed in the CD
spectrum of gramicidin S and of [DLys2]tuftsin, where
the type 2′ â-turn is present.33 The CD curves of
[MOrn2]tuftsin (25) in TFE (Figure 2B) and of [MLys2]-
tuftsin (26b) (not shown) in water and in TFE display
a broader negative maximum in the region 212-218 nm
and a positive maximum at 190-195 nm, suggesting
the presence of different types of â-turns.40 The inten-
sity of the positive maximum is smaller than expected
which may indicate the presence of another population
of conformations or some distortion of the â-turn.
[MVal3]Tuftsin (27a ) in water (Figure 2A), [MThr1]-
Methanotuftsin analogs were tested, as compared to
tuftsin, for their stability in human serum toward

Novel 2,4-Methano Amino Acids
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24 4837
Macrophages were incubated in the presence of
antigen keyhole limpet hemocyanin (KLH), with various
concentrations of tuftsin or methanotuftsin analogs.
Twenty hours later the supernatants were collected and
measured for IL-6, employing cytokine-dependent cell
(B9) assay.51 The experimental data are compared to
KLH alone. The results, average of three experiments
(Figure 4), indicate that tuftsin augments secretion of
IL-6 by macrophages by 78% at 2 × 10-7 M, by 42% at
2 × 10-8 M and by 32% at 2 × 10-9 M. [MVal3]Tuftsin
(27a ) and [MThr1]tuftsin (24) significantly augment
secretion of IL-6 (141% and 125%, respectively) at 2 ×
10-7 M and to a lesser extent at 2 × 10-8 M (95% and
29%, respectively). [MOrn2]Tuftsin (25) augments se-
cretion of IL-6 by 68% at 2 × 10-7 M and by 54% at 2 ×
10-8 M. [MVal3]Tuftsin (27b) and the diastereomers
mixture 27 hardly augment IL-6 secretion (30%) at 2 ×
10-7 M, do not affect the macrophages at 2 × 10-8 M,
and show inhibitory effect at 2 × 10-9 M. [MLys2]-
Tuftsin isomers 26a and 26b, as well as [MArg4]tuftsin
(28), have no effect on the secretion of IL-6 at any
concentration and even show inhibitory effect at 2 ×
10-9 M (not shown). All the peptides (except for 26b)
show a dose-dependent pattern with maximal potency
at 2 × 10-7 M. Peptides alone (without KLH) did not
show any stimulatory effect at the above concentrations.
According to the results, [MVal3]tuftsin (27a ) and
[MThr1]tuftsin (24) were more effective as compared to
tuftsin. [MOrn2]Tuftsin 25 was as effective as tuftsin,
while all the other analogs were less effective and even
showed some inhibitory effect in diluted concentration.
The higher capacity of [MThr1]tuftsin (24) as compared
to tuftsin, as well as that of [MOrn2]tuftsin (25), which
was similar to that of tuftsin, can be explained, perhaps,
by the lower sensitivity of the MThr-Lys and Thr-MOrn
bonds to enzymatic cleavage, thus a lower production
rate of the inhibitory fragment Lys-Pro-Arg. The higher
effectiveness of [MVal3]tuftsin (27a ), however, can be
explained both by the lower sensitivity of the “proline”-
centered bonds toward endo-peptidases as well as to
conformational constraints which may lead to a facili-
tated and more fruitful binding to the tuftsin’s receptor.
Moreover, enhanced affinity of the analog toward tuftsin’s
specific receptor should not be excluded. The inactivity
of all other analogs can be explained either by metabolic
instability or by structural constraints that prevent the
peptide from attaining the bioactive conformation.
En zym e-Lin k ed Im m u n osor ben t Assa y (ELISA).
Anti-tuftsin antibodies, raised in rabbits against a
conjugate of (p-aminophenylacetyl)tuftsin and tetanus
toxoid, were employed in competitive ELISA assays with
the MAA-tuftsin analogs. It is evident from the results
(not shown) that none of the peptides can compete with
tuftsin, indicating further structural dissimilarity.
F igu r e 3. Stability toward enzymatic degradation by human
serum of tuftsin and methano tuftsin analogs. Symbols cor-
respond to the following peptides: (~) tuftsin (T), ([) [MArg4]T
28, (‚‚9‚‚) [MLys2]T 26a , (]) [MVal3]T 27a , (- 9 -) [MThr1]T
24, (0) [MOrn2]T 25.
enzymatic degradation. Thus, tuftsin and the synthetic
analogs were incubated with human serum at 37 °C,
aliquots were taken at various time intervals, and the
amount of intact peptides was evaluated by analytical
HPLC. From the results obtained (Figure 3), it is clear
that tuftsin was totally degraded after 60 min with a
half-life (t1/2) of 16 min. [MThr1]Tuftsin (24) and
[MOrn2]tuftsin (25) underwent comparatively very slow
degradation with t1/2 of 90 and 420 min, respectively.
[MVal3]Tuftsin (27a ) was degraded with t1/2 of 25 min,
while [MVal3]tuftsin (27b ) (not shown) underwent
degradation similar to tuftsin with t1/2 of 15 min and
total degradation after 60 min. Total degradation was
revealed after 90 min with the two [MLys2]tuftsin
isomers 26a and 26b, with t1/2 of 13 min for both of
them. [MArg4]Tuftsin (28) was totally degraded, like
tuftsin, after 60 min with t1/2 of 11 min. These results
clearly show that only analogs 24 and 25, which possess
one methano amino acid at the Thr1-Lys2 bond of
tuftsin, are much more resistant to enzymatic hydrolysis
as compared to the parent peptide. This further indi-
cates that it is this bond which is indeed more prone to
the enzymatic cleavage of tuftsin to produce the tripep-
tide inhibitor. It is not clear, as yet, why analogs 26a
and 26b, which contain methanolysine (at the sensitive
Thr-Lys bond), show insignificant enhanced stability
toward enzymatic degradation. The analog [MArg4]-
tuftsin (28) was degraded exactly as tuftsin, indicating
that substitution by methano amino acid in this position
does not contribute to stability.
IL-6 Assa y. On the basis of previous findings that
the immunomodulatory capacity of tuftsin apparently
lies in mediation of interleukin production,17,50 the
relative potencies of tuftsin and its methano analogs
were evaluated, in vitro, in stimulating mouse perito-
neal macrophages to secrete interleukin-6 (IL-6). IL-6
plays a central role in host-defense mechanisms relevant
to the action of tuftsin51 (also R. Granoth, manuscript
in preparation), thus for example, it promotes the
differentiation of B cells into antibodies producing cells
and induces synthesis of acute phase proteins by hepa-
tocytes.
Con clu sion s
We have synthesized a series of 2,4-methano amino
acid analogs of lysine, ornithine, and arginine and of a
methano analog of valine related to proline and incor-
porated them into the peptide chain of tuftsin. The
results of these experiments confirm our working hy-
pothesis that substitution of amino acid residues in
tuftsin by methano amino acids, in positions which are
more susceptible to degradation, may lead to resistance
toward enzymatic degradation and thus to enhanced
metabolic stability. Consequently, biological potency

4838 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24
Gershonov et al.
F igu r e 4. Augmentation of IL-6 secretion from macrophages by tuftsin and methano tuftsin analogs. From left to right, the bars
correspond to the following peptides: tuftsin (T), [MOrn2]T 25, [MThr1]T 24, [MVal3]T 27a , [MVal3]T 27b, 27a +27b. A solid
black bar is used for KLH alone.
(for detection of guanidine function of Arg). The silica gel for
column chromatography was Merck Kieselgel 60 (70-230
mesh). Tetrahydrofuran (THF) was dried by distillation from
sodium diphenyl ketyl. Commercial BuLi in hexane was
titrated with diphenylacetic acid to determine its concentra-
tion. All reactions with BuLi were carried out under argon
atmosphere by using syringe and septum techniques for
handling of air-sensitive reagents. Preparative high-voltage
may be retained and even augmented. The hypothesis
that restriction of bond rotations, imposed by the
cyclobutane ring, may affect global conformation and
biopotency was realized in the case of methanovaline,
i.e. “proline-related” analog of tuftsin, which possesses
a conformation different from that of tuftsin and is
highly active. Introduction of more then one methano
amino acid into tuftsin might be beneficial and is
currently under investigation.
