Synthesis of reagents for the construction of hypusine and deoxyhypusine peptides and their application as peptidic antigens

Article abstract of DOI:10.1021/jm980389p

Two new synthetic methods which allow access to (2S)-deoxyhypusine, natural (2S,9R)-hypusine, (2S,9S)-hypusine, and deoxyhypusine- and hypusine- containing peptides are described. The methods involve both the construction of a deoxyhypusine reagent in which the α-nitrogen protecting group is orthogonal to the N-7 and N-12 protecting groups and an alternate synthesis of our previous hypusine reagent, a synthesis which provides for better stereochemical control at C-9. Synthetic hypusine and deoxyhypusine can be generated from these reagents. The hypusine-containing hexapeptide (Cys-Thr- Gly-Hpu-His-Gly) is conjugated to ovalbumin (OVA), keyhole limpet hemocyanin (KLH), and a bis-maleimide; KLH conjugates are also made with the deoxyhypusine- and lysine-containing hexapeptides. Monoclonal antibodies are generated to the hypusine-containing hexapeptide-OVA conjugate in mice. These are isolated and screened against the hypusine-containing hexapeptide-KLH and hypusine-containing hexapeptide-bis-maleimide conjugates, as well as against the deoxyhypusine-containing and lysine-containing hexapeptide-KLH conjugates. These antibodies may be useful in localizing intracellular hypusine-containing peptides as well as peptides containing hypusine analogues.

Full text of DOI:10.1021/jm980389p

3888
J . Med. Chem. 1998, 41, 3888-3900
Syn th esis of Rea gen ts for th e Con str u ction of Hyp u sin e a n d Deoxyh yp u sin e
P ep tid es a n d Th eir Ap p lica tion a s P ep tid ic An tigen s
Raymond J . Bergeron,* William R. Weimar, Ralf Mu¨ller, Curt O. Zimmerman, Bruce H. McCosar, Hua Yao, and
Richard E. Smith
Department of Medicinal Chemistry, University of Florida, Gainesville, Florida 32610-0485
Received J une 17, 1998
Two new synthetic methods which allow access to (2S)-deoxyhypusine, natural (2S,9R)-
hypusine, (2S,9S)-hypusine, and deoxyhypusine- and hypusine-containing peptides are de-
scribed. The methods involve both the construction of a deoxyhypusine reagent in which the
R-nitrogen protecting group is orthogonal to the N-7 and N-12 protecting groups and an
alternate synthesis of our previous hypusine reagent, a synthesis which provides for better
stereochemical control at C-9. Synthetic hypusine and deoxyhypusine can be generated from
these reagents. The hypusine-containing hexapeptide (Cys-Thr-Gly-Hpu-His-Gly) is conjugated
to ovalbumin (OVA), keyhole limpet hemocyanin (KLH), and a bis-maleimide; KLH conjugates
are also made with the deoxyhypusine- and lysine-containing hexapeptides. Monoclonal
antibodies are generated to the hypusine-containing hexapeptide-OVA conjugate in mice.
These are isolated and screened against the hypusine-containing hexapeptide-KLH and
hypusine-containing hexapeptide-bis-maleimide conjugates, as well as against the deoxyhy-
pusine-containing and lysine-containing hexapeptide-KLH conjugates. These antibodies may
be useful in localizing intracellular hypusine-containing peptides as well as peptides containing
hypusine analogues.
In tr od u ction
eIF-5A was a critical initiation factor in protein syn-
thesis.12 However, workers have since shown (e.g., in
Saccharomyces cerevisiae)13 that when the eIF-5A pro-
tein is rendered unavailable via genetic manipulation,
overall protein synthesis, rather than ceasing com-
pletely, is reduced to about 70% of control. This
suggests that eIF-5A is not responsible for protein
synthesis in a global sense. Instead, eIF-5A may be
associated with a subgroup of mRNAs; the specific class
still remains elusive.13 However, two issues are quite
clear: eIF-5A is critical to mitotic processes,14 and the
hypusine fragment of eIF-5A is required for the protein’s
function.15
The inhibitor of deoxyhypusine synthase, N1-guanyl-
1,7-diaminoheptane (CG7), diminishes the growth of
CHO cells without affecting polyamine metabolism.16
Site-directed mutagenesis experiments15 in which Lys-
50 was replaced with arginine resulted in a nonfunc-
tional protein in yeast cells; the arginine could not be
modified to form hypusine. Furthermore, yeast cells
that had the wild-type copy of the gene replaced with
the mutant copy failed to grow. The precise role of the
hypusine residue in eIF-5A activity remains elusive.
While it is clear that the N-terminal methionine of the
protein is replaced with an acetyl group and that
acetylation occurs at Lys-47, neither event seems critical
to the protein’s function;17,18 modification (e.g., phos-
phorylation) of the C-9(R)-hydroxyl of the hypusine
residue has not yet been demonstrated.
While hypusine [(2S,9R)-2,11-diamino-9-hydroxy-7-
azaundecanoic acid], an unusual amino acid, has been
found in its free form and as R-(â-alanyl) and R-(γ-
aminobutyryl) dipeptides in brain tissue,1,2 it has gained
most of its attention in association with eukaryotic
initiation factor 5A (eIF-5A, formerly eIF-4D). This 17-
kDa protein seems to be very highly conserved among
many eukaryotic species including yeast and higher
mammals, attesting to its importance from an evolu-
tionary perspective.3,4 In particular, the 12-amino acid
region surrounding the hypusine residue, L-Ser-L-Thr-
L-Ser-L-Lys-L-Thr-Gly-H p u -L-His-Gly-L-His-L-Ala-L-
Lys, is extremely well-conserved across species.5 Hy-
pusination of eIF-5A, or “maturation” of this protein,
occurs as a posttranslational event.6 An aminobutyl
group is removed from spermidine; deoxyhypusine syn-
thase catalyzes the attachment of the aminobutyl group
to the side chain of Lys-50 of the human protein.7,8
Next, deoxyhypusine hydroxylase, which appears to
be an iron-dependent enzyme,9 catalyzes the introduc-
tion of the hydroxyl group at C-9 in the (R)-configura-
tion.6
There appears to be some confusion as to the primary
intracellular localization of eIF-5Asthe cytoplasm, the
nucleus, the nuclear membrane, or some combination
of these.10,11 This controversy may derive from differ-
ences in the nature of the antibodies generated by two
different research groups.10,11 Because eIF-5A was
shown to stimulate methionyl-puromycin synthesis, a
model reaction for formation of the first peptide bond
during protein assembly, it was initially thought that
Probably the most intriguing aspect regarding eIF-
5A is its role in the replication of human immunodefi-
ciency virus (HIV); eIF-5A is a transactivating factor
during replication of HIV.10 The eIF-5A molecule binds
to a complex formed between the Rev response element
* To whom correspondence should be addressed.
S0022-2623(98)00389-6 CCC: $15.00 © 1998 American Chemical Society
Published on Web 08/27/1998

Construction of Hypusine and Deoxyhypusine Peptides
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20 3889
Sch em e 1. Synthesis of the Hypusine Reagenta
(RRE) in the stem-loop IIB of the viral mRNA and Rev,
a viral protein that serves as a nuclear export signal.19
Once eIF-5A binds to Rev-RRE, the now active eIF-5A-
Rev-RRE complex is able to be exported from the
nucleus; viral replication ensues. In experiments in
which antisense nucleotides were used to prevent eIF-
5A synthesis, viral replication was inhibited.10 It has
also been demonstrated in gel shift experiments that
the hypusine- or deoxyhypusine-containing fragments
were required for this binding of eIF-5A to Rev-RRE.20
Rev has domains which direct both nuclear import and
nuclear export.20,21 Certain eIF-5A mutants, while
capable of being transported into the nucleus and
binding to Rev-RRE, actually prevent nuclear export
and, thus, viral replication.20,21
The observations that eIF-5A is required for both
mitotic events and HIV viral replication and that
immature eIF-5A must be deoxyhypusinated or hypu-
sinated for activity render inhibition of eIF-5A deoxy-
hypusination or hypusination an interesting target in
therapeutic strategies for anticancer and antiviral drug
development. Another potential antiviral strategy in-
volves identifying the basic platform within eIF-5A
responsible for nuclear import that will permit Rev-RRE
binding but not nuclear export of viral message. The
current study develops a number of tools, including new
synthetic methods for accessing hypusine and deoxy-
hypusine as well as the corresponding peptides and
murine monoclonal antibodies to a hypusine-containing
hexapeptide. These antibodies should be useful in
monitoring potential migration of hypusine-containing
peptides into and out of the nucleus.
Syn th esis
In an earlier paper22 we described the synthesis of a
hypusine reagent (11 in that work; 10a , Scheme 1, in
the present paper) useful for accessing hypusine-
containing peptides. The two key steps in assembling
this reagent in the earlier study involved reaction of
Nꢀ-benzyl-NR-BOC-L-lysine tert-butyl ester with (S)-(+)-
epichlorohydrin to generate (2S,9S)-7-benzyl-2-[(tert-
butoxycarbonyl)amino]-10-chloro-9-hydroxy-7-azadecano-
ic acid tert-butyl ester. This chlorohydrin was next
treated with potassium cyanide in the presence of 18-
crown-6 to produce the corresponding nitrile, (2S,9R)-
7-benzyl-2-[(tert-butoxycarbonyl)amino]-10-cyano-9-hy-
droxy-7-azadecanoic acid tert-butyl ester. A series of
reductions and functional group protections ensued,
culminating in the final reagent 10a . The orthogonally
protected reagent was shown to be of utility in accessing
hypusine-containing peptides by either matrix synthesis
or standard solution methods. However, in attempting
scaleup of this reagent, we observed that the stereo-
chemistry of the cyanation product at C-9 is very
sensitive to reaction conditions, particularly to temper-
ature and the ratio of 18-crown-6 to potassium cyanide.
This problem may revolve around potential azetidinium
salt formation;23 this intermediate, on cyanide opening,
would result in racemization at C-9.24 Thus, the extent
of the racemization depends on the ratio of azetidinium
formation and cyanide opening to direct displacement
of the chloride by cyanide, a relationship affected by the
reaction conditions, which can be difficult to control.
Azetidinium formation likely occurred during cyanation
a
Reagents: (a) (BOC)2O, NaHCO3; (b) H2, Pd-C; (c) (S)-(+)-
or (R)-(-)-epichlorohydrin, respectively; (d) benzyl chloroformate,
triethylamine; (e) KCN, 18-crown-6; (f) H2, Pd-C, PtO2; (g) CBZ-
ONSu, KHCO3; (h) TFA, triethylsilane, CH2Cl2; (i) 3,4-dihydro-
2H-pyran; (j) FMOC-ONSu, Na2CO3; (k) (R)-(-)-Mosher’s acid
chloride, pyridine.
rather than spontaneously from the intermediate chlo-
rohydrin on standing. Upon standing for 1 week at
room temperature, the optical rotation of the chlorohy-
drin did not change.
Assuming that azetidinium formation was the origin
of the problem, we adopted an approach (Scheme 1)
which would prevent the generation of this compound.
The key step in overcoming the internal cyclization was
to vitiate the nucleophilicity of the N-7 electron pair by
construction of the halocarbamate 4a ,b. Utilizing this
new approach, we synthesized both the (2S,9R)-reagent
10a and the corresponding (2S,9S)-compound 10b.
Commercially available Nꢀ-CBZ-L-lysine tert-butyl ester
(Scheme 1) was protected with di-tert-butyl dicarbonate
to yield 1, which was hydrogenated to remove the CBZ
protecting group. The resulting primary amine 2 was
reacted with either (S)-(+)-epichlorohydrin to generate

