Development of a Hypusine Reagent for Peptide Synthesis

Article abstract of DOI:10.1021/jo970119z

The synthesis of a reagent that enables the incorporation of the unusual amino acid (2S,9R)-hypusine (Hpu) into peptide sequences is described.The reagent, (2S,9R)-11-<(benzyloxycarbonyl)amino>-7-(carbobenzyloxy)-2-<(9-fluorenylmethoxycarbonyl)amino>-9-(tetrahydropyran-2-yloxy)-7-azaundecanoic acid, is utilized in the synthesis of a hexapeptide containing the primary pentapeptide sequence of the eukaryotic initiation factor eIF-5A, L-Cys-L-Thr-Gly-Hpu-L-His-Gly.The reagent is shown to be effective for both solution phase and Merrifield resin synthesis.

Full text of DOI:10.1021/jo970119z

J . Org. Chem. 1997, 62, 3285-3290
3285
Develop m en t of a Hyp u sin e Rea gen t for P ep tid e Syn th esis
Raymond J . Bergeron,*,† Christian Ludin,‡ Ralf Mu¨ller,† Richard E. Smith,† and
Otto Phanstiel, IV§
Department of Medicinal Chemistry, University of Florida, Gainesville, Florida 32610-0485,
Institut fu¨r Organische Chemie, Freiest. 3, Bern, CH-3012, Switzerland, and Chemistry Department,
University of Central Florida, Orlando, Florida 32816-2366
Received J anuary 22, 1997X
The synthesis of a reagent that enables the incorporation of the unusual amino acid (2S,9R)-
hypusine (Hpu) into peptide sequences is described. The reagent, (2S,9R)-11-[(benzyloxycarbonyl)-
amino]-7-(carbobenzyloxy)-2-[(9-fluorenylmethoxycarbonyl)amino]-9-(tetrahydropyran-2-yloxy)-7-
azaundecanoic acid, is utilized in the synthesis of a hexapeptide containing the primary pentapeptide
sequence of the eukaryotic initiation factor eIF-5A, L-Cys-L-Th r -Gly-Hp u -L-His-Gly. The reagent
is shown to be effective for both solution phase and Merrifield resin synthesis.
In tr od u ction
a point of intervention in anti-HIV therapeutic strategies.7a
The concept, at least, is simple: prevent Rev-eIF-5A bind-
ing. Thus, methodologies that would allow the assembly
of hypusine-containing peptide fragments would be valu-
able in both unraveling the role of hypusine in Rev-eIF-
5A complex formation and for the assembly of potential
inhibitors.
(+)-Hypusine, 1, an unusual naturally occurring amino
acid, was first isolated from bovine brain extracts by T.
Shiba et al. in 1971.1 Since its discovery, the post-
translational formation of hypusine has been shown to
occur on a precursor protein of the eukaryotic initiation
factor 5A (eIF-5A; formerly called eIF-4D).2,3 This initia-
tion factor 5A is unique in that it is the only known
cellular protein that contains the amino acid hypusine
(Hpu). In the mid-1970s, eIF-5A was shown to stimulate
ribosomal subunit joining and to enhance 80 S-bound
Met-t-RNAi reactivity with puromycin.4,5 Later, in 1983,
Folk and co-workers suggested that a hypusine-modified
protein serves as an important initiation factor in all
growing eukaryotic cells.2 In 1986, Park et al. isolated
the eIF-5A protein from human red blood cells and
elucidated the amino acid sequence surrounding the
single hypusine residue as Thr-Gly-Hp u -His-Gly-His-
Ala-Lys.6
Furthermore, and most interesting, eIF-5A has been
shown to play a critical role in the replication of human
immunodeficiency virus-1 (HIV-1). The presence of Rev,
an active gene product of the virus, is required for HIV-1
genome transcription events, nuclear export of HIV-1
mRNA, and subsequent translation. This phosphopro-
tein consists of 116 amino acid residues and binds to a
fragment of viral mRNA located on the Stem-Loop IIB,
called the Rev response element (RRE). However, Rev
is not active in the absence of the hypusinated protein
eIF-5A.7a In fact, antisense and missense DNA experi-
ments have revealed that in the absence of eIF-5A there
is no viral replication.7a,b Because of these observations,
there is considerable interest in Rev-eIF-5A binding as
We previously described a flexible synthetic approach
to hypusine itself.8 The key step in this method involved
the Nꢀ-alkylation of Nꢀ-benzyl-NR-(carbobenzyloxy)-L-
lysine benzyl ester with (R)- or (S)-epichlorohydrin to give
the respective (2S,9R)- and (2S,9S)-chlorohydrins. Sub-
sequent displacement of the respective chlorides by
cyanide ion provided the protected hypusine skeletons.
The final step, hydrogenation over PtO2 in acetic acid
followed by neutralization and reacidification, yielded the
respective (2S,9S)- or (2S,9R)-hypusine dihydrochlorides
in excess of 50% overall yield. A comparison of the
reported hypusine optical rotation with that of our
synthetic (2S,9R)-hypusine, 1, confirmed the stereochem-
ical integrity of both chiral centers throughout the
synthesis.
Having developed synthetic methodology for accessing
hypusine itself, we now focus on developing a selectively
protected hypusine reagent that can be used to incorpo-
rate this unusual amino acid into peptides. The reagent
is designed to be utilized both in solution and Merrifield
resin-based synthesis.
Resu lts a n d Discu ssion
Inspection of hypusine (1) (Figure 1) reveals five
potentially reactive centers: two primary amino groups,
a secondary amino group, a secondary hydroxyl, and a
carboxyl group. In eIF-5A, the R-amino nitrogen and the
carboxyl group are coupled to other amino acids. We
envisioned the target hypusine reagent with all three
nitrogens (N2, N7, and N12) protected while the carboxyl
group remained free for peptide coupling. Additionally,
the R-amino nitrogen (N2) required a protecting group
orthogonal to those masking the other two potentially
* To whom correspondence should be addressed.
† University of Florida.
‡ Institut fu¨r Organische Chemie.
§ University of Central Florida.
X Abstract published in Advance ACS Abstracts, April 15, 1997.
(1) Shiba, T.; Mizote, H.; Kaneko, T.; Nakajima, T.; Kakimoto, Y.;
Sano, I. Biochim. Biophys. Acta 1971, 244, 523-531.
(2) Cooper, H. L.; Park, M. H.; Folk, J . E.; Safer, B.; Braverman, R.
Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 1854-1857.
(3) Safer, B. Eur. J . Biochem. 1989, 186, 1-3.
(4) Anderson, W. F.; Bosch, L.; Cohn, W. E.; Lodish, H.; Merrick,
W. C.; Weissbach, H.; Wittmann, H. G.; Wool, I. G. FEBS Lett. 1977,
76, 1-10.
(7) (a) Ruhl, M.; Himmelspach, M.; Bahr, G. M.; Hammerschmid,
F.; J aksche, H.; Wolff, B.; Aschauer, H.; Farrington, G. K.; Probst, H.;
Bevec, D.; Hauber, J . J . Cell Biol. 1993, 123, 1309-1320 and references
cited therein. (b) Bevec, D.; J aksche, H.; Oft, M.; Wo¨hl, T.; Himmel-
spach, M.; Pacher, A.; Schebesta, M.; Koettnitz, K.; Dobrovnik, M.;
Csonga, R.; Lottspeich, F.; Hauber, J . Science 1996, 271, 1858-1860.
(8) Bergeron, R. J .; Xia, M. X. B.; Phanstiel IV, O. J . Org. Chem.
1993, 58, 6804-6806.
(5) Kemper, W. M.; Berry, K. W.; Merrick, W. C. J . Biol. Chem. 1976,
251, 5551-5557.
(6) Park, M. H.; Liu, T.-Y.; Neece, S. H.; Swiggard, W. J . J . Biol.
Chem. 1986, 261, 14515-14519.
S0022-3263(97)00119-9 CCC: $14.00 © 1997 American Chemical Society