The basic amino acids lysine and arginine, as well as
the cyclic species proline, constitute the “active site” of
many and versatile bioactive peptides, e.g. neurotensin,
adrenocorticotropin hormone, and bradykinin. Thus,
incorporation of methano-analogs into these peptides
may lead to stable and long-acting potent analogs.
paper electrophoresis was performed on a Whatman No.
3
paper for 60 min at 33.3 V/cm in 1.6 M formic acid (pH 1.65).
Products were extracted from paper by 10% solution of acetic
acid. Methano amino acids were analyzed with a Dionex
automatic amino acid analyzer.
Syn th esis of 2,4-Meth a n o Am in o Acid s. tr a n s-1-(Ben -
zoylam in o)-3-[[(m eth ylsu lfon yl)oxy]m eth yl]cyclobu tan e-
N,N-p en ta m eth ylen eca r boxa m id e (2). To solution of al-
cohol 1 (1.14 g, 3.6 mmol; prepared according to published
procedure12) in dichloromethane (DCM, 36 mL) under Ar, at
0 °C, was added triethylamine (365 mg, 3.6 mmol). This was
followed by dropwise addition of methanesulfonyl chloride (412
mg, 3.6 mmol). The solution was then slightly warmed to
initialize the reaction and set aside at room temperature for
1 h. An extractive workup with acidified water yielded a crude
product which was further purified by recrystallization from
chloroform-hexane to give 1.0 g (70%) of mesylate 2: TLC
system A, Rf 0.25; mp 193-194 °C; NMR (400 MHz, CDCl3) δ
1.52 and 1.60 (two br, 4 H and 2 H, piperidine protons), 2.71-
2.73 (d, J ) 7.45, 4 H, cyclobutane (CB) protons), 2.90-2.92
(m, 1 H, CB proton), 3.03 (s, 3 H, MsCH3), 3.50 (br, 4 H,
piperidine protons), 4.23-4.25 (d, J ) 6.8, 2 H, CH2OMs),
7.43-7.77 (m, 5 H, aromatic protons). The product was not
characterized further and was used as such for the next step.
Exp er im en ta l Section
Ch em istr y. Gen er a l P r oced u r es. Melting points were
taken on a Fisher-J ohns apparatus and were not corrected.
Proton NMR spectra were measured on a Bruker 270 or
Bruker AMX 400 spectrometer. Chemical shifts are reported
in δ units downfield from internal Me4Si when measurements
were taken in deuterated chloroform, from DHO (δ 4.80) when
measurements were taken in deuterium oxide, and from
CHD2OH (δ 3.30) when measurements were taken in deuter-
ated methanol. All J values are given in hertz. Mass spectra
were determined with a Finnigan 4500 or Finnigan TSQ-70B
triple stage quadropole spectrometer. Chemical ionization was
performed with isobutane as reagent gas. Fast atom bom-
bardment (FAB) was determined, unless indicated otherwise,
in magic bullet matrix (dithiothreitol-dithioerythritol, 5:1, v/v)
and with Xenon as the bombarding atom. TLC was done on
Merck Kieselgel 60-F254, or on Riedel-de Hae¨n DC-Karten CE
F cellulose, precoated aluminum plates, using the following
solvent systems: (A) dichloromethane (DCM)-ethyl acetate-
ether (2:1:1); (B) DCM-methanol (9:1); (C) 1-butanol-pyri-
dine-H2O-AcOH (15:10:12:3); (D) CHCl3-methanol-AcOH
(5:3:1); (E) 1-butanol-AcOH-H2O (2:1:1); (F) CHCl3-metha-
nol-AcOH (90:8:2); (G) DCM-methanol (19:1); (H) CH3CN-
H2O (9:1). The products were detected on the TLC plates by
one of the following methods or by a combination of them:
Ultraviolet light, iodine vapor, ninhydrin, and Sakaguchi’s test
tr a n s-1-(Ben zoyla m in o)-3-(a zid om eth yl)cyclobu ta n e-
N,N-p en ta m eth ylen eca r boxa m id e (3). To a solution of
mesylate 2 (1.0 g, 2.5 mmol) in 1-methyl-2-pyrrolidone (NMP;
12 mL), was added N,N,N′,N′-tetramethylguanidinium azide
(TMGA; 0.6 g, 3.8 mmol). The solution was then warmed to
80 °C for 2 h. Acidified water (120 mL) was added, and the
solution was repeatedly extracted with ethyl acetate. The
combined ethyl acetate extracts were washed three times with
water to remove NMP. After evaporation of the solvent, the
crude material was triturated with ether to remove excess
NMP, yielding 0.6 g (70%) of azide 3. TLC system A, Rf 0.35;
system B, Rf 0.75; mp 194-195 °C; NMR (400 MHz, CDCl3) δ

Novel 2,4-Methano Amino Acids
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24 4839
1.53 and 1.60 (two br, 4 H and 2 H, piperidine protons), 2.61-
2.80 (two m, 1 H and 4 H, cyclobutane (CB) protons), 3.36 (d,
J ) 6.8, 2 H, CH2N3), 3.52 (br, 4 H, piperidine protons), 6.64
(s, 1 H, NH), 7.43-7.79 (m, 5 H, aromatic protons); mass
spectrum (CI, positive mode) m/ z 342 (100, MH+), 299 (9, MH+
- HN3).
1-(Ben zoyla m in o)-3-(p h en ylsu lfon yl)-3-(2-h yd r oxyeth -
yl)cyclobu ta n e-N,N-p en ta m eth ylen eca r boxa m id e (9). To
a stirred solution of sulfone 8 (5 g, 11.7 mmol; prepared
according to published procedure12) in THF (120 mL) at -50
°C was added a solution of BuLi in hexane (2 equiv). The
mixture was allowed to warm to -20 °C for about 45 min
before ethylene oxide was added in excess. Stirring was
continued for 1 h before the reaction was quenched with water.
The THF was evaporated, and the aqueous solution was
extracted with DCM. During extraction, part of the product
precipitated and was collected and triturated with ether to give
1.8 g (33%) of the cis alcohol 9. The DCM layer was
evaporated, and the resulting crude was chromatographed on
silica gel (120 g, elution: DCM-ethyl acetate-ether, 1:1:1) to
give 0.9 g of the starting material and 0.8 g (14%) of mixture
of cis and trans alcohol 9: TLC system B, Rf 0.60; system F,
Rf 0.42; NMR (270 MHz, CDCl3 + TFA) of the cis isomer δ
1.57 (br, 6 H, piperidine protons), 2.22 (t, J ) 7.1, 2 H, CH2-
CH2OH), 2.94 and 3.74 (ABq, J ) 14.1, 4 H, CB protons), 3.48
and 3.58 (two br, 2 H each, piperidine protons), 4.07 (t, J )
7.1, 2 H, CH2CH2OH), 7.36-7.89 (m, 10 H, aromatic protons);
mass spectrum (CI, positive mode) m/ z 471 (29, MH+), 427
(100, MH+ - C2H3OH), 143 (89, PhSO2H2+).
1-(Ben zoyla m in o)-3-(p h en ylsu lfon yl)-3-[2-(tetr a h yd r o-
p yr a n yloxy)eth yl]cyclobu ta n e-N,N-p en ta m eth ylen eca r -
boxa m id e (10). Alcohol 9 (4 g, 8.5 mmol), 3,4-dihydro-2H-
pyran (1.5 g, 17.8 mmol), and pyridinium tosylate (PPTS; 220
mg, 0.88 mmol) were dissolved in DCM (60 mL) and stirred
at room temperature for 3 days (alcohol 9 was not completely
soluble in DCM and dissolved during the reaction). An
additional 1 equiv of dihydropyran and 0.5 equiv of PPTS were
added after 2 days for the completion of the reaction (TLC
monitoring, system G). The crude mixture was then washed
with water and brine, dried, and evaporated. The residue was
triturated with ether to give 5.2 g of crude solid 10, which was
utilized in the following step without further purification: TLC
system G, Rf 0.73; the NMR (270 MHz, CDCl3) spectrum was
a superposition of the two isomers and was too complexes for
interpretation. At δ 4.55 and 4.47, two br were observed (ratio
9:1) related to OCH(CH2)O proton of the cis and trans isomers.
tr a n s-1-(Ben zoyla m in o)-3-(a m in om eth yl)cyclobu ta n e-
N,N-p en ta m eth ylen eca r boxa m id e (4). Azide 3 (510 mg,
1.5 mmol) was stirred in ethanol (25 mL) with 10% Pd/C
catalyst (100 mg) under hydrogen, at atmospheric pressure
and room temperature, for 20 h (extra catalyst and fresh
hydrogen might be added if the starting material is still
detected by TLC: system B; the amine product is more polar
then the azide). After filtration of the catalyst through Celite,
the solvent was evaporated and the residue was recrystallized
from DCM-hexane to yield 440 mg (93%) of impure amine 4.