3890 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20
Bergeron et al.
Sch em e 2. Verification of Stereochemical Integrity of the Hypusine Reagenta
a
Reagents: (a) L-valine tert-butyl ester hydrochloride, BOP, DIEA; (b) piperidine; (c) pTosOH; (d) (BOC)2O, NaHCO3; (e) (R)-(-)-
Mosher’s acid chloride, pyridine; (f) HBr/HOAc in TFA, phenol, pentamethylbenzene, triisopropylsilane; (g) (BOC)2O, NaOH.
the (9S)-chlorohydrin (3a ) or with (R)-(-)-epichlorohy-
drin to produce the (9R)-chlorohydrin (3b).25 Benzyl
chloroformate was condensed with N-7 of the chlorohy-
drins 3a ,b to yield the N7-CBZ compounds 4a ,b. Again,
because of the carbamate linkage at N-7, azetidinium
formation is precluded. Reaction of 4a ,b with KCN in
the presence of 18-crown-626 resulted in the correspond-
ing nitriles 5a ,b. In each instance, the (9R)- and (9S)-
hydroxy nitriles were converted to the Mosher esters
11a ,b, respectively,27 in order to verify chiral integrity
hydroxyl groups were next converted to the THP ethers
9a ,b. Finally, both 9a ,b were transformed to their
FMOC derivatives 10a ,b.29
An additional verification of the stereochemical in-
tegrity of the reagent and its peptide products was
undertaken (Scheme 2). In the course of earlier syn-
thetic studies we demonstrated that the extent of
racemization at the R-methine of the hypusine fragment
may be quantified by preparing the corresponding
L-valine dipeptide.22 Proton NMR spectra clearly dis-
tinguished between the L-lysyl-L-valine and D-lysyl-L-
valine as well as between the related (2S,9R)-hypusinyl-
L-valine and (2R,9R)-hypusinyl-L-valine dipeptide
diastereomers. The protected hypusine reagent 10a
(2S,9R) was condensed with valine tert-butyl ester
resulting in the R-N-FMOC-protected dipeptide 12
(2S,9R). The FMOC protecting group was next removed
by exposure to piperidine, yielding 13, followed by
p-toluenesulfonic acid in acetone-water to remove the
THP ether (dipeptide 14). Protection of the free nitro-
gen with BOC anhydride provided NR-BOC dipeptide
15a (2S,9R). This dipeptide 15a was subjected to two
separate reactions: conversion to Mosher ester 16a
(2S,9R) or deprotection using HBr in acetic acid/TFA/
phenol/pentamethylbenzene/triisopropylsilane to dipep-
tide 17a (2S,9R). To generate the other set of diaster-
1
at C-9. The methoxy resonance in the H NMR spec-
trum of 11a was observed at 3.60 ppm and that of 11b
at 3.46 ppm. Both resonances were easily distinguish-
able in a mixture of the two compounds. The inability
to observe the 3.46 ppm resonance of 11b in the proton
NMR of 11a or the 3.60 ppm resonance of 11a in that
of 11b confirmed that chiral integrity at C-9 had been
maintained to this stage in the synthetic sequence.
Nitriles 5a ,b were next reduced with hydrogen and
a mixed catalyst. The resulting diamine diacetates 6a ,b
were then reacted with N-(benzyloxycarbonyloxy)suc-
cinimide (CBZ-ONSu) to produce the N7,N12-di-CBZ
hydroxy derivatives 7a ,b. Treatment of these systems
with trifluoroacetic acid and triethylsilane28 released
both the BOC and tert-butyl ester protecting groups,
resulting in the di-CBZ amino acids 8a ,b . The C-9

Construction of Hypusine and Deoxyhypusine Peptides
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20 3891
Sch em e 3. Synthesis of the Deoxyhypusine Reagent
Sch em e 4. Polymer-Bound Synthesis of a
and Free Deoxyhypusinea
Deoxyhypusine Hexapeptidea
a
Reagents: (a) HBr/acetic acid in TFA, phenol, pentamethyl-
benzene, triisopropylsilane, 1,2-ethanedithiol.
deoxyhypusine reagent involved a molecular sieve-
promoted condensation of 3-cyanopropanal (19)30 with
the NR-BOC-tert-butyl ester of L-lysine (2) and reduction
31
of the resulting intermediate imine over H2/PtO2 to
the deoxyhypusine framework 20. Further reduction
of the nitrile functionality on 20 was done using H2 over
Pd/C and PtO2 in acetic acid to 21; the N-7 and N-12
positions were reacted with CBZ-ONSu to produce
tetraprotected deoxyhypusine 22. Both tert-butyl pro-
tecting groups were removed with trifluoroacetic acid
and triethylsilane,28 and the amino acid 23 was con-
verted to the NR-FMOC-di-CBZ-protected compound 24.
This reagent was both deprotected to yield free deoxy-
hypusine and utilized to generate a deoxyhypusine-
containing hexapeptide.
a
Reagents: (a) 2, benzene, molecular sieves; (b) H2, PtO2, THF;
For free deoxyhypusine (25), the deoxyhypusine re-
agent 24 was first treated with piperidine to remove the
FMOC protecting group and then deprotected using 30%
HBr in trifluoroacetic acid with a “cocktail” of cation
scavengers (Scheme 3). Once the reaction was complete,
the reactants were dissolved in water, and the nonsalts
were extracted into methyl tert-butyl ether. After
concentration, the product was chromatographed on
silica, eluting with methylene chloride/methanol/am-
monium hydroxide. The free amine was converted to
the dihydrochloride salt 25. Analytical data of the final
compound were in agreement with the literature val-
ues.31 The same procedure can be used to convert
hypusine reagent 10a to hypusine (data not shown).
The deoxyhypusine reagent was utilized to prepare
Cys-Thr-Gly-deoxyhypusine-His-Gly. The synthesis of
the hexapeptide 27 (Scheme 4) was performed on a
2-chlorotrityl resin using SPPS and FMOC chemistry
with HBTU as an activating agent. The cysteine and
histidine residues of the hexapeptide were protected as
4-methoxytrityl (Mmt) and 4-methyltrityl- (Mtt) group
derivatives, respectively, while the threonine was pro-
tected as its tert-butyl ether. Deprotection of 26 was
then achieved with HBr/acetic acid in TFA, 1,2-
ethanedithiol to prevent disulfide bond formation, and
a “cocktail” of carbocation scavengers (phenol, pentam-
ethylbenzene, triisopropylsilane) at room temperature.
(c) H2, Pd/C, PtO2, HOAc; (d) CBZ-ONSu, KHCO3; (e) TFA,
triethylsilane, CH2Cl2; (f) FMOC-ONSu, Na2CO3; (g) piperidine;
(h) HBr/HOAc in TFA, phenol, pentamethylbenzene, triisopropyl-
silane.
eomers (2S,9S), the R-amino group of the di-CBZ
intermediate 8b (2S,9S) was reacted with BOC anhy-
dride; the acid 18 thus generated was condensed with
valine tert-butyl ester. As with 15a , two separate
conversions were initiated: 15b was transformed to the
corresponding Mosher ester 16b (2S,9S) and depro-
tected to yield dipeptide 17b (2S,9S). Thus, three sets
of diastereomers were available for comparison: 15a ,b,
16a ,b, and 17a ,b. Proton NMR spectra of 15a ,b and
17a ,b did not reveal the presence of any racemization
at C-2 of the hypusine moiety as no (2R)-hypusinyl-L-
valine diastereomers were detected. Furthermore, a
comparison of both the 1H and 19F NMR analyses of 16a
(9R) and 16b (9S) clearly supported the optical integrity
at C-9. The Mosher ester methoxy protons and fluorine
signals of 16a (9R) occurred at δ 3.50 and -71.78,
respectively, while the signals for 16b (9S) were at δ
3.44 and -71.40.
In keeping with the ease of peptide synthesis using
hypusine reagent 10a on a Merrifield resin,22 we elected
to synthesize the analogous deoxyhypusine reagent 24
(Scheme 3). The initial step in our synthesis of the

3892 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20
Bergeron et al.
Sch em e 5. Coupling the Hypusine Peptide Hapten to
The final peptide 27 was purified by reverse-phase
HPLC. Both high-field (600-MHz) 1H NMR at two
different temperatures and amino acid analysis revealed
the correct structure for the hexapeptide.
Macromolecular Carriersa
Hyp u sin e Mon oclon a l An tibod y Gen er a tion
To assess whether small hypusine- or deoxyhypusine-
containing peptide fragments are transported into or out
of the nucleus under the conditions of microinjection
experiments, it was clear that antibodies to the peptides
would be valuable as an analytical tool. This would
allow us to confirm, using a different method, the work
of Bevec et al.20 in the determination of nuclear import/
export properties of these peptides. Microinjection of
these peptides into the nucleus or cytoplasm of Xenopus
oocytes, time-dependent fixation and treatment of cells
with the appropriate hypusine/deoxyhypusine peptide-
directed murine IgG, and use of a FITC-labeled anti-
mouse antibody should make it possible to localize the
peptides. In so doing, the identification of the minimal
nuclear import/export structures should be possible.
Recall that the mutant studies20 have demonstrated
that eIF-5A fragments with nuclear import signals, once
transported into the nucleus, bind to the HIV Rev-RRE
complex and prevent nuclear export and thus viral
replication. Given the conflicting views of eIF-5A
localization using polyclonal antisera,10,11 we instead
developed monoclonal antibodies to the hypusine-
containing fragment of eIF-5A.
a
The OVA conjugate 31 was used to immunize mice for
antibody production; the KLH conjugate 32 was used as an antigen
in an ELISA. Reagents: (a) pH 7.4 0.1 M Na phosphate buffer, 3
h; (b) after determination of maleimide content; 90% of the
theoretical amount of 31.
To produce antibodies in animals to haptens such as
synthetic peptides (e.g., a peptide containing hypusine),
conjugation to a larger carrier protein (e.g., ovalbumin
(OVA)) is generally required.32 The hypusine reagent
10a was employed for synthesis of a hypusine-contain-
ing hexapeptide; cysteine replaced the lysine residue on
the amino terminus (L-Cys-L-Thr-Gly-Hpu-L-His-Gly-
OH, 28, Scheme 5).22 The peptide was prepared in this
manner in order to facilitate conjugation to a maleimide-
derivatized OVA.33 The amino terminal end of OVA was
covalently linked to a bifunctional cross-linking agent
via an amide linkage to produce a derivatized OVA
containing maleimide coupling sites. After reaction
with 28, the final product was the 28-OVA conjugate
31.
Sch em e 6. Formation of the Bis-Hypusinyl
Hexapeptide 33 for Use as an ELISA Antigena
a
Reagents: (a) 28, 2 mmol.
The conjugate 31 combined with Ribi adjuvant was
used to immunize four Balb/c BYJ mice subcutaneously.
Sera were obtained after four immunizations and tested
in a series of enzyme-linked immunosorbent assays
(ELISAs). Because ELISA screens of antibodies elicited
by 31 could be directed to either the OVA- or the
hypusine-containing peptide regions of the conjugate
molecule, we needed to be sure that there was a
response against 28. Therefore, suitable ELISA anti-
gens containing the hapten 28 were required. The
hexapeptide 28 itself is too small to reliably remain
attached to the 96-well plate as required by the ELISA.
Thus, 28 was (1) attached to an antigenically neutral
macromolecular carrier, keyhole limpet hemocyanin
(KLH), and (2) cross-linked to itself to form a bis-
hexapeptide, now large enough to remain attached for
ELISA. The 28-KLH conjugate 32 was made utilizing
procedures identical with those for 31 (Scheme 5).
Formation of the bis-maleimide complex 33 employed
a sulfhydryl-specific homobifunctional cross-linking re-
agent (Scheme 6).
The murine sera were assayed by ELISA for reactivity
to the following: unconjugated KLH (neutral carrier
control), 32 (to check for reactivity of the sera to the
peptide coupled to a neutral carrier), and 33. In the
ELISA assay employed, a test serum absorbance value
3 times above that of the normal serum background was
considered a positive response at the dilution of serum
tested. For example, testing of the normal serum
control at a 1:800 dilution yielded an absorbance value
of 0.1 against the bis-hexapeptide conjugate; the serum
from mouse #4 at a 1:800 dilution gave a greater than
10-fold increase. By the use of class-specific secondary
antibodies in the ELISA, it was also possible to deter-
mine whether the class of antibody elicited by im-
munization has switched from the lower-affinity IgM
to the higher-affinity IgG; such a class switch was
detected in these immunized mice.
Once sufficient and specific reactivity to the hypusine
peptide in the sera of the immunized mice was estab-
lished, a fusion could be performed. The fusion was
performed by standard procedures34 followed by a 10-