The compound was further purified by dissolving in diluted
HCl, extraction of aqueous solution with DCM, addition of base
to pH ∼10 and re-extraction of the amine with DCM: TLC
system C, Rf 0.67; system D, Rf 0.65; mp 180-182 °C; NMR
(400 MHz, CDCl3) δ 1.50 and 1.57 (two br, 6 H, piperidine
protons), 2.52-2.68 (m, 5 H, CB protons), 2.74 (d, J ) 4.9, 2
H, CH2NH2), 3.50 (br, 4 H, piperidine protons), 7.27-7.79 (m,
5 H, aromatic protons); mass spectrum (CI, positive mode) m/ z
316 (100, MH+), 231 (41, MH+ - piperidine).
tr a n s-1-Am in o-3-(a m in om eth yl)cyclobu ta n e-1-ca r box-
ylic Acid (tr a n s-Meth a n oor n ith in e, 5). (Aminomethyl)-
cyclobutane 4 (450 mg, 1.43 mmol) was dissolved in 6 N HCl
(20 mL), and the solution was heated at 130 °C for 20 h. Water
was added to the reaction mixture, and the solution was
extracted with ether. The aqueous layer was evaporated, and
the residue was dissolved in water and evaporated, repeating
this process for three times. The residue was then dissolved
in ethanol, treated with propylene oxide, and triturated to
furnish 200 mg (97%) of pure trans-methanoornithine 5: TLC
system C (cellulose), Rf 0.18; system E, Rf 0.25; NMR (400
MHz, D2O) δ 2.51 (d, J ) 8.5, 4 H, CB protons), 2.88 (m, 1 H,
CB proton), 3.23 (d, J ) 7.3, 2 H, CH2NH2); mass spectrum
(CI, positive mode) m/ z 145 (100, MH+), 127 (77, MH+ - H2O);
amino acid analysis, tR 36.62 min.
1-(Ben zoyla m in o)-3-[2-(tetr a h yd r op yr a n yloxy)eth yl]-
cyclobu ta n e-N,N-p en ta m eth ylen eca r boxa m id e (11) was
prepared from 10 by treatment with 6% sodium amalgam, as
described previously.12 After evaporation of the solvents, the
residue was dissolved in ethyl acetate, washed with water,
dried, and evaporated. The resulting crude mixture was
chromatographed on silica gel (200 g, elution DCM-ether-
ethyl acetate, 16:8:3), but separation of the cis and trans
isomers could not be achieved. The overall yield from alcohol
9 was 72%: TLC system A, Rf 0.44; system B, Rf 0.69; the
NMR (270 MHz, CDCl3) spectrum is complex, δ 1.35-1.62 (m,
12 H, piperidine + THP protons), 1.63-1.83 (m, superimposed
of two dt, 2 H, CHCH2CH2O), 2.37-2.67 (m, 2 H, CB trans
protons) and 2.10-2.32 and 3.05-3.13 (two m, 3 H, CB cis
protons), 3.40-3.58 (br, 6 H, piperidine + THP protons), 3.33
and 3.70 (two dt, J AB ) 9.8, J AX ) 6.3, 1 H each, ABX of
CH2CHAHBO), 4.53 (br, 1 H, OCH(CH2)O), 7.38-7.80 (m, 5 H,
aromatic protons); mass spectrum (CI, positive mode) m/ z 415
(49, MH+), 330 (62, MH+ - THP), 287 (100, MH+ - C2H3-
OTHP).
tr a n s-1-Am in o-3-(gu a n id in om eth yl)cyclobu ta n e-1-ca r -
boxylic Acid (tr a n s-Meth a n oa r gin in e, 6). To a solution
of amine 4 (200 mg, 0.63 mmol) and triethylamine (667 mg,
6.6 mmol) in dried DMF (10 mL) was added 3,5-dimethylpyra-
zole-1-carboxamidine nitrate (1.27 g, 6.3 mmol), and the
reaction mixture was stirred, protected from moisture, at room
temperature for 24 h. TLC (system H, Sakaguchi and ninhy-
drin detection) showed disappearance of starting material. The
DMF was removed entirely in vacuo, and the crude residue
was hydrolyzed, in the same procedure used for methanoor-
nithine 5, without any purification. The resultant product was
dissolved in water and passed through strong acidic cation
exchange column (Dowex 50W×8). The eluent was concen-
trated and freeze-dried to give still impure product. Prepara-
tive purification by high-voltage paper electrophoresis was
then performed, providing 94 mg (80%) of trans-methanoargi-
nine 6: TLC system C (cellulose), Rf 0.27; system E, Rf 0.31;
NMR (400 MHz, D2O) δ 2.45 (m, 4 H), 2.84 (m, 1 H), 3.40 (d,
J ) 7.1, 2 H, CH2NH); mass spectrum (CI, positive mode) m/ z
187 (20, MH+), 169 (15.4, MH+ - H2O), 145 (48, MH+
-
(NHCdNH)), 127 (100, M - guanidine+); mass spectrum (CI,
negative mode) m/ z 185 (100, [M - H]-), 143 (70, [M - H -
(NHCdNH)]-; mass spectrum (FAB, glycerol matrix, positive
mode) m/ z 187 (100, MH+); amino acid analysis, tR 46.32 min.
1-(Ben zoyla m in o)-3-(2-h yd r oxyeth yl)cyclobu ta n e-N,N-
p en ta m eth ylen eca r boxa m id e (12). Pyranyl ether 11 (2.3
g, 5.5 mmol) and PPTS (138 mg, 0.55 mmol) were dissolved in
ethanol (44 mL), and the mixture was stirred at 55 °C for 20
h. The mixture was evaporated under reduced pressure and
chromatographed on silica gel (30 g, elution: DCM-methanol,
19:1), providing 1.53 g (84%) of alcohol 12. TLC system G, Rf
0.45; NMR (270 MHz, CDCl3 + CD3OD) revealed some
tr a n s-1-Am in o-3-(h yd r oxym et h yl)cyclob u t a n e-1-ca r -
boxylic Acid (tr a n s-Meth a n oth r eon in e, 7). Alcohol 1 (475
mg, 1.5 mmol) was hydrolyzed in the same procedure used for
methanoornithine 5, providing 190 mg (87%) of the free amino
acid 7: TLC system C (cellulose), Rf 0.36; system E, Rf 0.45;
mp 250 °C dec (lit. mp 255 °C dec) with similar 1H NMR
spectra to that of an authentic sample;23 NMR (400 MHz, D2O)
δ 2.35-2.41 and 2.45-2.50 (two symmetrical m, 2 H each),
2.74-2.80 (m, 1 H), 3.73 (d, J ) 7.1, 2 H, CH2OH); mass
spectrum (FAB, positive mode) m/ z 146 (8.6, MH+); amino acid
analysis, tR, 17.28 min.
impurities δ 1.53 (br, 6 H, piperidine protons), 1.66 (dt, J 1
)
∼6.3, J 2 ) ∼10.2, 2 H, CH2CH2OH), 2.06-2.28 and 2.99-3.06
(two m, 3 H, CB cis protons), and 2.42-2.55 (m, 2 H, CB trans
protons), 3.48 (br, 4 H, piperidine protons), 3.54 (t, J ) 6.3, 2
H, CH2CH2OH), 7.39-7.83 (m, 5 H, aromatic protons). The
product was not characterized further and was used as such
for the next step.

4840 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24
Gershonov et al.