Construction of Hypusine and Deoxyhypusine Peptides
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20 3893
Ta ble 1. Reactivity of Monoclonal Antibodies with
Hypusine-Containing Peptide Conjugates and Related Antigens
as Measured by ELISA
antigens 32 and 33 with A410 values of greater than 2.0
to 32 and greater than 1.4 to 33. However, clone 2A3
is possibly the most useful; the antibodies did not
appreciably bind to the 27-KLH conjugate, yet the A410
values of 2.6 to 32 and 2.3 to 33 indicate the secretion
of high-affinity antibodies specific to the hypusine-
containing peptide. It is important to point out that
none of the antibodies cross-reacted with the eIF-5A
parent polypeptides as tested by ELISA against a
recombinant human eIF-5A precursor polypeptide in
which Lys-50 had not been posttranslationally modified
and against a protein fraction from CHO cells known
to contain hypusinated eIF-5A. Since the isolated
antibodies do not cross-react with these constitutively
expressed cellular proteins, the antibodies should prove
to be useful in experiments tracking the localization of
microinjected hypusine-containing peptides.
A410 versus antigena
clone
OVA KLH Lys-KLH 27-KLH
32
33
medium
imm mouse 2.529 0.192
serum
0.104 0.109
0.101
2.575
0.100
2.575
0.108 0.104
2.684 2.751
1D9
1F6
0.112 0.116
0.111 0.120
0.108 0.115
0.110 0.129
0.102 0.109
0.116 0.115
0.111 0.207
0.104 0.125
0.102 0.114
0.103 0.113
0.101 0.113
0.111 0.150
0.105 0.112
0.097 0.116
0.099 0.106
0.107 0.116
0.115 0.111
0.117 0.111
0.111 0.120
0.235
0.112
2.108
0.106
0.102
0.107
0.128
0.108
0.109
0.102
0.104
0.242
0.106
0.937
0.098
2.561
0.106
0.114
0.112
0.261
0.114
1.760
0.112
1.522
2.442
0.137
0.267
1.452
0.704
1.580
0.489
1.355
0.928
0.122
2.285
2.474
2.196
1.298
0.244 0.226
0.150 0.128
2.523 2.108
0.130 0.105
2.233 1.902
2.777 2.710
0.150 0.129
2.611 2.265
2.452 1.592
2.383 1.848
2.575 1.600
2.078 1.521
2.411 1.435
0.835 0.737
0.168 0.172
2.670 2.474
2.670 2.612
2.520 2.219
1.530 0.657
4H3
4A6
1F12
5A4
4E3
2A3
5E10
4E12
3C9
5F10
4D4
1B3
4D11
1C3
4E9
1E6
4F4
Discu ssion
The synthetic methods described in this paper allow
for the assembly of both deoxyhypusine- and hypusine-
containing peptides. Although a synthetic approach for
the hypusine reagent was previously described,22 in the
course of scaleup racemization problems were noted at
C-9. The key step in the current approach, N-7-
carbobenzoxylation of the halohydrin intermediate 3a ,b
(Scheme 1), prevented any racemization at C-9. This
is in keeping with the idea that azetidinium ring
formation was the source of C-9 epimerization. Thus,
the stereochemical integrity of both C-2 and C-9 in the
hypusine reagent and the ensuing peptides is now easily
controllable. The deoxyhypusine reagent, lacking the
C-9 problem, also lends itself nicely to matrix method-
ologies. Both reagents were successfully employed in
Merrifield syntheses of hexapeptides (27, Scheme 4, and
2822). Furthermore, the schemes allowed access to the
parent amino acids deoxyhypusine and hypusine.
Finally, the availability of deoxyhypusine- and hypu-
sine-containing peptides with an N-terminal cysteine
residue has made it possible to conjugate such peptide
fragments to OVA for monoclonal antibody production.
The resulting monoclonal antibodies will now make it
possible to detect deoxyhypusine and hypusine and
determine whether the deoxyhypusine- or hypusine-
containing peptide fragments accessible via the methods
described herein contain the necessary structural in-
formation for nuclear import, nuclear export, or both.
a
A410 determined after 1-h incubation with p-nitrophenyl
phosphate substrate; murine immune serum (positive control),
medium alone (negative control), and hybridoma culture super-
natants were undiluted. The antibody-containing hybridoma
culture supernatants were obtained 10 days after fusion of spleen
cells (from a mouse immunized with 31) with the SP2/0 cell line.
day incubation of the cultures. A primary ELISA screen
was then done on the hybridoma culture supernatants
to detect whether cells were secreting antibody to 28,
in the form of either 32 or 33. Those wells that were
positive in this assay were screened again 1 week later
to determine the class of antibody (IgG or IgM). All but
4 of the 19 positive clones secreted only IgG; this
indicates that the antibodies secreted were of high
affinity and should therefore be useful in immunode-
tection systems.
To assess whether the monoclonal antibodies can
differentiate between eIF-5A peptide fragments con-
taining unmodified lysine, eIF-5A peptide fragments
containing deoxyhypusine, and eIF-5A peptide frag-
ments containing hypusine, we have also synthesized
the lysine- and deoxyhypusine-containing hexapeptides
with the same sequence as 28, including the cysteine
residue at the amino terminus. Recall that substitution
of the cysteine residue for serine is to facilitate coupling
by the procedures already described for 28. The panel
of monoclonal antibodies was screened against these
conjugates in the ELISA (Table 1). Most of the clones
tested positive with more than one of the peptide
antigens but not with either of the carriers. Specifically,
those antibodies reactive with the lysine-containing
peptide (Lys-KLH) were also reactive with the deoxy-
hypusine- and hypusine-containing peptides; the clones
that recognized the deoxyhypusine-containing peptide
27-KLH conjugate also recognized 32 and 33. These
antibodies were likely binding to epitopes common to
all three peptide antigens. Of interest were clones 4E12
and 5F10 which, while somewhat reactive with the 27-
KLH conjugate with A410 values of 0.7 and 0.5, respec-
tively (versus a medium control value of 0.1), gave a
strong signal against the hypusine-containing peptide
Exp er im en ta l Section
Chemical reagents were purchased from Aldrich, Fluka, or
Sigma Chemical Co. and used without further purification. Nꢀ-
CBZ-L-lysine tert-butyl ester hydrochloride was obtained from
BACHEM Bioscience Inc. Fisher Optima grade solvents were
routinely used, and organic extracts were dried with anhy-
drous magnesium sulfate. Acetonitrile was distilled from
NaH. THF was distilled from sodium and benzophenone
immediately before use. KCN was finely ground and dried
for 20 h at 145 °C in vacuo. Silica gel 32-63 (40-µm “flash”)
from Selecto, Inc. (Kennesaw, GA) was used for flash column
chromatography, and Supelclean LC-18 gel from Supelco, Inc.
(Bellefonte, PA) was used for reversed-phase column chroma-
tography. 1H NMR spectra were recorded at 300 MHz and
13C NMR spectra at 75 MHz in CD3OD and at 25 °C, unless
otherwise specified. Chemical shifts for proton NMR are given
in parts per million downfield from an internal tetramethyl-
silane or 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid, sodium
salt, standard, unless otherwise specified. 13C NMR chemical

3894 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20
Bergeron et al.
shifts in CD3OD are calibrated on the CD3OD resonance at
49.0 ppm. Coupling constants (J ) are in Hz. Melting points
were determined on a Fisher-J ohns melting point apparatus
were coated with 50 µL/well of the antigens at a concentration
of 10 µg/mL. Pooled sera from strain-, age-, and sex-matched
unimmunized mice were run as a background control when
immune sera were screened; a test serum absorbance value 3
times above that of the normal serum background was
considered a positive response at the dilution of serum tested.
Postimmunization sera were tested at 2-fold dilutions ranging
from 1:200 to 1:1600. When hybridoma culture supernatants
were analyzed, undiluted medium was the background control,
and test hybridoma culture supernatants were also undiluted.
Rabbit anti-mouse IgG (whole molecule), goat anti-mouse IgG
(γ-chain-specific), and goat anti-mouse IgM (µ-chain-specific),
all alkaline phosphatase-conjugated (Sigma), were utilized at
1:1000, 1:8000, and 1:8000 dilutions, respectively. The ab-
sorbance at 410 nm (A410) was read after a 1-h incubation at
room temperature with p-nitrophenyl phosphate (Sigma).
and are uncorrected. FAB mass spectra were run in
a
3-nitrobenzyl alcohol matrix. Elemental analyses were per-
formed by Atlantic Microlabs, Norcross, GA.
Con ju ga tion of Hyp u sin e-Con ta in in g P ep tid e to Ca r -
r ier s. We conjugated the hypusine-containing peptide 28 to
the carrier protein ovalbumin (OVA) via a maleimide proce-
dure as pictured in Scheme 5.33 Reagent 10a was employed
for synthesis of a hypusine-containing hexapeptide with cys-
teine instead of the normal lysine on the amino terminus (28)
in order to facilitate conjugation to OVA. In the first coupling
step, the amino terminal end of OVA was covalently linked to
the cross-linking agent via an amide linkage to produce a
derivatized OVA containing maleimide moieties. The coupling
reaction was run in phosphate buffer (pH 7.4) with OVA at a
concentration of 10 mg/mL. The bifunctional cross-linking
agent 4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid
3-sulfo-N-hydroxysuccinimide ester (sodium salt) was added
at a final concentration of 4 mM (Scheme 5). After a 3-h
reaction the OVA maleimide derivative 29 was immediately
purified by gel filtration chromatography (Sephadex G-25) to
remove starting materials which could interfere with the
sulfhydryl peptide coupling reaction. The maleimide content
of the purified OVA derivative was then quantitated by
Isola tion of Recom bin a n t eIF -5A. A plasmid, containing
the eIF-5A cDNA sequences (pBKS(-)/4D), was a gift from Dr.
J ohn Hershey, University of California at Davis.37 Escherichia
coli HB101 competent cells were from Gibco/BRL (Gaithers-
burg, MD). E. coli competent cells were transformed; the
plasmid was isolated and subjected to restriction enzyme
(Promega, LaJ olla, CA) mapping and sequencing using a T3
primer (DNA Sequencing Core Laboratory, University of
Florida). Amplification of the cDNA fragment was performed
using a polymerase chain reaction (PCR) kit (Perkin-Elmer,
Norwalk, CT). The PCR primers were from Gibco: (1, 5′ end)
5′-CGT GGA TCC ATG GCA GAT GAC TTG GAC TTC-3′, (2,
3′ end) 5′-AGA GAA TTC TTA TTT TGC CAT GGC CTT GAT
TGC-3′, (3, 5′ end) 5′-CGT GGA TCC GCA GAT GAC TTG
GAC TTC GAG-3′. For amplification, 29 cycles of 95 °C for 2
min, 52 °C for 2 min, and 72 °C for 2 min, followed by a final
extension at 72 °C for 7.5 min, were run. An expression vector,
pGEX-2T, was obtained from Pharmacia (Piscataway, NJ ), and
the PCR-amplified fragment and expression plasmid were
digested with EcoRI and BamHI. The digested nucleic acids
were purified, combined in a fragment-to-vector molar ratio
of 1:4, and ligated using T4 DNA ligase (Gibco/BRL). The
ligation product was transformed into competent HB101, E.
coli J M 109, or E. coli BL 21 cells. Sequence confirmation was
performed by PCR sequencing (using primers 1 and 2 de-
scribed above) of DNA isolated from transformed cells using
a Wizard megapreps DNA purification kit (Promega, Madison,
WI). One clone containing the correct DNA sequence for eIF-
5A except for a deleted methionine codon and the addition of
codons for Gly-Ser at the amino terminus was utilized for
large-scale expression in either J M109 or BL21 cells under
the following conditions. Cultures were incubated at 30 °C
with shaking at 300 rpm until late log phase, 0.1 mM
isopropylthio-â-D-galactoside was added, and the culture was
incubated 6 h. The glutathione S-transferase (GST)-eIF-5A
protein was isolated from cell extracts in the following manner.
After centrifugation at 7700g for 10 min at 4 °C and resus-
pension of the pellet in 1/20 volume of PBS (140 mM NaCl,
2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3), the
cells were lysed by sonication (5 × 40 s) (Sonifier 450, Branson
Ultrasonics Corp., Danbury, CT). The crude homogenate was
centrifuged at 23000g for 10 min at 4 °C; the supernatant was
centrifuged at 26 000 rpm (Beckman SW 28 tubes) for at least
6 h at 4 °C.
reacting with
a known excess of a sulfhydryl-containing
peptide (glutathione), and the remaining sulfhydryl content
was measured using Ellman’s reagent.35 Derivative 29,
containing about 1.8 maleimides/mol of OVA, was then incu-
bated with 90% of the theoretical amount of peptide 28 (the
theoretical amount determined on the basis of sulfhydryl
content) overnight to allow the cysteine-SH of 28 to be
alkylated by the maleimide double bond. The OVA-hexapep-
tide conjugate 31 was then purified by Sephadex G-25 chro-
matography, lyophilized, and stored desiccated at -20 °C.
Additional conjugations were performed to generate antigens
for the ELISA, i.e., 28 to keyhole limpet hemocyanin (KLH)
and 28 to itself. The KLH conjugate 32 was made utilizing
procedures and conditions identical with those for 31 (Scheme
5). Formation of the bis-maleimide complex 33 employed the
sulfhydryl-specific homobifunctional cross-linking reagent N,N′-
bis(3-maleimidopropionyl)-2-hydroxy-1,3-propanediamine(Scheme
6) at a concentration of 1 mM in phosphate buffer, pH 7.4.
The theoretical amount (2 mmol) of 28 was added, and the
reaction mixture was stirred overnight. The product was
passed though a P-4 column.
Mon oclon a l An tibod y P r od u ction . The conjugate 31
combined with Ribi adjuvant (Ribi Immunochemicals, Hamil-
ton, MT) was used to immunize four Balb/c BYJ mice subcu-
taneously with 50, 100, 150, or 200 µg of the peptide conjugate.
Sera were obtained after four immunizations spaced 2 weeks
apart and tested in an ELISA (described below) for reactivity
to the following: unconjugated KLH (neutral carrier control),
32 (to check for reactivity of the sera to the peptide coupled to
a neutral carrier), and 33. Three days before fusion, the mice
were immunized intraperitoneally with 100 µg of 31 without
adjuvant. On the day of the fusion, one mouse was sacrificed;
the spleen was removed. Half of the spleen was used in the
fusion. The fusion was performed by procedures described in
Simrell and Klein34 except the fusion partner was SP2/0 (a
murine aminopterin-resistant cell line with a defect in purine
metabolism). After a 10-day incubation, a primary ELISA
screen was done on the culture supernatants to detect whether
cells were secreting antibody to 28, presented as either 32 or
33. Those wells that were positive in this assay were screened
again 1 week later to determine the class of antibody (IgG or
IgM). These clones were also tested for reactivity to the
deoxyhypusine-containing peptide (Cys-Thr-Gly-dHpu-His-
Gly)- and lysine-containing peptide (Cys-Thr-Gly-Lys-His-
Gly)-KLH conjugates as well as to carrier controls.
The GST fusion protein (43 kDa) was purified from the
supernatant by affinity chromatography on glutathione
Sepharose 4B. The GST (26 kDa) was cleaved from the
N-terminal end of the eIF-5A fusion protein by thrombin
protease. The product, recombinant human eIF-5A precursor
polypeptide (17 kDa), was purified to homogeneity by FPLC
on MonoQ Sepharose.
P r ep a r a tion of Cell Extr a cts Con ta in in g eIF -5A. Ap-
proximately 1 × 108 CHO cells in midlogarithmic growth were
homogenized in 50 mM sodium phosphate buffer, pH 7.4,
containing 0.9% NaCl, 0.1 mM EDTA, 0.1 mM dithiothreitol,
and 1 mM phenylmethanesulfonyl fluoride (1.5 mL). This
homogenate was fractionated by ammonium sulfate precipita-
tion as described by Abbruzzese et al.38 and dialyzed.
En zym e-Lin k ed Im m u n osor ben t Assa y (ELISA). The
ELISA was performed essentially as described by Kao and
Klein.36 Briefly, Nunc Maxisorp plates (Nunc, Naperville, IL)