1-(Ben zoyla m in o)-3-[2-[(m eth ylsu lfon yl)oxy]eth yl]cy-
clobu ta n e-N,N-p en ta m eth ylen eca r boxa m id e (13). Me-
sylation of 12 (1.53 g, 4.6 mmol) was carried out as described
before for 2, but since the reaction was not homogeneous, the
mixture was stirred for 20 h. The crude product was triturated
with ether, providing 1.56 g (83%) of a solid, further purified
by recrystallization from DCM-hexane to give pure 13: TLC
system B, Rf 0.69; system F, Rf 0.44; mp 138-140 °C; NMR
(400 MHz, CDCl3) δ 1.51-1.59 (3 br, 6 H, piperidine protons),
1.90 and 1.98 (2q, J ) 6.2 and 6.4, respectively, 2 H, CH2CH2-
OMs of cis and trans isomers), 2.23-2.36 (m, 2 H, CB trans
protons) and 2.48-2.67 and 3.05-3.10 (2m, 3 H, CB cis
protons), 3.00 (s, 3 H, MsCH3), 3.60 (br, 4 H, piperidine
protons), 4.20 and 4.21 (m, superimposed of 2t, J ) 6.1 and
6.3 respectively, 2 H, CH2CH2OMs of cis and trans isomers),
7.38-7.81 (m, aromatic protons); mass spectrum (CI, positive
mode) m/ z 409 (28.3, MH+), 324 (100, M - piperidine+), 319
(39.1, M - OMs+).
1-(Ben zoyla m in o)-3-(2-a zid oet h yl)cyclob u t a n e-N,N-
p en ta m eth ylen eca r boxa m id e (14) was prepared from 13
(1.54 g, 3.8 mmol) as described for 3. The crude product (800
mg) was chromatographed on silica gel (20 g, elution; DCM-
methanol, 19:1), providing 450 mg (34%) of azide 14: TLC
system B, Rf 0.73; system F, Rf 0.51; mp 165-167 °C dec; NMR
(400 MHz, CDCl3) δ 1.47 and 1.56 (2 br, 6 H, piperidine
protons), 1.69 and 1.76 (2q, J ) 6.7 and 6.8, respectively, 2 H,
CH2CH2N3 of cis and trans isomers), 2.17-2.24 (m, 2 H, CB
trans protons) and 2.45-2.65 and 3.03-3.07 (2m, 3 H, CB cis
protons), 3.19 and 3.20 (m, superimposed of 2t, J ) 6.9 and
6.7 respectively, 2 H, CH2CH2N3 of cis and trans isomers), 3.48
(br, 4 H, piperidine protons), 7.35-7.80 (m, aromatic protons);
mass spectrum (CI, positive mode) m/ z 356 (100, MH+), 328
(14.7, MH+ - N2), 271 (73, M - piperidine+), 105 (43.5,
COPh+).
1-(Ben zoyla m in o)-3-(2-a m in oet h yl)cyclob u t a n e-N,N-
p en ta m eth ylen eca r boxa m id e (15) was prepared from 14
(420 mg, 1.2 mmol) as described for 4. The crude product was
recrystallized from DCM-hexane to give 370 mg (94%) of the
amine 15 and was not further purified: TLC system D, Rf 0.66;
system E, Rf 0.68; mp 170-175 °C (not sharp); NMR (400 MHz,
CD3OD) δ 1.42 and 1.51 and 1.60 (3 br, 6 H, piperidine
protons), 1.76-1.86 (m, 2 H, CH2CH2NH2 of cis and trans
isomers), 2.05-2.22 and 3.02-3.07 (2m, 3 H, CB cis protons)
and 2.48-2.60 (m, 2 H, CB trans protons), 2.84-2.90 (m, 2 H,
CH2CH2NH2 of cis and trans isomers), 3.44 and 3.54 (2br, 4
H, piperidine protons), 7.45-7.87 (m, aromatic protons); mass
spectrum (CI, positive mode) m/ z 330 (100, MH+), 245 (43.9,
M - piperidine+).
1-Am in o-3-(2-am in oeth yl)cyclobu tan e-1-car boxylic acid
(m et h a n olysin e, 16) was prepared from 15 (320 mg, 0.97
mmol) as described for 5. The product did not crystallize when
treated with propylene oxide. After evaporation of the solvent,
the residue was triturated with ether, collected, dissolved in
water, and freeze-dried to give 142 mg (90%) of the free amino
acid 16: TLC system C (cellulose), Rf 0.12; system E, Rf 0.26;
mp 210-213 °C dec; NMR (400 MHz, D2O) δ 1.84 and 1.92
(2q, J ) 7.6 and 7.8, respectively, 2 H, CH2CH2NH2 of cis and
trans isomers), 2.02-2.08 and 2.52-2.68 (2m, 3 H, CB cis
protons) and 2.36-2.42 (m, 2 H, CB trans protons), 2.89-2.95
(q, J ) ∼7.8, 2H, ABX spectrum of CH2CHAHBNH2); mass
spectrum (CI, positive mode) m/ z 159 (100, MH+), 142 (21.3,
M - NH2+), 141 (12, MH+ - H2O); mass spectrum (EI) showed
typical fragmentation, 159 (11.3, MH+), 87 (92.1, CH2dC-
(COOH)NH2+), 71 (61.3, CH2dCHCH2CH2NH2+), 72 (100,
CH2dCHCH2CH2NH3+); amino acid analysis, one peak with
retention time of 38.95 min.
5 H, aromatic protons); mass spectrum (FAB, positive mode)
m/ z 293 (100, MH+), 585 (44.6, [2M + H]+).
Nr-Boc-NE-ca r boben zoxym eth a n olysin e (18). Nꢀ-CBZ-
methanolysine (17; 250 mg, 0.86 mmol), sodium hydroxide (420
mg, 10.5 mmol), and di-tert-butyl dicarbonate (2.81 g, 12.9
mmol) were dissolved in tert-butyl alcohol and water (30 mL
each) and stirred at room temperature for 24 h. The tert-butyl
alcohol was removed in vacuo, and the aqueous solution was
extracted with ether. The aqueous layer was then cooled and
acidified to pH ∼2 and repeatedly extracted with ethyl acetate.
The combined ethyl acetate extracts were evaporated to give
a mixture of the starting material 17 and the Boc derivative
18 (TLC monitoring, system E). The mixture was, therefore,
reacted again with di-tert-butyl dicarbonate as described below
for 19, using only DMF as the solvent and stirring only for 2
h. The resulting crude was triturated with petroleum ether
to give 135 mg (40% yield from 16) of a sticky product: TLC
system E, Rf 0.92; system B, Rf 0.94; NMR (400 MHz, CDCl3)
δ 1.42 (s, 9 H, t-BOC), 1.62-1.69 (m, 2 H, CH2CH2NH), 2.00-
2.88 (3m, 5 H, CB protons of both isomers), 3.10-3.15 (m, 2
H, CH2CH2NH), 5.08 (s, 2 H, CH2OCO), 7.34 (br, 5 H, aromatic
protons); NMR also indicated the presence of some impurities;
mass spectrum (FAB, positive mode) m/ z 392 (41.5, M+), 293
(100, MH+ - t-BOC); mass spectrum (FAB, negative mode)
m/ z 391 (86.1, [M - H]-), 317 (100, [M - H - CO(CH3)3]-).
tr a n s-NR-Boc-m eth a n oth r eon in e (19). To an ice-cooled,
stirred solution of methanothreonine 7 (180 mg, 1.24 mmol)
and triethylamine (250 mg, 2.48 mmol) in DMF (10 mL) and
H2O (10 mL) was added di-tert-butyl dicarbonate (810 mg, 3.71
mmol). The reaction was allowed to warm to room tempera-
ture and stirred for 24-30 h, until no starting material was
observed by TLC. The DMF was removed in vacuo, and the
aqueous solution was extracted with ether. The aqueous layer
was then cooled to 0 °C, acidified to pH ∼2, and repeatedly
extracted with ethyl acetate. The combined ethyl acetate
extracts were dried (MgSO4) and concentrated, and the product
was crystallized with petroleum ether to give 240 mg (79%) of
solid 19: TLC system B, Rf 0.20; system H, Rf 0.35; NMR (400
MHz, D2O) δ 1.40 (s, 9 H, t-Boc), 2.17-2.23 and 2.34-2.39 (two
symmetrical m, 2 H each, CB protons), 2.60-2.68 (m, 1 H, CB
proton), 3.60 (d, J ) 6.8, 2 H, CH2OH); mass spectrum (FAB,
positive mode) m/ z 246 (11.5, MH+); mass spectrum (FAB,
negative mode) m/ z 244, (100, [M - H]-).
tr a n s-Nδ-Ca r boben zoxym eth a n oor n ith in e (20). Excess
basic CuCO3 (∼5 equiv) was added gradually to a boiling
solution of methanoornithine 5 (210 mg, 1.46 mmol) in water
(20 mL). After being cooled to room temperature, the mixture
was filtered. The resulting deep-blue solution was stirred at
0 °C and treated portionwise during 0.5 h with benzyl
chloroformate (683 mg, 4 mmol) in the presence of magnesium
oxide (300 mg, 7.5 mmol). Stirring was continued for 2 h at
room temperature, and the precipitated copper complex was
collected and washed on the filter, first with water and then
with ethanol. The air-dried precipitate was suspended in 0.9
N HCl and decomposed with excess sodium sulfide. The
mixture was then boiled for a few minutes and rapidly filtered
from the CuS which was further washed with boiling water.
On cooling, the combined filtrate and washings were brought
to pH 4-5 with 3 N HCl, and the product (152 mg, 37.5%)
crystallized out and was collected. The mother liquors yielded
on concentration in vacuum a mixture of 20 with salts (∼1 g):
TLC system D, Rf 0.66; system E, Rf 0.70; mp 232-235 °C dec;
NMR (400 MHz, CDCl3) δ 2.52-2.63 (m, 4 H, CB protons)
2.84-2.89 (m, 1 H, CB proton) 3.39 (d, J ) 6.6, 2 H, CH2NH)
5.10 (br, 2 H, CH2OCO) 7.33 (br, 5 H, aromatic protons); mass
spectrum (CI, positive mode) m/ z 279 (100, MH+), 171 (4.5,
M - PhCH2O+); mass spectrum (CI, negative mode) m/ z 277
(100, [M - H]-).
NE-Ca r boben zoxym eth a n olysin e (17) was prepared from
16 (137 mg, 0.87 mmol) as described below for 20, but the
product did not crystallize from the final water solution and
was isolated by freeze-drying. The crude product was dis-
solved in absolute ethanol and filtered to remove most of the
salts, providing 250 mg of 17, which was utilized without
further purification: TLC system E, Rf 0.80; NMR (400 MHz,
CD3OD) δ 1.73-1.78 (m, 2 H, CH2CH2NH), 2.12 and 2.69 (2m,
3 H, CB cis protons) and 2.44 (m, 2 H, CB trans protons), 3.09
(br, 2 H, CH2CH2NH), 5.14 (br, 2 H, CH2OCO), 7.31-7.35 (m,
tr a n s-Nr-Boc-Nδ-ca r boben zoxym eth a n oor n ith in e (21)
was prepared from 20 as described for 19, except for using
only DMF as the solvent: yield 87%; TLC system B, Rf 0.55;
system E, Rf 0.94; mass spectrum (CI, positive mode) m/ z 379
(8.5, MH+), 323 (100, MH+ - C(CH3)3); mass spectrum (CI,
negative mode) m/ z 377 (100, [M - H]-), 243 (13.1, M -
CBZ+). The second batch of 20, which contained salts (see
above), was dissolved in absolute ethanol and filtered to

Novel 2,4-Methano Amino Acids
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24 4841
remove most of the salts. It was then treated as described for
19, adding (dimethylamino)pyridine (0.3 equiv) for the comple-
tion of the reaction. The product (yellow oil) contained
impurities and did not crystallize. It was used later in tuftsin
analog synthesis only in the second coupling of methanoorni-
thine.
r a c-tr a n s-1-(Nr-Boc-a m in o)-2-m et h ylcyclob u t a n e-1-
ca r boxylic a cid (23) was prepared from 2229 (340 mg, 2.63
mmol) as described for 19, but using only DMF as the solvent,
to give 400 mg (66%). Product 23: TLC system E, Rf 0.92;
system H, Rf 0.83; mp 157-160 °C; NMR (400 MHz, CD3OD)
δ 1.01 (d, J ) 7.00, 3 H, Me), 1.41 (s, 9 H, t-Boc) 1.63-1.68
and 2.59-2.62 (2m, 1 H each, ring-CH2CH2CHMe), 1.90-2.04
(m, 2 H, ring-CH2CH2CHMe), 2.69-2.75 (m, 1 H, ring-
CH2CHMe); mass spectrum (FAB, positive mode) m/ z 230
(100, MH+), 459 (63.8, [2M + H]+; mass spectrum (FAB,
negative mode) m/ z 228 (90, [M - H]-).
ature a solution of MArg (1 equiv) and KHCO3 (2 equiv) in
H2O (1 mL). The reaction mixture was stirred until all
starting material was consumed (∼3-4 h, TLC monitoring in
system E). Water (15 mL) was then added, and the solution
was acidified (pH ∼2) with 0.5 N H2SO4 at 0 °C before
extracting three times with ethyl acetate. The aqueous layer
was then cooled, brought to pH ∼5 with 0.5 N NaOH, and
evaporated under reduced pressure. Water was removed by
dissolving the residue in absolute ethanol and evaporation,
following four similar repetitions. The residue was then
dissolved in TFA, for deprotection, and allowed to stand at
room temperature for 15 min. After TFA was evaporated, the
peptide was dissolved in water, lyophilized, and purified by
HPLC: amino acid analysis, Thr (1.00), Lys (0.96), Pro (0.94),
MArg (1.04); mass spectrum (FAB, positive mode) m/ z 513
(100, MH+), 339 (23, MH+ - Thr - (Guanidine-CH2)); mass
spectrum (FAB, negative mode) m/ z 511 (73, [M - H]-), 409
(100, [M - H - Guanidine - CH2CH2NH2]-); TLC system C,
Rf 0.17; system E, Rf 0.15; HPLC (isocratic, 1% solution B, RP-
Syn th esis of Meth a n otu ftsin An a logs. All protected
amino acids derivatives were obtained from Bachem, CA. The
R-amino functional groups were protected by tert-butyloxy-
carbonyl (t-Boc). Side-chain protecting groups were as fol-
lows: Lys, Nꢀ-benzyloxycarbonyl; Arg, Nγ-tosyl; Thr, O-benzyl.
All protected methano amino acids were synthesized as
described previously. Chloromethylated polystyrene, Merri-
field resin, with 0.4 mequiv of active chlorine per 1 g was
purchased from Nova, Switzerland. Tuftsin was synthesized
in our laboratory using conventional methods.17 The metha-
noarginine (MArg) analog of tuftsin was synthesized by liquid
phase coupling, while all other tuftsin analogs were synthe-
sized by a solid phase method using a manual procedure.30
Coupling of the first amino acid Arg to the resin was achieved
through its cesium salt. All coupling stages were performed
in NMP with a 2-fold excess of protected amino acids deriva-
tives using N,N′-dicyclohexylcarbodimide (DCC) as a coupling
agent in the presence of 1-hydroxybenzotriazole (HOBT).
Usually the coupling stage was repeated twice. Coupling
stages employing methano amino acids were performed in the
same manner. After the first coupling stage, the protected
methano amino acids were recovered from the reaction solution
and used for the repeated coupling stage. All stages of
coupling and of R-amino deprotection were monitored with
ninhydrin and/or isatin for the presence or absence of free
amino groups. After synthesis, the peptides were cleaved from
the resin and deprotected simultaneously by treatment with
anhydrous HF containing 10% anisole (used as scavenger) for
1 h at 0 °C. Evaporation of HF followed by addition of dried
ether yielded solid residue. The crude peptide was filtered,
washed with dried ether, and extracted with 50% acetic acid
solution. The solution was concentrated, diluted with water,
and lyophilized to give the crude peptides. The peptides were
then purified by preparative HPLC. Reversed phase HPLC
was performed on a Spectra-Physics SP8800 liquid chroma-
tography system equipped with an Applied Biosystems 757
variable-wavelength absorbance detector. The column ef-
fluents were monitored by UV absorbance at 220 nm, and
chromatograms were recorded on a Chrom-J et integrator.