Construction of Hypusine and Deoxyhypusine Peptides
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20 3895
Nr-BOC-NE-CBZ-L-lysin e ter t-Bu tyl Ester (1). Sodium
hydrogen carbonate (2.81 g, 33.47 mmol) in water (75 mL) was
added to Nꢀ-CBZ-L-lysine tert-butyl ester hydrochloride (12.00
g, 32.18 mmol) in chloroform (100 mL). The mixture was
stirred at room temperature for 5 min under an N2 atmo-
sphere. Di-tert-butyl dicarbonate (7.02 g, 32.18 mmol) in
chloroform (50 mL) was added; the mixture was refluxed for
1.5 h and allowed to cool to room temperature. The layers
were separated, the aqueous layer was extracted with chlo-
roform (3 × 100 mL), and the combined organic layers were
dried. Concentration in vacuo followed by flash chromatog-
raphy (25% ethyl acetate/hexane) gave 1 (13.82 g, 98%) as a
colorless oil: 1H NMR δ 1.30-1.83 (m, 6 H), 1.43 (s, 9 H), 1.45
(s, 9 H), 3.11 (t, 2 H, J ) 6.7), 3.94 (dd, 1 H, J ) 8.3, 5.1), 5.06
(s, 2 H), 7.24-7.37 (m, 5 H); 13C NMR δ 24.0, 28.3, 28.7, 30.4,
32.4, 41.4, 55.7, 67.3, 80.4, 82.5, 128.7, 128.9, 129.4, 138.4,
158.1, 158.8, 173.8; HRMS m/z calcd for C23H37N2O6 437.2652,
(s, 2 H), 7.26-7.40 (m, 5 H); 13C NMR δ 24.1, 28.3, 28.8, 32.5,
49.4, 51.4, 52.2, 55.7, 68.4, 71.0, 80.4, 82.5, 129.0, 129.1, 129.6,
138.0, 157.5-158.4 (br), 158.1, 173.7; HRMS m/z calcd for
C26H42ClN2O7 529.2681, found 529.2694. Anal. (C26H41
-
ClN2O7) C, H, N. [R]23 -21.8° (c 1.00, CH3OH).
D
(2S,9R)-2-[(ter t-Bu t oxyca r bon yl)a m in o]-7-(ca r b ob en -
zyloxy)-10-ch lor o-9-h yd r oxy-7-a za d eca n oic Acid ter t-Bu -
t yl E st er (4b). According to the method described for the
preparation of 4a , 3b (2.93 g, 7.42 mmol) was reacted with
benzyl chloroformate (1.60 g, 9.38 mmol) and triethylamine
(1.50 g, 14.81 mmol) to obtain 4b (3.35 g, 85%) as a colorless
oil: 1H NMR δ 1.24-1.82 (m, 6 H), 1.44 (s, 9 H), 1.45 (s, 9 H),
3.15-3.64 (m, 6 H), 3.92 (m, 1 H), 4.00 (m, 1 H), 5.12 (m, 2
H), 7.25-7.40 (m, 5 H); 13C NMR δ 24.0, 28.3, 28.8, 32.5, 42.4,
51.3, 52.3, 55.7, 68.4, 71.0, 71.1, 80.4, 82.5, 129.0, 129.1, 129.6,
138.0, 158.1, 158.4, 173.7; HRMS m/z calcd for C26H42ClN2O7
529.2681, found 529.2691. Anal. (C26H41ClN2O7) C, H, N.
found 437.2643. Anal. (C23H36N2O6) C, H, N. [R]27 +5.0° (c
D
[R]23 -1.7°(c 0.98, CH3OH).
D
2.00, CHCl3).
(2S,9R)-2-[(ter t-Bu t oxyca r b on yl)a m in o]-7-(ca r b ob en -
zyloxy)-10-cya n o-9-h yd r oxy-7-a za d eca n oic Acid ter t-Bu -
tyl Ester (5a ). A mixture of 4a (2.65 g, 5.02 mmol), dry KCN
(3.45 g, 53.0 mmol), and 18-crown-6 (279 mg, 1.05 mmol) in
dry acetonitrile (100 mL) was stirred at 60 °C for 16 h under
an Ar atmosphere. The reaction mixture was cooled, filtered
through Celite, and concentrated. The residue was purified
by flash chromatography (33% ethyl acetate/hexane, then 50%
ethyl acetate/hexane) to give 5a (1.84 g, 70%) as a colorless
oil: 1H NMR δ 1.25-1.80 (m, 6 H), 1.44 (s, 9 H), 1.45 (s, 9 H),
2.42-2.76 (m, 2 H), 3.20-3.58 (m, 4 H), 3.92 (m, 1 H), 4.08
(m, 1 H), 5.13 (s, 2 H), 7.26-7.48 (m, 5 H); 13C NMR δ 24.0,
24.2, 28.2, 28.7, 32.4, 52.9, 53.8, 55.7, 67.1, 68.4, 80.4, 82.5,
118.9, 129.0, 129.2, 129.6, 138.0, 157.5-158.4 (br), 158.1, 173.7;
HRMS m/z calcd for C27H42N3O7 520.3023, found 520.3013.
Nr-BOC-L-lysin e ter t-Bu tyl Ester Hyd r och lor id e (2).
To a solution of 1 (34.51 g, 79.05 mmol) in ethanol (300 mL)
and 1 N HCl (88 mL) was added 10% Pd-C (2.95 g), and H2
gas was introduced. Additional catalyst (1.0 g) was added 7
h later. After 5 h the black suspension was filtered through
Celite and washed with ethanol. The filtrate was concen-
trated, and the residue was dried in vacuo to give 2 as the
hydrochloride salt (26.59 g, 99%): 1H NMR δ 1.30-1.84 (m, 6
H), 1.43 (s, 9 H), 1.45 (s, 9 H), 2.93 (t, 2 H, J ) 7.7), 3.95 (dd,
1 H, J ) 8.8, 5.0); 13C NMR δ 23.9, 28.3, 28.7, 32.1, 40.6, 55.5,
79.5, 80.5, 82.7, 158.2, 173.5; HRMS m/z calcd for C15H31N2O4
303.2284, found 303.2272. [R]23 -21.2° (c 1.00, CH3OH).
D
(2S,9S)-2-[(ter t-Bu t oxyca r b on yl)a m in o]-10-ch lor o-9-
h yd r oxy-7-a za d eca n oic Acid ter t-Bu tyl Ester (3a ).
A
solution of 2 (4.83 g, 14.3 mmol) in chloroform (100 mL) was
extracted with saturated NaHCO3 solution (2 × 100 mL) and
water (100 mL). The organic layer was dried, concentrated,
and further dried in vacuo. The resulting oil was dissolved in
cyclohexane (26 mL), and (S)-(+)-epichlorohydrin (1.58 g, 17.1
mmol) was added under an Ar atmosphere. The precipitated
product was filtered after 27 h, washed with cold cyclohexane,
and dried in vacuo to give 3a (2.20 g, 39%) as a fine, white
powder: mp 87-88 °C; 1H NMR δ 1.30-1.83 (m, 6 H), 1.44 (s,
9 H), 1.46 (s, 9 H), 2.57-2.66 (m, 3 H), 2.77 (dd, 1 H, J ) 12.2,
3.9), 3.51 (dd, 1 H, J ) 11.2, 5.7), 3.56 (dd, 1 H, J ) 11.2, 5.2),
3.84-3.91 (m, 1 H), 3.94 (dd, 1 H, J ) 8.6, 5.3); 13C NMR δ
24.6, 28.3, 28.7, 30.0, 32.6, 48.2, 50.3, 53.5, 55.8, 71.1, 80.4,
82.5, 158.1, 173.8; HRMS m/z calcd for C18H36ClN2O5 395.2313,
[R]22 -19.8° (c 1.00, CH3OH).
D
(2S,9S)-2-[(ter t-Bu t oxyca r b on yl)a m in o]-7-(ca r b ob en -
zyloxy)-10-cya n o-9-h yd r oxy-7-a za d eca n oic Acid ter t-Bu -
t yl E st er (5b). According to the method described for the
preparation of 5a , 4b (3.23 g, 6.10 mmol) was reacted with
KCN (3.96 g, 60.82 mmol) and 18-crown-6 (242 mg, 0.92 mmol)
to obtain 5b (1.62 g, 51%) as a colorless oil: 1H NMR δ 1.20-
1.84 (m, 6 H), 1.44 (s, 9 H), 1.45 (s, 9 H), 2.55 (m, 2 H), 3.20-
3.50 (m, 4 H), 3.92 (dd, 1 H, J ) 8.2, 5.1), 4.07 (m, 1 H), 5.13
(s, 2 H), 7.26-7.41 (m, 5 H); 13C NMR δ 24.1, 24.2, 28.3, 28.8,
32.4, 55.7, 67.1, 68.4, 80.4, 82.5, 118.9, 129.0, 129.2, 129.6,
138.0, 158.1, 173.7; HRMS m/z calcd for C27H42N3O7 520.3023,
found 520.3048. Anal. (C27H41N3O7) C, H, N. [R]22 -5.3° (c
D
found 395.2303. Anal. (C18H35ClN2O5) C, H, Cl, N. [R]22
-26.1° (c 1.00, CH3OH).
1.15, CH3OH).
D
(2S,9R)-2-[(ter t-Bu t oxyca r b on yl)a m in o]-11-a m in o-9-
h yd r oxy-7-a za u n d eca n oic Acid ter t-Bu tyl Ester , Dia c-
eta te Sa lt (6a ). To a solution of 5a (1.74 g, 3.34 mmol) in
glacial acetic acid (42 mL) were added 10% Pd-C (0.17 g) and
PtO2 (0.34 g), and H2 gas was introduced. The catalyst was
removed after 22 h by filtration through Celite. The filtrate
was concentrated in vacuo. Azeotropic removal of acetic acid
with toluene provided 6a as a colorless oil (1.70 g, 100%): 1H
NMR (D2O) δ 1.44 (s, 9 H), 1.47 (s, 9 H), 1.50-1.90 (m, 8 H),
1.97 (s, 6 H), 3.00-3.25 (m, 6 H), 3.97 (dd, 1 H, J ) 8.8, 5.2),
4.06 (tt, 1 H, J ) 9.6, 3.0); 13C NMR (D2O, internal standard
CH3OH ) 49.5 ppm39) δ 24.3, 24.5, 26.9, 29.3, 29.7, 32.1, 33.5,
38.5, 54.1, 56.5, 66.8, 83.5, 85.8, 159.8, 176.0, 182.0; HRMS
(2S,9R)-2-[(ter t-Bu t oxyca r b on yl)a m in o]-10-ch lor o-9-
h yd r oxy-7-a za d eca n oic Acid ter t-Bu tyl Ester (3b). Com-
pound 3b was prepared from reaction of 2 (5.93 g, 17.50 mmol)
with (R)-(-)-epichlorohydrin (1.62 g, 17.50 mmol) in 44% yield
using the same procedure as described for 3a . 3b: mp 101
°C; 1H NMR δ 1.30-1.80 (m, 6 H), 1.44 (s, 9 H), 1.46 (s, 9 H),
2.61 (m, 3 H), 2.77 (dd, 1 H, J ) 12.3, 4.0), 3.49 (dd, 1 H, J )
11.3, 5.8), 3.57 (dd, 1 H, J ) 11.3, 5.3), 3.88 (m, 1 H), 3.94 (dd,
1 H, J ) 8.6, 5.0); HRMS m/z calcd for C18H36ClN2O5 395.2313,
found 395.2303. Anal. (C18H35ClN2O5) C, H, N. [R]22D -15.5°
(c 0.96, CH3OH).
(2S,9S)-2-[(ter t-Bu t oxyca r b on yl)a m in o]-7-(ca r b ob en -
zyloxy)-10-ch lor o-9-h yd r oxy-7-a za d eca n oic Acid ter t-Bu -
tyl Ester (4a ). A solution of benzyl chloroformate (1.21 g,
7.09 mmol) in chloroform (10 mL) was added over a period of
15 min to an ice-cold solution of 3a (2.16 g, 5.46 mmol) in
chloroform (40 mL) under an Ar atmosphere. After dropwise
additon of triethylamine (1.11 g, 11.0 mmol) in chloroform (10
mL), the reaction mixture was stirred for 4.5 h at room
temperature. The reaction mixture was extracted with 1 N
HCl (60 mL) and water (60 mL), dried, and concentrated. The
residue was purified by flash chromatography (33% ethyl
acetate/hexane) to give 4a (2.69 g, 93%) as a colorless oil: 1H
NMR δ 1.30-1.83 (m, 6 H), 1.44 (s, 9 H), 1.45 (s, 9 H), 3.20-
3.64 (m, 6 H), 3.93 (dd, 1 H, J ) 8.2, 5.5), 4.00 (m, 1 H), 5.12
m/z calcd for C19H40N3O5 390.2968, found 390.2965. [R]21
-16.1° (c 1.00, CH3OH).
D
(2S,9S)-2-[(ter t-Bu t oxyca r b on yl)a m in o]-11-a m in o-9-
h yd r oxy-7-a za u n d eca n oic Acid ter t-Bu tyl Ester , Dia c-
eta te Sa lt (6b). According to the method described for the
preparation of 6a , to a solution of 5b (172 mg, 0.33 mmol) in
glacial acetic acid (5 mL) were added 10% Pd-C (17 mg) and
PtO2 (35 mg) under an H2 atmosphere to obtain 6b in
quantitative yield as a colorless oil: 1H NMR (D2O) δ 1.30-
2.00 (m, 8 H), 1.45 (s, 9 H), 1.48 (s, 9 H), 1.92 (s, 6 H), 2.99-
3.28 (m, 6 H), 3.99 (dd, 1 H, J ) 9.0, 5.1), 4.06 (tt, 1 H, J )
9.5, 3.3); HRMS m/z calcd for C19H40N3O5 390.2968, found
390.2967. [R]22 -7.5° (c 1.03, CH3OH).
D

3896 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20
Bergeron et al.
(2S,9R)-11-[(Ben zyloxyca r bon yl)a m in o]-2-[(ter t-bu tox-
yca r bon yl)a m in o]-9-h yd r oxy-7-(ca r boben zyloxy)-7-a za -
u n d eca n oic Acid ter t-Bu tyl Ester (7a ). A solution of 6a
(1.20 g, 2.35 mmol) in water (35 mL) and diethyl ether (35
mL) was vigorously stirred and cooled to 0 °C under an Ar
atmosphere. KHCO3 (3.08 g, 30.8 mmol) was added, and then
N-(benzyloxycarbonyloxy)succinimide (CBZ-ONSu; 1.70 g, 6.82
mmol) was added in five portions over 20 min. The reaction
mixture was warmed to room temperature and stirred for 4
h. The layers were separated; the aqueous layer was extracted
with diethyl ether (2 × 35 mL). The combined ether layers
were dried and concentrated. The residue was purified by
flash chromatography (50% ethyl acetate/hexane) to give 7a
(772 mg, 50%) as a colorless oil: 1H NMR δ 1.22-1.82 (m, 8
H), 1.43 (s, 9 H), 1.44 (s, 9 H), 3.08-3.45 (m, 6 H), 3.81 (m, 1
H), 3.92 (m, 1 H), 5.06 (s, 2 H), 5.10 (s, 2 H), 7.24-7.38 (m, 10
H); 13C NMR δ 24.1, 28.3, 28.8, 32.5, 35.9, 38.6, 54.0, 54.7,
55.7, 67.4, 68.3, 69.0, 80.4, 82.5, 128.8, 128.9, 129.1, 129.4,
129.6, 138.1, 138.4, 158.1, 158.4, 158.9, 173.7; HRMS m/z calcd
for C35H52N3O9 658.3704, found 658.3767. Anal. (C35H51N3O9)
C, H, N.
added over the next 31 h. The reaction mixture was stirred
for an additional 16 h and concentrated in vacuo. The oil was
dissolved in water (4 mL) and methanol (8 mL) and then
adjusted to pH 7 with saturated NaHCO3 solution. The
solution was concentrated, and the crude oil was purified by
chromatography on a C18 column (30% acetone/water, followed
by 55% acetone/water) to give 9a (219 mg, 52%) as a colorless
oil: 1H NMR δ 1.20-1.98 (m, 14 H), 3.06-3.56 (m, 8 H), 3.68-
4.02 (m, 2 H), 4.33-4.62 (m, 1 H), 5.06 (s, 2 H), 5.11 (s, 2 H),
7.24-7.36 (m, 10 H); 13C NMR δ 20.7, 21.1, 21.7, 23.6, 26.3,
26.5, 32.0, 32.5, 34.2, 38.1, 38.5, 52.0, 56.2, 64.2, 65.2, 67.3,
68.3, 74.9, 100.1, 101.6, 128.79, 128.96, 129.02, 129.10, 129.20,
129.47, 129.56, 129.62, 138.3, 138.5, 158.0, 158.7, 174.8; HRMS
m/z calcd for C31H43N3NaO8 608.2948, found 608.2954.
(2S,9S)-2-Am in o-11-[(ben zyloxycar bon yl)am in o]-7-(car -
b ob en zyloxy)-9-(t et r a h yd r op yr a n -2-yloxy)-7-a za u n d e-
ca n oic Acid (9b). According to the method described for the
preparation of 9a , 8b (37 mg, 73.8 µmol) was reacted with
trifluoroacetic acid (17 mg, 0.15 mmol) and 3,4-dihydro-2H-
pyran (46 mg, 50 µL, 0.55 mmol) in CH2Cl2 (2 mL) to give 9b
(25 mg, 58%) as a colorless oil: 1H NMR δ 1.26-1.96 (m, 14
H), 3.02-4.04 (m, 10 H), 4.32-4.63 (m, 1 H), 5.06 (s, 2 H),
5.11 (s br, 2 H), 7.22-7.40 (m, 10 H); HRMS m/z calcd for
(2S,9S)-11-[(Ben zyloxyca r bon yl)a m in o]-2-[(ter t-bu tox-
yca r bon yl)a m in o]-9-h yd r oxy-7-(ca r boben zyloxy)-7-a za -
u n d eca n oic Acid ter t-Bu tyl Ester (7b). According to the
method described for the preparation of 7a , 6b (165 mg, 0.32
mmol) was reacted with CBZ-ONSu (178 mg, 0.71 mmol) to
give 7b (90 mg, 43%) as a colorless oil: 1H NMR δ 1.18-1.82
(m, 8 H), 1.43 (s, 9 H), 1.44 (s, 9 H), 3.06-3.48 (m, 6 H), 3.83
(m, 1 H), 3.92 (m, 1 H), 5.05 (s, 2 H), 5.10 (s, 2 H), 7.22-7.40
(m, 10 H); 13C NMR δ 24.1, 28.3, 28.8, 32.5, 35.9, 38.6, 49.3,
54.0, 54.7, 55.8, 67.4, 68.3, 69.0, 80.4, 82.5, 128.9, 128.9, 129.1,
129.4, 129.6, 138.1, 138.4, 158.0, 158.4, 158.9, 173.7; HRMS
m/z calcd for C35H52N3O9 658.3704, found 658.3702. Anal.
C
31H44N3O8 586.3128, found 586.3137.
(2S,9R)-11-[(Ben zyloxyca r bon yl)a m in o]-7-(ca r boben -
zyloxy)-2-[(9-flu or en ylm et h oxyca r b on yl)a m in o]-9-(t et -
r a h yd r op yr a n -2-yloxy)-7-a za u n d eca n oic Acid (10a ).
A
solution of 9-fluorenylmethyl N-succinimidyl carbonate (203
mg, 0.60 mmol) in DMF (3.0 mL) was added to a solution of
9a (219 mg, 0.37 mmol) in 9% Na2CO3 (803 mg, 0.68 mmol) at
0 °C and stirred overnight at room temperature. The pH was
adjusted to 7.0 with 0.1 N HCl. The mixture was concentrated
to an oil and purified by flash chromatography (CHCl3, then
95% CHCl3/MeOH) to give 10a (150 mg, 50%) as a colorless
oil: 1H NMR δ 1.15-1.94 (m, 14 H), 3.10-3.52 (m, 7 H), 3.66-
3.98 (m, 2 H), 4.05-4.21 (m, 2 H), 4.24-4.61 (m, 3 H), 5.04 (s,
2 H), 5.08 (s, 2 H), 7.21-7.40 (m, 14 H), 7.65 (dd, 2 H, J ) 6.9,
4.2), 7.77 (d, 2 H, J ) 7.2); 13C NMR δ 21.1, 21.7, 24.1, 26.4,
26.5, 28.2, 28.9, 32.1, 32.5, 32.6, 33.2, 34.3, 38.1, 38.5, 51.9,
52.4, 55.5, 64.1, 65.2, 67.4, 67.9, 68.3, 74.9, 79.5, 100.0, 101.5,
120.9, 126.3, 128.2, 128.80, 128.97, 129.10, 129.20, 129.48,
129.57, 129.62, 138.2, 138.5, 142.6, 145.2, 145.4, 158.1, 158.6,
176.8; HRMS m/z calcd for C46H54N3O10 808.3809, found
808.3833.
(C35H51N3O9) C, H, N. [R]23 -5.2° (c 1.00, CH3OH).
D
(2S,9R)-2-Am in o-11-[(ben zyloxycar bon yl)am in o]-7-(car -
boben zyloxy)-9-h yd r oxy-7-a za u n d eca n oic Acid (8a ). The
ester 7a (766 mg, 1.16 mmol) was added to a mixture of
trifluoroacetic acid (1.73 g, 15.1 mmol), CH2Cl2 (1.2 mL), and
triethylsilane (4.37 g, 37.5 mmol) and stirred at room tem-
perature for 21 h under an Ar atmosphere. The reaction
mixure was concentrated to dryness and stirred again in the
mixture as described above for an additional 18 h. The
reaction mixture was concentrated; the resultant oil was
dissolved in water (4.0 mL) and adjusted to pH 7 with
saturated NaHCO3 solution. The solution was concentrated,
and the residue was purified by chromatography on a C18
column (30% acetone/water, followed by 55% acetone/water)
to give 8a (421 mg, 72%) as a colorless oil: 1H NMR (45 °C) δ
1.31-1.99 (m, 8 H), 3.10-3.46 (m, 6 H), 3.52 (t, 1 H, J ) 5.9),
3.83 (m, 1 H), 5.06 (s, 2 H), 5.10 (s, 2 H), 7.22-7.36 (m, 10 H);
13C NMR δ 23.5, 28.4, 29.2, 32.0, 35.9, 38.6, 54.2, 54.9, 56.0,
67.4, 68.3, 69.0, 128.7, 128.8, 128.9, 129.1, 129.4, 129.5, 138.1,
138.4, 158.0, 158.3, 158.9, 174.5; HRMS m/z calcd for
(2S,9S)-11-[(Ben zyloxyca r b on yl)a m in o]-7-(ca r b ob en -
zyloxy)-2-[(9-flu or en ylm et h oxyca r b on yl)a m in o]-9-(t et -
r a h yd r op yr a n -2-yloxy)-7-a za u n d eca n oic Acid (10b). Ac-
cording to the method described for the preparation of 10a ,
9b (26 mg, 44 µmol) was reacted with 9-fluorenylmethyl
N-succinimidyl carbonate (22 mg, 66 µmol) and Na2CO3 (9 mg,
88 µmol) to give 10b (20 mg, 55%) as a colorless oil: 1H NMR
δ 1.14-1.96 (m, 14 H), 3.04-3.55 (m, 6 H), 3.64-3.98 (m, 3
H), 4.14 (m, 1 H), 4.20 (t, 1 H, J ) 6.9), 4.34 (d, 2 H, J ) 6.9),
4.38-4.61 (m, 1 H), 5.05 (s, 2 H), 5.09 (s, 2 H), 7.20-7.42 (m,
14 H), 7.65 (m, 2 H), 7.78 (d, 2 H, J ) 7.4); HRMS m/z calcd
for C46H54N3O10 808.3809, found 808.3839. Anal. (C46H53N3O10)
C, H, N.
C
26H36N3O7 502.2553, found 502.2517. [R]24 +5.0° (c 1.00,
D
CH3OH).
(2S,9S)-2-Am in o-11-[(ben zyloxycar bon yl)am in o]-7-(car -
boben zyloxy)-9-h yd r oxy-7-a za u n d eca n oic Acid (8b). Ac-
cording to the method described for the preparation of 8a , 7b
(78 mg, 0.12 mmol) was reacted with trifluoroacetic acid (704
mg, 6.20 mmol) and triethylsilane (140 mg, 11.92 mmol) in
CH2Cl2 (0.98 mL) to give 8b (37 mg, 62%) as a colorless oil:
1H NMR δ 1.20-2.10 (m, 8 H), 3.05-3.70 (m, 7 H), 3.82 (m, 1
H), 5.05 (s, 2 H), 5.10 (s, 2 H), 7.20-7.60 (m, 10 H); HRMS
(2S,9R)-2-[(ter t-Bu t oxyca r b on yl)a m in o]-7-(ca r b ob en -
zyloxy)-10-cya n o-9-[(S)-r-m et h oxy-r-(t r iflu or om et h yl)-
ph en ylacetoxy]-7-azadecan oic Acid ter t-Bu tyl Ester (11a).
The reaction was carried out in an oven-dried 5- × 175-mm
NMR tube, fitted with a rubber septum, under an Ar atmo-
sphere. The reagents were injected via syringe in the following
order: anhydrous pyridine (300 µL), (R)-(-)-R-methoxy-R-
(trifluoromethyl)phenylacetic acid (Mosher’s acid chloride; 13
µL, 70 µmol), anhydrous carbon tetrachloride (200 µL), and
then a solution of 5a (27 mg, 52 µmol) in anhydrous carbon
tetrachloride (500 µL). The reaction mixture was shaken and
allowed to stand at room temperature for 18 h. The reaction
mixture was then taken up in chloroform (20 mL), extracted
with saturated NaHCO3 solution, and then extracted with
saturated NaCl solution. The organic layer was dried and
concentrated in vacuo. The residue was purified by flash
m/z calcd for C26H36N3O7 502.2553, found 502.2546. [R]23
+3.6° (c 1.00, CH3OH).
D
(2S,9R)-2-Am in o-11-[(ben zyloxycar bon yl)am in o]-7-(car -
b ob en zyloxy)-9-(t et r a h yd r op yr a n -2-yloxy)-7-a za u n d e-
ca n oic Acid (9a ). Trifluoroacetic acid (164 mg, 1.44 mmol)
was added to a solution of 8a (360 mg, 0.72 mmol) in CHCl3
(7 mL). The solution was concentrated in vacuo. The result-
ant oil was dissolved in CH2Cl2 (20 mL), and 3,4-dihydro-2H-
pyran (69 mg, 75 µL, 0.83 mmol) was added at room temper-
ature. The reaction progress was monitored by TLC, and six
additional portions of 3,4-dihydro-2H-pyran (69 mg each) were