HPLC prepacked columns used are (1) Merck, LiChroCART
250-10 mm containing LiChrosorb RP-8 (7 µm); (2) Merck,
LiChrospher 100 RP-8 (5 µm), 250 × 4 mm (3); Merck,
LiChrospher 100 RP-18 (5 µm), 250 × 4 mm. HPLC purifica-
tion was achieved using a gradient formed from 0.1% tri-
flouroacetic acid (TFA) in H2O (solution A) and 0.1% TFA in
acetonitrile-H2O, 75:25 (solution B), run at the following
times: tmin ) 0, A ) 100%, B ) 0%; tmin ) 5, A ) 100%, B )
0%; tmin ) 30, A ) 75%, B ) 25%. The desired fraction was
then concentrated and lyophilized to give pure peptide. For
amino acid composition analysis, peptides were hydrolyzed in
6 N HCl at 110 °C for 24 h under vacuum and analyzed with
a Dionex automatic amino acid analyzer. Methano amino
acids were evaluated separately. Optical rotations were
measured with a Perkin-Elmer model 141 polarimeter and
were taken in 5% acetic acid.
8) tR 18.2 min; [R]23 -34 (c 0.1).
D
The following tuftsin analogs were prepared by solid phase
peptide synthesis following the general procedure described
previously.
[MTh r 1]Lys-P r o-Ar g (24): amino acid analysis, MThr
(1.26), Lys (1.37), Pro (1.17), Arg (1.00); mass spectrum (FAB,
positive mode) m/ z 527 (15, MH+), 491 (18, M+ - H2O - NH3),
481 (21, M+ - COOH); TLC system C, Rf 0.16; system E, Rf
0.22; HPLC (isocratic, 1% solution B, RP-8) tR 17.8 min; [R]23
-5.5 (c 0.2).
D
Th r -[MOr n 2]P r o-Ar g (25): amino acid analysis, Thr (1.04),
MOrn (0.99), Pro (0.89), Arg (1.00); mass spectrum (FAB,
positive mode) m/ z 499 (55, MH+), 309 (100, M+ - NH2 - Arg);
TLC system C, Rf 0.14; system E, Rf 0.10; HPLC (isocratic,
1% solution B, RP-18) tR 14.0 min; [R]23 -57 (c 0.1).
D
Th r -[MLys2]P r o-Ar g (26). Isomers 26a and 26b were
obtained in a 10:7 ratio from 26 by HPLC separation, and their
configurations at C3 of MLys have not been established yet.
26a : amino acid analysis, Thr (1.03), MLys (0.99), Pro (1.14),
Arg (1.00); mass spectrum (FAB, positive mode) m/ z 513 (26,
MH+), 481 (50, MH+ - 2NH2), 373 (100, M+ - H2O - NH2
-
OH - C(NH)NH2 - Thr side chain), 395 (36, M+ - Thr - NH);
TLC system C, Rf 0.25; system E, Rf 0.20; HPLC (isocratic,
4% solution B, RP-18) tR 9.3 min; [R]23 -62 (c 0.1). 26b:
D
amino acid analysis, Thr (1.32), MLys (0.94), Pro (0.96), Arg
(1.00); mass spectrum (FAB, positive mode) m/ z 513 (42,
MH+), 481 (50, MH+ - 2NH2), 427 (45, MH+ - Arg side chain),
373 (63, M+ - H2O - NH2 - OH - C(NH)NH2 - Thr side
chain), 339 (73, M - Arg+); TLC system C, Rf 0.31; system E,
Rf 0.33; HPLC (isocratic, 4% solution B, RP-18) tR 18.5 min;
[R]23 -62 (c 0.1).
D
Th r -Lys-[MVa l3]Ar g (27). Diastereomers 27a and 27b
were obtained in 3:2 ratio from 27 by HPLC separation, using
analytical column RP-8. Their configurations at C1 of MVal
have not been established as yet. 27a : amino acid analysis,
Thr (0.96), Lys (1.05), MVal (0.98), Arg (1.00); mass spectrum
(FAB, positive mode) m/ z 515 (100, MH+), 481 (30, MH+
-
NH2 - H2O), 453 (47, M+ - NH2 - Thr side chain), 411 (75,
M+ - Thr side chain - guanidine), 369 (87, M+ - Thr and
Arg side chains); mass spectrum (FAB, negative mode) m/ z
513 (45, [M - H]-), 419 (100, [M - H - H2O - NH2 - OH -
C(NH)NH2]-); TLC system C, Rf 0.21; system E, Rf 0.18; HPLC
(isocratic, 8% solution B, RP-18) tR 13.2 min; [R]23D -39 (c 0.1).
27b: amino acid analysis, Thr (0.94), Lys (1.01), MVal (0.96),
Arg (1.00); mass spectrum (FAB, positive mode) m/ z 515 (100,
MH+), 469 (11, M+ - Thr side chain); TLC system C, Rf 0.23;
system E, Rf 0.20; HPLC (isocratic, 8% solution B, RP-18) tR
16.4 min; [R]23 -110 (c 0.05).
D
Cir cu la r Dich r oism (CD). CD were recorded on a J asco
J -500C spectropolarimeter equipped with a J asco DP-500N
data processor. A path length of 0.1 mm was used. Each
spectrum represents the average of four scans. The dried,
weighed peptides were dissolved in double-distilled water or
in 98% 2,2,2-trifluoroethanol (TFE), in concentration of 1 mg/
mL. The pH of the solutions was ∼5.0. The data are
presented as molar ellipticity, [θ].
Syn th esis of Th r -Lys-P r o-[MAr g4] (28). Boc-Thr-Lys-
(Boc)-Pro-OSu was synthesized in our laboratory via a solution
procedure.17 To a stirred solution of Boc-Thr-Lys(Boc)-Pro-
OSu (3 equiv) in dioxane (3 mL) was added at room temper-

4842 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24
Gershonov et al.
Biologica l a ssa ys. Sta bility in Ser u m . Blood from
human donors was incubated at room temperature. The
serum was separated by centrifugation at 2500 rpm for 10 min.
Tuftsin (0.5 mg) and 0.5 mg of each of the tested tuftsin
analogs were added to 0.5 mL of serum, and the samples were
then incubated at 37 °C. Aliquots of 50 µL were taken at the
following times: 0, 5, 15, 30, 60, 90, 120, 180, and 240 min.
The samples were denatured, in tightly closed microtubes, by
boiling at 90-95 °C for 5 min. H2O (950 µL) was then added,
and the mixture was thoroughly stirred and then centrifuged
twice. Recovery of tuftsin, following these manipulations, is
rather quantitative.17 Supernatants were analyzed by HPLC.
Each sample (200 µL, containing approximately 10 µg of
peptide at time 0) was eluted at isocratic conditions at flow
rate of 1 mL/min. The area corresponding to the peptide peak
(in percent, calculated by the integrator) was always referred
to an internal peak of the serum (eluted after about 5-7 min,
depending on the above conditions). The peptide’s peak area
at time 0 was always assigned as 100%.
IL-6 P r od u ction Assa y. The methods were described in
detail elsewhere.21 In brief, (BALB/c x C3H.eb)F1 mice were
bred in our rodent facilities and used at the age of 6-8 weeks.
The parents, BALB/c and C3H.eb, were purchased from
J ackson Laboratories (Bar Harbor, ME). Mice were injected
intraperitoneally (3 mL/mouse) with thioglycollate broth (Difco
Laboratories, Detroit, MI). Four days later the macrophages
were aspirated in phosphate buffer saline (PBS, pH 7.4, Gibco,
Grand Island, NY) supplemented with 5 IU/mL heparin,
centrifuged and resuspended in Dulbecco’s modified Eagle
medium (DMEM, Gibco, Grand Island, NY) containing 40 µg/
mL gentamycin. Cell preparation consistently contained
>90% macrophages, of which more than 90% were viable cells.
Macrophages (106/mL) were cultured in 1 mL of DMEM
(Nunclon, 24-well plates) at 37 °C in a humidifier atmosphere
of 5% CO2 in air for 48 h prior to the assay. After 24 and 48
h the cells were washed twice with warm medium. The
cultures were then incubated in triplicate in the presence of
KLH (Calbiochem, CA) alone, KLH + tuftsin or tuftsin analogs
at concentrations range of 2 × 10-7 to 2 × 10-9 M, and tuftsin
or tuftsin analogs alone for 20 h. The supernatants were then
collected, and triplicates were combined and centrifuged at
900g for 10 min. Supernatants were then assayed for IL-6.