Construction of Hypusine and Deoxyhypusine Peptides
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20 3897
chromatography (33% ethyl acetate/hexane) to give 11a (31
mg, 81%) as a colorless oil: 1H NMR (CDCl3, 45 °C) δ 1.08-
1.80 (m, 6 H), 1.447 (s, 9 H), 1.453 (s, 9 H), 2.52-3.12 (m, 4
H), 3.38 (dd, 1 H, J ) 14.7, 6.7), 3.52 (dd, 1 H, J ) 14.7, 4.6),
3.60 (s br, 3 H), 4.08 (m, 1 H), 4.92 (m, 1 H), 5.10 (s, 2 H), 5.38
(m, 1 H), 7.28-7.53 (m, 10 H); 19F NMR (282 MHz, CDCl3,
CFCl3 as internal standard, 45 °C) δ -71.86; HRMS m/z calcd
BOC-(2S,9R)-Hp u (N7,N12-d i-CBZ)-Va l-O-t-Bu (15a ). Un-
der argon, 14 (50.9 mg, 77.5 mmol) was dissolved in dioxane
(2 mL) and H2O (1 mL) and cooled to 0 °C. Di-tert-butyl
dicarbonate (30 mg, 0.13 mmol) was added; the solution was
allowed to stir for 5 min, then warmed to room temperature,
and stirred for 5 h. Concentration followed by flash chroma-
tography (2.5% MeOH/CHCl3) gave 15a (58 mg, 99%) as a
colorless oil: 1H NMR δ 0.94 (d, 6 H, J ) 6.8), 1.22-1.83 (m,
8 H), 1.43 (s, 9 H), 1.45 (s, 9 H), 2.12 (m, 1 H), 3.10-3.42 (m,
6 H), 3.82 (m, 1 H), 4.06 (m, 1 H), 4.20 (d, 1 H, J ) 5.7), 5.06
(s, 2 H), 5.10 (s, 2 H), 7.23-7.37 (m, 10 H); 13C NMR δ 18.4,
19.5, 24.0, 27.5, 28.3, 28.7, 32.0, 35.8, 38.6, 54.4, 55.8, 59.6,
67.4, 68.3, 68.8, 80.6, 82.8, 128.80, 128.87, 128.94, 129.06,
129.44, 129.56, 138.1, 138.4, 157.9, 158.3, 158.9, 172.0, 175.2;
HRMS m/z calcd for C40H61N4O10 757.4387, found 757.4396.
BOC-(2S,9R)-H p u [N7,N12-d i-CBZ, 9-(S)-r-m et h oxy-r-
(tr iflu or om eth yl)p h en yla cetoxy]-Va l-O-t-Bu (16a ). Un-
der argon, anhydrous pyridine (900 µL), anhydrous CCl4 (600
µL), and (R)-(-)-Mosher’s acid chloride (20 µL, 27 mg, 107
µmol) were mixed with vigorous stirring. A solution of 15a
(55 mg, 73 µmol) in CCl4 (1.5 mL) was added. After 17 h of
stirring at room temperature, the solution was diluted with
chloroform (25 mL) and extracted with 25 mL of each of the
following: brine, 1:1 1 N HCl/brine, brine, 1:1 saturated
NaHCO3/brine, and brine. The organic layer was dried and
concentrated. Purification by flash chromatography (2:1 hex-
ane/ethyl acetate) gave 16a (55 mg, 78%) as a colorless oil:
1H NMR (CDCl3, 45.0 °C) δ 0.91 (d, 3 H, J ) 6.8), 0.92 (d, 3 H,
J ) 6.8), 1.10-1.94 (m, 8 H), 1.44 (s, 9 H), 1.46 (s, 9 H), 2.14
(m, 1 H), 2.72-3.54 (m, 7 H), 3.50 (s, 3 H), 4.01 (m, 1 H), 4.38
(dd, 1 H, J ) 8.7, 4.7), 4.83-5.37 (m, 2 H), 5.09 (s, 2 H), 5.10
(s, 2 H), 6.54 (m, 1 H), 7.24-7.52 (m, 15 H); 13C NMR (CDCl3,
20 °C) δ 17.5, 18.8, 22.5, 26.7, 28.0, 28.3, 31.3, 32.3, 37.1, 54.3,
55.5, 57.4, 66.7, 67.3, 73.3, 79.9, 81.9, 84.6, 127.05, 127.88,
128.06, 128.23, 128.47, 128.53, 129.6, 131.8, 136.2, 136.5,
155.6, 155.9, 156.3, 166.6, 170.7, 171.8; 19F NMR (282 MHz,
CDCl3, CFCl3 ) 0 ppm) δ -71.78; HRMS m/z calcd for
C50H68F3N4O12 973.4786, found 973.4867. [R]21D -16.0° (c 0.50,
CHCl3).
for C37H49F3N3O9 736.3421, found 736.3367. [R]22 -29.2° (c
D
1.00, CHCl3).
(2S,9S)-2-[(ter t-Bu t oxyca r b on yl)a m in o]-7-(ca r b ob en -
zyloxy)-10-cya n o-9-[(S)-r-m et h oxy-r-(t r iflu or om et h yl)-
ph en ylacetoxy]-7-azadecan oic Acid ter t-Bu tyl Ester (11b).
According to the method described for the preparation of 11a ,
5b (24 mg, 46 µmol) was reacted with (R)-(-)-Mosher’s acid
chloride (14 mg, 55 µmol) to give 11b (23 mg, 69%) as a
colorless oil: 1H NMR (CDCl3, 45 °C) δ 1.14-1.80 (m, 6 H),
1.44 (s, 9 H), 1.45 (s, 9 H), 2.54-2.92 (m, 2 H), 3.09 (m, 1 H),
3.27 (m, 1 H), 3.46 (s br, 3 H), 3.48 (dd, 1 H, J ) 14.7, 6.9),
3.61 (dd, 1 H, J ) 14.7, 5.1), 4.10 (m, 1 H), 4.94 (m, 1 H), 5.13
(s, 2 H), 5.38 (m, 1 H), 7.28-7.52 (m, 10 H); 19F NMR (282
MHz, CDCl3, CFCl3 as internal standard, 45 °C) δ -71.85;
HRMS m/z calcd for C37H49F3N3O9 736.3421, found 736.3443.
[R]22 +16.1° (c 1.08, CHCl3).
D
FMOC-(2S,9R)-Hpu [N7,N12-di-CBZ, 9-(tetr ah ydr opyr an -
2-yloxy)]-Va l-O-t-Bu (12). Under argon, 10a (66.8 mg, 82.7
µmol) and L-valine tert-butyl ester hydrochloride (21.6 mg, 103
µmol) were dissolved in DMF (9.3 mL) and stirred at 0 °C.
BOP reagent (42.4 mg, 95.9 mmol) was added, and the reaction
mixture was stirred for 30 min before adding diisopropyleth-
ylamine (21.4 mg, 165 µmol) and allowing to warm to room
temperature. After 21 h, the DMF was diluted with brine (40
mL) and extracted with ethyl acetate (3 × 30 mL). The ethyl
acetate layer was then washed with 20 mL of each of the
following: 1:1 10% citric acid/brine, brine, 1:1 saturated
NaHCO3/brine, and brine. The ethyl acetate was dried and
evaporated, and the residue was purified by flash chromatog-
raphy (2% MeOH/CHCl3) to give 12 (66.0 mg, 83%) as a
colorless oil: 1H NMR δ 0.94 (d, 6 H, J ) 6.8), 1.20-1.88 (m,
14 H), 1.44 (s, 9 H), 2.11 (m, 1 H), 3.13-3.49 (m, 8 H), 3.66-
3.98 (m, 2 H), 4.05-4.24 (m, 2 H), 4.28-4.62 (m, 3 H), 5.05 (s,
2 H), 5.09 (s, 2 H), 7.22-7.41 (m, 14 H), 7.64 (m, 2 H), 7.77 (d,
2 H, J ) 7.5).
(2S,9R)-Hyp u sin yl-L-va lin e (17a ). Under argon, phenol
(270 mg), pentamethylbenzene (250 mg), and 15a (10 mg, 13
µmol) were dissolved in TFA (5 mL) at 0 °C. With vigorous
stirring, triisopropylsilane (200 µL) and 30% HBr/HOAc (200
µL) were added, and the solution was allowed to stir for 5 min
before being warmed to room temperature and stirred for an
additional 55 min. After concentration, the reaction mixture
was diluted with 10% HOAc/H2O (10 mL) and extracted three
times with methyl tert-butyl ether (25 mL). Evaporation of
the aqueous layer gave 17a (8 mg) as an oil: 1H NMR (D2O,
NaTSP external reference) δ 0.98 (d, 3 H, J ) 6.8), 0.99 (d, 3
H, J ) 6.8), 1.49 (m, 2 H), 1.69-2.01 (m, 6 H), 2.22 (m, 1 H),
3.01-3.26 (m, 6 H), 4.07 (tt, 1 H, J ) 9.7, 3.1), 4.13 (t, 1 H, J
) 6.6), 4.32 (d, 1 H, J ) 5.7); HRMS m/z calcd for C15H33N4O4
333.2502, found 333.2506.
(2S,9R)-H p u [N7,N12-d i-CBZ,
9-(t et r a h yd r op yr a n -2-
yloxy)]-Va l-O-t-Bu (13). The fully protected dipeptide 12
(141.6 mg, 147.0 µmol) was dissolved in a solution of piperidine
(2.0 mL) and DMF (20 mL) and stirred under argon for 4.5 h.
Concentration and purification by flash chromatography (2%
MeOH/CHCl3) gave 13 (97 mg, 89%) as a colorless oil: 1H NMR
δ 0.96 (d, 6 H, J ) 6.8), 1.22-1.84 (m, 14 H), 1.44 (s, 9 H),
2.13 (m, 1 H), 3.04-3.49 (m, 8 H), 3.68-4.00 (m, 2 H), 4.20 (d,
1 H, J ) 5.7), 4.35-4.63 (m, 1 H), 5.06 (s, 2 H), 5.11 (s, 2 H),
7.24-7.38 (m, 10 H); 13C NMR δ 18.5, 19.5, 21.7, 23.8, 26.4,
28.3, 32.0, 36.2, 36.9, 38.5, 55.7, 59.6, 64.0, 67.3, 68.2, 74.7,
82.8, 99.8, 101.5, 128.77, 128.94, 129.18, 129.46, 129.54,
129.60, 138.2, 138.5, 158.0, 158.6, 172.2, 177.6; HRMS m/z
calcd for C40H61N4O9 741.4438, found 741.4458.
(2S,9R)-Hp u (N7,N12-d i-CBZ)-Va l-O-t-Bu (14). The free
amine 13 (92 mg, 124 µmol) was dissolved in acetone (10 mL)
and H2O (1.0 mL). Under argon, p-toluenesulfonic acid
monohydrate (64 mg, 0.34 µmol) was added, and the solution
was heated and stirred at 45 °C for 2 h. The reaction mixture
was diluted with H2O (10 mL), and the pH was adjusted to
7-8 with saturated NaHCO3. The acetone was evaporated
under vacuum, and the remaining aqueous layer was extracted
with chloroform (2 × 10 mL). Drying, evaporation, and high-
vacuum drying of the organic layer gave 14 (66.7 mg, 82%) as
a colorless oil: 1H NMR δ 0.95 (d, 6 H, J ) 6.8), 1.22-1.83
(m, 8 H), 1.45 (s, 9 H), 2.12 (m, 1 H), 3.10-3.46 (m, 7 H), 3.82
(m, 1 H), 4.20 (d, 1 H, J ) 5.7), 5.05 (s, 2 H), 5.10 (s, 2 H),
7.24-7.38 (m, 10 H); 13C NMR δ 18.4, 19.5, 23.8, 28.3, 29.4,
31.9, 36.0, 36.9, 38.6, 54.6, 55.7, 59.6, 67.3, 68.2, 69.0, 82.8,
128.78, 128.87, 128.91, 129.05, 129.44, 129.55, 138.1, 138.4,
158.1, 158.9, 172.2, 177.7; HRMS m/z calcd for C31H53N4O8
657.3863, found 657.3928.
(2S,9S)-11-[(Ben zyloxyca r bon yl)a m in o]-2-[(ter t-bu tox-
yca r bon yl)a m in o]-9-h yd r oxy-7-(ca r boben zyloxy)-7-a za -
u n d eca n oic Acid (18). A solution of 8b (37 mg, 74.0 µmol)
in 1,4-dioxane (2 mL) and water (1 mL) was cooled to 0 °C,
and 1 N NaOH (74 µmol) and di-tert-butyl dicarbonate (18 mg,
82.5 µmol) were added. The reaction mixture was stirred at
room temperature for 2.5 h. The organic solvent was removed
under reduced pressure; the residue was taken up in ethyl
acetate (4 mL) and water (4 mL) and cooled to 0 °C. This
mixture was acidified to pH 2-3 with 1 N KHSO4 solution
under stirring. The layers were separated, the aqueous layer
was extracted with ethyl acetate (3 × 5 mL), and the combined
organic layers were dried. Concentration in vacuo followed
by flash chromatography (10% methanol/chloroform) gave 18
(33 mg, 74%) as a colorless oil: 1H NMR δ 1.25-1.95 (m, 8
H), 1.44 (s, 9 H), 3.06-3.50 (m, 6 H), 3.83 (br m, 1 H), 4.04 (br
m, 1 H), 5.06 (s, 2 H), 5.10 (s, 2 H), 7.22-7.44 (m, 10 H); HRMS
m/z calcd for C31H44N3O9 602.3078, found 602.3053. [R]21
+3.6° (c 1.00, CH3OH).
D