The IL-6 dependent murine hybridoma cell line, B9, was used
to determine IL-6 levels.51 B9 cells were cultured in RPMI
containing 5% fetal calf serum (FCS), 5 × 10-5 M 2-mercap-
toethanol, 40 µg/mL gentamycin, and 10 IU/mL human rIL-6
(Genzyme, Boston, MA). The B9 cells were starved in IL-6-
free medium 24 h prior to the assay. B9 cells (5000/200 µL)
were cultured in 96-well plates (flat-bottom, Falcon 3072, NJ )
in triplicate wells in the presence of samples to be tested in
eight sequential 2-fold dilutions. Proliferation was measured
by a [3H]thymidine pulse (1 µCi/well) from 48 h for 16 h. All
results are expressed as arbitrary units. Supernatants ob-
tained from macrophages cultured in presence of KLH alone
were assigned to 1000 units. All samples were calculated
based on corresponding cpm at different dilutions and the slope
of the obtained curve.
thiazoline-6-sulfonic acid)), Sigma, USA) solution (dissolved
in 0.1 M citric acid, 28 mL; 0.2 M Na2HPO4, 22 mL; H2O, 50
mL; H2O2, 6 µL) was added, and the samples were then
incubated at room temperature. After 15 min the reaction was
stopped with 100 µL of 0.2 M citric acid. Optical density (OD)
was recorded at 630 nm using Microplate autoreader instru-
ment (Bio-tek, Vermont).
Refer en ces
(1) Veber, D. F.; Freidinger, R. M. The design of metabolically-stable
peptide analogs. Trends Neurosci. 1985, 8, 392-396.
(2) Mezo, G.; Szekerke, M.; Sarmay, G.; Gergely, J . Synthesis and
functional studies of tuftsin analogs containing isopeptide bond.
Peptides 1990, 11, 405-415.
(3) Hruby, V. J . Conformational restrictions of biologically active
peptides via amino acid side chain groups. Life Sci. 1982, 31,
189-199.
(4) Spatola, A. F. In Chemistry and Biochemistry of Amino Acids,
Peptides and Proteins; Weinstein, B., Ed.; Marcel Dekker: New
York, 1983; pp 267-357.
(5) Al-Obeidi, F.; Castrucci, A. M.; Hadley, M. E.; Hruby, V. J . Potent
and prolonged acting cyclic lactam analogues of alpha-
melanotropin: design based on molecular dynamics. J . Med.
Chem. 1989, 32, 2555-2561.
(6) Gilon, C.; Halle, D.; Chorev, M.; Selinger, Z.; Byk, G. Backbone
cyclization: A new method for conferring conformational con-
straint on peptides. Biopolymers 1991, 31, 745-750.
(7) Hruby, V. J .; Al-Obeidi, F.; Kazmierski, W. Emerging approaches
in the molecular design of receptor selective peptide ligands:
Conformational, topographical and dynamic considerations.
Biochem. J . 1990, 268, 249-262.
(8) Veber, D. F.; Freidinger, R. M.; Perlow, D. S.; Palevda, W. J .;
Holly, F. W.; Strachan, R. G.; Nutt, R. F.; Arison, B. H.; Homnick,
C.; Randall, W. C.; Glitzer, M. S.; Saperstein, R.; Hirschmann,
R. A potent cyclic hexapeptide analogue of somatostatin. Nature
1981, 292, 55-58.
(9) Rizo, J .; Gierasch, L. M. Constrained Peptides: Models of
bioactive peptides and protein substructures. Annu. Rev. Bio-
chem. 1992, 61, 387-418.
(10) Kraus, B. L.; Ferry, G.; Combe, P. V.; Visalli, M.; Remond, G.;
Vincent, M.; Boutin, J . A. Approaches to some biochemical
mechanisms of action of tuftsin and analogues. Biochem. Phar-
macol. 1991, 41, 1411-1418.
(11) Benedetti, E. X-ray crystallography of peptides: the contribu-
tions of the Italian laboratories. Biopolymers 1996, 40, 3-44.
(12) Gaoni, Y.; Chapman, A. G.; Parvez, N.; Pook, P. C.; J ane, D. E.;
Watkins, J . C. Synthesis, NMDA receptor antagonist activity,
and anticonvulsant action of 1-aminocyclobutanecarboxylic acid
derivatives. J . Med. Chem. 1994, 37, 4288-4296.
(13) Lanthorn, T. H.; Hood, W. F.; Watson, G. B.; Compton, R. P.;
Rader, R. K.; Gaoni, Y.; Monahan, J . B. Cis-2,4-methano-
glutamate is a potent and selective N-methyl-D-aspartate recep-
tor agonist. Eur. J . Pharmacol. 1990, 182, 397-404.
(14) Allan, R. D.; Hanrahan, J . R.; Hambley, T. W.; J ohnston, G. A.;
Mewett, K. N.; Mitrovic, A. D. Synthesis and activity of a potent
N-methyl-D-aspartic acid agonist, trans-1-aminocyclobutane-1,3-
dicarboxylic acid, and related phosphonic and carboxylic acids.
J . Med. Chem. 1990, 33, 2905-2915.
(15) Tsang, J . W.; Schmied, B.; Nyfeler, R.; Goodman, M. Peptide
sweeteners. 6. Structural studies on the C-terminal amino acid
of L-aspartyl dipeptide sweeteners. J . Med. Chem. 1984, 27,
1663-1668.
(16) Talluri, S.; Montelione, G. T.; van Duyne, G.; Piela, L.; Clardy,
J .; Scheraga, H. A. Conformational properties of 2,4-methano-
proline (2-carboxy-2,4-methanopyrrolidone) in peptides: Evi-
dence for 2,4-methanopyrrolidine asymmetry based on solid-
state X-ray crystallography, 1H NMR in aqueous solution, and
CNDO/2 conformational energy calculations. J . Am. Chem. Soc.
1987, 109, 4473-4477.
(17) Fridkin, M.; Najjar, V. A. Tuftsin: its chemistry, biology, and
clinical potential. Crit. Rev. Biochem. Mol. Biol. 1989, 24, 1-40.
(18) Bump, N. J .; Lee, J .; Wleklik, M.; Reichler, J .; Najjar, V. A.
Isolation and subunit composition of tuftsin receptor [published
erratum appears in Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 963].
Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 7187-7191.
(19) Bar-Shavit, Z.; Stabinsky, Y.; Fridkin, M.; Goldman, R. Tuftsin-
macrophage interaction: specific binding and augmentation of
phagocytosis. J . Cell. Physiol. 1979, 100, 55-62.
En zym e-Lin ked Im m u n osor ben t Assay (ELISA). ELISA
was performed using the conventional procedure.52 Briefly,
96 wells of microtiter plates (Nunc, Roskilde, Denmark) were
coated with 100 µL of ovalbumin (Sigma, USA)-tuftsin
conjugate in PBS (0.1 mg/mL) at 4 °C for 20 h and then washed
three times with PBS containing 0.05% Tween 20 (Sigma,
USA). The wells were incubated for an additional 1 h at 37
°C with 200 µL of 1% bovine serum albumin (BSA, Sigma,
USA) in PBS to saturate free sites (in order to prevent non
specific binding). Following three more washes, 100 µL of
preincubated (20 h, 4 °C) tuftsin and tuftsin analogs at
different concentrations with tuftsin antisera (1:1000) in PBS
were applied to duplicate wells and incubated for 2 h at 37
°C. After three washes, 100 µL samples of affinity-purified
enzyme-labeled (Horse-reddish peroxidase) goat anti-rabbit
IgG (H+L, Biomakor, Israel) at a dilution of 1:1000 in PBS
were added, and the samples were incubated for another 2 h
at 37 °C. Finally, the plates were washed three times, 100
µL of substrate (1 mg/mL, ABTS (2,2′-azino-bis(3-ethylbenz-
(20) Spirer, Z.; Zakuth, V.; Tzehoval, E.; Dagan, S.; Fridkin, M.;
Golander, A.; Melamed, I. Tuftsin stimulates IL-1 production
by human mononuclear cells, human spleen cells and mouse
spleen cells in vitro. J . Clin. Lab. Immunol. 1989, 28, 27-31.