3898 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20
BOC-(2S,9S)-Hp u (N7,N12-d i-CBZ)-Va l-O-t-Bu (15b).
Bergeron et al.
A
H2 gas for 17 h. The black suspension was filtered through
Celite to give 20. The filtrate was concentrated, and the
residue was immediately dissolved in glacial acetic acid (25
mL). Under argon, PtO2 (152 mg) and 10% Pd-C catalyst (82
mg) were added; the suspension was stirred under H2 gas for
23 h and then filtered through Celite and washed with acetic
acid. The filtrate was concentrated in vacuo. Azeotropic
removal of acetic acid with toluene provided 21 as a colorless
oil (650 mg, 53% calculated from the free amine): 1H NMR
(D2O) δ 1.20-2.00 (m, 10 H), 1.40 (s, 9 H), 1.47 (s, 9 H), 1.97
(s, 6 H), 2.97-3.13 (m, 6 H), 3.97 (dd, 1 H, J ) 9.0, 5.5).
solution of 18 (31 mg, 51.5 µmol) and L-valine tert-butyl ester
hydrochloride (12 mg, 57 µmol) in dry DMF (3 mL) was cooled
to 0 °C under an Ar atmosphere. BOP (25 mg, 57 µmol) was
added, and after 45 min DIEA (18 µL, 103 µmol) was added.
The solution was warmed to room temperature and stirred
overnight. The reaction mixture was diluted with saturated
NaCl solution and extracted with ethyl acetate (3 × 30 mL).
The combined organic layers were successively washed with
ice-cold 10% citric acid/brine (1:1, 20 mL), brine (20 mL),
saturated NaHCO3 solution/brine solution (1:1, 20 mL), and
brine (20 mL). The organic layer was dried and concentrated
in vacuo. The residue was purified by flash chromatography
(98% chloroform/methanol, then 95% chloroform/methanol) to
yield 15b (23 mg, 59%) as a colorless oil: 1H NMR δ 0.95 (d,
6 H, J ) 6.8), 1.25-1.85 (m, 8 H), 1.43 (s, 9 H), 1.45 (s, 9 H),
2.12 (m, 1 H), 3.08-3.48 (m, 6 H), 3.82 (m, 1 H), 4.05 (m, 1
H), 4.20 (d, 1 H, J ) 5.7), 5.06 (s, 2 H), 5.11 (s, 2 H), 7.22-
7.42 (m, 10 H); HRMS m/z calcd for C40H61N4O10 757.4388,
(2S)-11-[(Ben zyloxyca r b on yl)a m in o]-2-[(ter t-b u t oxy-
ca r b on yl)a m in o]-7-(ca r b ob en zyloxy)-7-a za u n d eca n oic
Acid ter t-Bu tyl Ester (22). Under argon, 21 (556 mg, 1.13
mmol) was dissolved in H2O (15 mL) and diethyl ether (30 mL).
The biphasic mixture was stirred and cooled to 0 °C, at which
point KHCO3 (1.19 g, 11.9 mmol) was added. Over 20 min,
CBZ-ONSu (660.0 mg, 2.648 mmol) was added in five portions.
The mixture was allowed to warm to room temperature and
stirred for 21 h. The organic layer was separated, the aqueous
layer was washed with ether, and the combined organic layers
were dried. Concentration and purification by flash chroma-
tography (2:1 hexane/ethyl acetate) gave 22 (271 mg, 37%) as
a colorless oil: 1H NMR δ 1.20-1.80 (m, 10 H), 1.43 (s, 9 H),
1.44 (s, 9 H), 3.09 (m, 2 H), 3.18-3.32 (m, 4 H), 3.91 (m, 1 H),
5.06 (s, 2 H), 5.09 (s, 2 H), 7.26-7.37 (m, 10 H); 13C NMR
(CDCl3) δ 22.4, 25.4, 25.8, 27.2, 28.0, 28.3, 32.5, 40.7, 46.5,
47.0, 53.8, 66.6, 66.9, 79.6, 81.7, 127.9, 128.0, 128.5, 136.9,
155.4, 156.1, 156.4, 171.9; HRMS m/z calcd for C35H52N3O8
found 757.4387. [R]21 -13.9° (c 1.17, CH3OH).
D
BOC-(2S,9S)-Hp u (N7,N12-d i-CBZ, 9-[(S)-r-m et h oxy-r-
(tr iflu or om eth yl)p h en yla cetoxy])-Va l-O-t-Bu (16b). The
reaction was carried out according to the procedure described
for the synthesis of 16a , starting with 15b (18.6 mg, 24.6 µmol)
and (R)-(-)-Mosher’s acid chloride (10 µL, 54 µmol). After
workup, the residue was purified by flash chromatography
(33% ethyl acetate/hexane) to give 16b (15 mg, 63%) as a
colorless oil: 1H NMR (CDCl3, 45 °C) δ 0.90 (d, 3 H, J ) 6.8),
0.93 (d, 3 H, J ) 6.8), 1.20-1.90 (m, 8 H), 1.44 (s, 9 H), 1.45
(s, 9 H), 2.14 (m, 1 H), 3.02 (m, 2 H), 3.14-3.58 (m, 4 H), 3.44
(s, 3 H), 4.02 (m, 1 H), 4.38 (dd, 1 H, J ) 8.8, 4.6), 4.80-5.20
(m, 2 H), 5.09 (s, 2 H), 5.09 (s, 2 H), 6.52 (m, 1 H), 7.14-7.54
(m, 15 H); 19F NMR (282 MHz, CDCl3, CFCl3 as internal
standard, 45 °C) δ -71.40; HRMS m/z calcd for C50H68F3N4O12
642.3754, found 642.3746. [R]23 ) -9.3° (c 1.01, CH3OH).
D
2(S)-Am in o-11-[(ben zyloxyca r bon yl)a m in o]-7-(ca r bo-
ben zyloxy)-7-a za u n d eca n oic Acid (23). Under argon, 22
(271 mg, 0.422 mmol) was added to a mixture of trifluoroacetic
acid (770 mg, 6.7 mmol), CH2Cl2 (3.0 mL), and triethylsilane
(330 mg, 2.8 mmol); this was stirred at room temperature for
23 h. The reaction mixture was concentrated; the resultant
oil was dissolved in water (5.0 mL) and adjusted to pH 7 with
saturated NaHCO3 solution. This solution was then concen-
trated; the residue was purified by chromatography on a C18
column (30% acetone/water, followed by 55% acetone/water)
to give 23 (137 mg, 67%) as a colorless oil: 1H NMR (19.7 °C)
δ 1.30-1.66 (m, 8 H), 1.78 (m, 2 H), 3.09 (m, 2 H), 3.20-3.33
(m, 4 H), 3.45 (m, 1 H), 5.05 (s, 2 H), 5.09 (s, 2 H), 7.26-7.37
(m, 10 H); HRMS m/z calcd for C26H36N3O6 486.2604, found
486.2614.
973.4786, found 973.4739. [R]23 -21.6° (c 0.50, CHCl3).
D
(2S,9S)-Hyp u sin yl-L-va lin e (17b). An aliquot of 15b (7.3
mg, 9.6 µmol), phenol (250 mg), and pentamethylbenzene (250
mg) were dissolved in degassed TFA (5.0 mL) at 0 °C.
Triisopropylsilane (0.1 mL) and a saturated solution of HBr
in acetic acid (0.2 mL) were added under an argon atmosphere.
The solution was stirred at room temperature for 1 h and
concentrated under reduced pressure. The residue was dis-
solved in 10% acetic acid (10 mL) and extracted with methyl
tert-butyl ether (3 × 25 mL). The aqueous layer was concen-
trated and dried in vacuo to give 17b (6.0 mg) as a colorless
oil: 1H NMR δ 0.96 (d, 3 H, J ) 6.8), 0.97 (d, 3 H, J ) 6.8),
1.47 (m, 2 H), 1.68-2.01 (m, 6 H), 2.20 (m, 1 H), 2.99-3.25
(m, 6 H), 4.06 (tt, 1 H, J ) 9.7, 3.1), 4.11 (t, 1 H, J ) 6.4), 4.26
(d, 1 H, J ) 5.7); HRMS m/z calcd for C15H33N4O4 333.2502,
found 333.2499.
11-[(Ben zyloxyca r b on yl)a m in o]-7-(ca r b ob en zyloxy)-
2(S)-[(9-flu or en ylm et h oxyca r b on yl)a m in o]-7-a za u n d e-
ca n oic Acid (24). A solution of 9-fluorenylmethyl N-succin-
imidyl carbonate (143 mg, 0.42 mmol) in DMF (1.60 mL) was
added to a solution of 23 (135 mg, 0.278 mmol) in 9% Na2CO3
(655.9 mg, 0.557 mmol) at 0 °C and stirred at room temper-
ature for 21 h under argon. The pH was adjusted to 7.0 with
0.1 N HCl. The mixture was concentrated to an oil and
purified by flash chromatography (CHCl3, then 95% CHCl3/
MeOH) to give 24 (115 mg, 58%) as a colorless oil: 1H NMR
(19.2 °C) δ 1.22-1.92 (m, 10 H), 3.08 (m, 2 H), 3.16-3.30 (m,
4 H), 4.12 (m, 1 H), 4.21 (t, 1 H, J ) 6.9), 4.30-4.45 (m, 2 H),
5.04 (s, 2 H), 5.08 (s, 2 H), 7.22-7.42 (m, 14 H), 7.66 (m, 2 H),
7.78 (d, 2 H, J ) 7.5); HRMS m/z calcd for C41H46N3O8
3-Cya n op r op a n a l (19).30 A mixture of water (50 mL) and
3-cyanopropionaldehyde diethyl acetal (10.0 g, 63.6 mmol) was
refluxed for 4 h under a N2 atmosphere and then distilled at
ambient pressure to remove water and ethanol. Glassware
utilized for the reflux and initial distillation was acid-washed
immediately prior to use by submerging in 3 N HCl for 15 min
and then rinsing twice with water. The remaining oil was
transferred to a dry, short-path distillation apparatus and
distilled under reduced pressure to give 19 (4.15 g, 78%): bp
708.3285, found 708.3284. [R]22 ) +9.3° (c 1.00, CHCl3).
1
80-82 °C (1.3 mmHg) [lit.40 bp 85.1-85.5 °C (3 mmHg)]; H
D
NMR (CDCl3) 2.63 (t, 2 H, J ) 7.05), 2.91 (t, 2 H, J ) 7.05),
(S)-Deoxyh yp u sin e Dih yd r och lor id e (25). Reagent 24
(53 mg, 75 µmol) was deprotected according to the method
described for the preparation of 17a . The crude product was
purified by flash chromatography (1:2:1 CH2Cl2/MeOH/NH3-
(aq), Kieselgel 60), concentrated, dissolved in 1 mL of H2O,
and adjusted to pH ) 4.5 with 0.1 N HCl. Concentration and
recrystallization from 1:9:15 H2O/MeOH/Et2O gave 25 as the
dihydrochloride salt (6.6 mg, 30%): 1H NMR (D2O) δ 1.53 (m,
2 H), 1.76 (m, 6 H), 1.97 (m, 2 H), 3.01-3.15 (m, 6 H), 3.98 (t,
1 H, J ) 6.3); HRMS m/z calcd for C10H24N3O2 218.1868, found
9.80 (s, 1 H).
(2S)-11-Am in o-2-[(ter t-b u t oxyca r b on yl)a m in o]-7-a za -
u n d eca n oic Acid ter t-Bu tyl Ester , Dia ceta te Sa lt (21). A
solution of 2 (1.00 g, 2.95 mmol) in chloroform (30 mL) was
extracted with saturated NaHCO3 (2 × 30 mL) and then water
(30 mL). The organic layer was dried, evaporated, and dried
overnight in vacuo to give the free amine of 2 (749 mg, 84%).
This oil was dissolved in benzene (100 mL) containing 19
(230.5 mg, 2.77 mmol) and activated 3-Å molecular sieves
(20.30 g). The reaction mixture was allowed to stir under
argon for 4.5 h, yielding the imine of 20. The solution was
filtered and concentrated in vacuo. Under argon, dry THF
(100 mL) and PtO2 (150 mg) were added; this was stirred under
218.1900. [R]21 ) +17.0° (c 0.44, 6 N HCl).
D
Cys-Th r -Gly-d eoxyh yp u sin e-His-Gly (27). The polymer-
bound peptide 26 (87.4 mg), phenol (250 mg), and penta-
methylbenzene (250 mg) were dissolved in degassed TFA (5.0

Construction of Hypusine and Deoxyhypusine Peptides
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20 3899
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J aksche, H.; Wolff, B.; Aschauer, H.; Farrington, G. K.; Probst,
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Cellular Target of the Human Immunodeficiency Virus Type 1
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Subcellular Distribution of Eukaryotic Translation Initiation
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Detection of a Subset of Polysomal mRNAs Associated with
Modulation of Hypusine Formation at the G1-S Boundary -
Proposal of a Role for eIF-5A in Onset of DNA Replication. FEBS
Lett. 1995, 366, 92-98.
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Hershey, J . W. B. Translation Initiation Factor 5A and Its
Hypusine Modification Are Essential for Cell Viability in the
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5A from HeLa Cells. Biochemistry 1995, 34, 14693-14702.
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Cytoplasm. Cell 1995, 82, 341-344.
(20) Bevec, D.; J acksche, H.; Oft, M.; Wo¨hl, T.; Himmelspach, M.;
Pacher, A.; Schebesta, M.; Koettnitz, K.; Dobrovnik, M.; Csonga,
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mL) at 0 °C. Triisopropylsilane (100 µL), 1,2-ethanedithiol
(100 µL), and saturated HBr in acetic acid solution (200 µL)
were added under an argon atmosphere. The solution was
stirred at room temperature for 1 h and concentrated under
reduced pressure. The residue was dissolved in 10% acetic
acid (10 mL) and extracted with methyl tert-butyl ether (3 ×
25 mL). The aqueous layer was concentrated in vacuo, and
the residue was purified on a preparative HPLC (solvent
systems A, aqueous 0.1% TFA; and B, 0.1% TFA in CH3CN;
linear gradient of 0-20% B in 85 min; flow rate 12 mL/min;
detection at 214 nm; retention time ) 13.7 min) using a C18
reverse-phase column (Vydac Protein & Peptide C18) to give
27 as a colorless oil (8.0 mg, 28%, calculated as tetrakis-
trifluoroacetate salt): 1H NMR (600 MHz, 6.0 °C) δ 1.26 (d, 3
H, J ) 6.2), 1.38 (m, 2 H), 1.63-1.81 (m, 8 H), 2.98-3.11 (m,
7 H), 3.15 (dd, 1 H, J ) 14.9, 5.6), 3.19 (dd, 1 H, J ) 15.4, 8.1),
3.31 (dd, 1 H, J ) 15.4, 6.2), 3.94-4.06 (m, 4 H), 4.23 (m, 1
H), 4.30 (m, 1 H), 4.35 (m, 1 H), 4.42 (d, 1 H, J ) 4.8), 4.77 (m,
1 H), 7.32 (s, 1 H), 8.64 (s, 1 H). Amino acid analysis: Thr
0.65, Gly 2.34, His 1.02.
Ack n ow led gm en t. The authors appreciate the gen-
erosity of Dr. J ohn Hershey of the University of Cali-
fornia, Davis, for the plasmid containing the eIF-5A
cDNA and the assistance of Dr. J enq-Kuen Huang in
expression and isolation of recombinant human eIF-5A
from this source. We also thank Dr. Eileen E. Hughes
for her editorial comments and Brian Raisler for his
technical support. The authors recognize A. Y. K. Chung
of the Department of Biochemistry and Protein Core,
Interdisciplinary Center for Biotechnology Research
(ICBR), University of Florida, for performing the SPPS
and HPLC purifications; H. D. Plant and J . R. Rocca of
the Center for Structural Biology - Brain Institute,
University of Florida, for high-field and 2D NMR
spectral analyses; and L. Green and D. Duke of the
Hybridoma Core Facility, ICBR, University of Florida,
for generation of monoclonal antibodies to the hypusine-
containing peptide.
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