(21) Dagan, S.; Tzehoval, E.; Fridkin, M.; Feldman, M. Tuftsin and
tuftsin conjugates potentiate immunogenic processes: effects and
possible mechanisms. J . Biol. Response Mod. 1987, 6, 625-636.
(22) Fridkin, M.; Gottlieb, P. Tuftsin, Thr-Lys-Pro-Arg. Anatomy of
an immunologically active peptide. Mol. Cell. Biochem. 1981,
41, 73-97.

Novel 2,4-Methano Amino Acids
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24 4843
(23) Fleet, G. W. J .; Seijas, J . A.; Vazquez Tato, M. P. Synthesis of
cis- and trans-1-amino-3-hydroxymethyl-cyclobutane-1-carbox-
ylic acids. Tetrahedron 1988, 44, 2077-2080.
(24) Hughes, P.; Clardy, J . Total synthesis of cyclobutane amino acids
from Atelia herbert smithii. J . Org. Chem. 1988, 53, 4793-4796.
(25) Gaoni, Y. Preparation of ring-substituted (arylsulfonyl)cyclo-
propanes and (arylsulfonyl)bicyclobutanes from γ,δ-epoxy sul-
fones. J . Org. Chem. 1982, 47, 2564-2571.
(26) Gaoni, Y. Regiospecific additions of hydrazoic acid and benzy-
lamine to 1-(arylsulfonyl)bicyclo[1.1.0]butanes. Application to the
synthesis of cis and trans 2,4-methanoglutamic acids. Tetrahe-
dron Lett. 1988, 29, 1591-1594.
(27) Gaoni, Y. New bridgehead-substituted 1-(arylsullfonyl)bicyclo-
[1.1.0]butanes and some novel addition reactions of the bicyclic
system. Tetrahedron 1989, 45, 2819-2840.
(28) Habeeb, A. F. Guanidination of proteins. Methods Enzymol.
1972, 25B, 558-566.
(29) Gaoni, Y. Synthesis of aminocyclobutane mono-and dicarboxylic
acids and derivatives thereof from (phenylsulfonyl)bicyclobu-
tanes. Org. Prep. Proced. Int. 1995, 27, 185-212.
(30) Barany, G.; Merrifield, R. B. Solid phase peptide synthesis. In
The peptides, analysis, synthesis, biology; Gross, E., Meienhofer,
J ., Eds.; Academic Press: New York, 1980; pp 1-284.
(31) Konopinska, D.; Nawrocka, E.; Siemion, I. Z.; Szymaniecz, S.;
Slopek, S. Synthetic and conformational studies with tuftsin and
its analogues. Peptides, Proceedings of the 14th European Peptide
Symposium; Universite de Bruxelle: Brussels, 1976; pp 535-
539.
(32) Sucharda, S. A.; Siemion, I. Z.; Konopinska, D. Infrared spec-
troscopic investigations of tuftsin and its analogs. Eur. J .
Biochem. 1979, 96, 131-139.
(33) Siemion, I. Z.; Lisowski, M.; Konopinska, D.; Nawrocka, E. 13C
nuclear magnetic resonance and circular dichroism studies of
the tuftsin conformation in water. Eur. J . Biochem. 1980, 112,
339-343.
(34) Fitzwater, S.; Hodes, Z. I.; Scheraga, H. A. Conformational
energy study of tuftsin. Macromolecules 1978, 11, 805-811.
(35) Nikiforovich, G. V. Calculation of stable conformations of tuftsin.
Bioorg. Khim 1978, 4, 1427-1431.
(36) Vicar, J .; Gut, V.; Fric, I.; Blaha, K. Synthesis and properties of
tuftsin, L-Threonyl-L-lysyl-L-prolyl-L-arginine. Collect. Czech.
Chem. Commun. 1976, 41, 3467-3473.
(37) Blumenstein, M.; Layne, P. P.; Najjar, V. A. Nuclear magnetic
resonance studies on the structure of the tetrapeptide tuftsin,
L-threonyl-L-lysyl-L-prolyl-L-arginine, and its pentapeptide ana-
logue L-threonyl-L-lysyl-L-prolyl-L-prolyl-L-arginine. Biochem-
istry 1979, 18, 5247-5253.
(38) O’Connor, S. D.; Smith, P. E.; Al-Obeidi, F.; Pettitt, B. M.
Quenched molecular dynamics simulations of tuftsin and pro-
posed cyclic analogues. J . Med. Chem. 1992, 35, 2870-2881.
(39) Rae, I. D.; Leach, S. J .; Minasian, E.; Smith, J . A.; Zimmerman,
S. S.; Weigold, J . A.; Hodes, Z. I.; Neˇmethy, G.; Woody, R. W.;
Scheraga, H. A. Conformational characteristics of the N-acetyl-
N′-methylamides of the four (Lys, Tyr) dipeptides. Int. J . Pept.
Protein. Res. 1981, 17, 575-592.
(40) Rose, G. D.; Gierasch, L. M.; Smith, J . A. Turns in peptides and
proteins. Adv. Protein Chem. 1985, 37, 1-109.
(41) Nishioka, K.; Constantopoulos, A.; Satoh, P.; Mitchell, W.; Najjar,
V. A. Characteristics and isolation of the phagocytosis-stimulat-
ing peptide tuftsin. Biochim. Biophys. Acta. 1973, 310, 217-
229.
(42) Nagaoka, I.; Yamashita, T. Inactivation of phagocytosis-
stimulating activity of tuftsin by polymorphonuclear neutrophils.
A possible role of leucine aminopeptidase as an ecto-enzyme.
Biochim. Biophys. Acta 1981, 675, 85-93.
(43) Fridkin, M.; Stabinsky, Y.; Zakuth, V.; Spirer, Z. Tuftsin and
some analogs. Synthesis and interaction with human polymor-
phonuclear leukocytes. Biochim. Biophys. Acta 1977, 496, 203-
211.
(44) Najjar, V. A.; Constantopoulos, A. A new phagocytosis stimulat-
ing tetrapeptide hormone, tuftsin and its role in disease. J .
Retinucleoendothel. Soc. 1972, 12, 197-215.
(45) Stabinsky, Y.; Fridkin, M.; Zakuth, V.; Spirer, Z. Synthesis and
biological activity of tuftsin and of [OdC)Thr1]-tuftsin. A novel
synthetic route to peptides containing N-terminal L-OdC)Thr
and L-OdC)Ser residues. Int. J . Pept. Protein. Res. 1978, 12,
130-138.
(46) Stabinsky, Y.; Gottlieb, P.; Fridkin, M. The phagocytosis stimu-
lating peptide tuftsin: further look into structure- function
relationships. Mol. Cell. Biochem. 1980, 30, 165-170.
(47) Nawrocka, E.; Siemion, I. Z.; Slopek, S.; Szymaniec, S. Tuftsin
analogs with D-amino acid residues. Int. J . Pept. Protein Res.
1980, 16, 200-207.
(48) Konopinska, D.; Luczak, M.; Gomulka, S. Synthesis and biologi-
cal investigations of new tuftsin analogs with elongated peptide
chain. Int. J . Pept. Protein Res. 1983, 22, 223-230.
(49) Dagan, S.; Gottlieb, P.; Tzehoval, E.; Feldman, M.; Fridkin, M.;
Yasumura, K.; Okamoto, K.; Yajima, H. Tuftsin analogues:
synthesis, structure-function relationships, and implications for
specificity of tuftsin’s bioactivity. J . Med. Chem. 1986, 29, 1961-
1968.
(50) Florentin, I.; Chung, V.; Martinez, J .; Maral, J .; Le, G. Y.; Mathe,
G. In vivo immunopharmacological properties of tuftsin (Thr-
Lys-Pro-Arg) and some analogues. Methods Find Exp. Clin.
Pharmacol. 1986, 8, 73-80.
(51) Helle, M.; Boeije, L.; Aarden, L. A. Functional discrimination
between interleukin 6 and interleukin 1. Eur. J . Immunol. 1988,
18, 1535-1540.
(52) Engvall, E. Enzyme immunoassay ELISA and EMIT. Methods
Enzymol. 1980, 70, 419-439.
J M960390T