3286 J . Org. Chem., Vol. 62, No. 10, 1997
Bergeron et al.
Sch em e 1.a Syn th esis of th e Hyp u sin e Rea gen t
F igu r e 1. (2S,9R)-Hypusine, 1.
reactive amines (N7 and N12). Therefore, the N2 nitro-
gen was protected as the FMOC derivative, while the N7
and N12 amines were protected as carbobenzyloxy (CBZ)
moieties. The 9-hydroxyl was masked as a tetrahydro-
pyranyl ether (THP). This protection was necessary as
the poorly reactive secondary hydroxyl was still expected
to cause difficulty with the anticipated N-acylating agents
used in solid phase synthesis.9
Syn th esis of th e Hyp u sin e Rea gen t. As shown in
Scheme 1, the synthesis began with the tert-butoxycar-
bonylation of Nꢀ-CBZ-L-lysine t-Bu ester and gave 2 in
98% yield.10 We had initially prepared 2 by esterification
of Nꢀ-CBZ-NR-BOC-L-lysine with tert-butyl alcohol, 1,3-
dicyclohexylcarbodiimide, and 4-(dimethylamino)pyri-
dine,11,12 but this resulted in partial racemization as
suggested by comparison of the optical rotations of
samples of 2 prepared by the two methods. This was
confirmed by analysis of the 1H NMR spectra of two
samples of L-lysyl-L-valine that were independently
prepared from similarly protected lysines after each had
been subjected to one of the above methods; 10% of the
D-lysyl-L-valine diastereomer was observed in the dipep-
tide sample obtained from the esterification method while
the sample that had been subjected to the tert-butoxy-
carbonylation method proved to be free of racemized
compound (data not shown).
Having solved the initial racemization problem, the Nꢀ-
CBZ group of 2 (Scheme 1) was removed by hydrogena-
tion over 10% Pd-C in ethanol and aqueous HCl to give
3 in 99% yield.15 Conversion of 3 to the Nꢀ-benzyl-NR-
BOC-L-lysine t-Bu ester 4 was accomplished by reductive
amination of the liberated Nꢀ amine with benzaldehyde
and sodium cyanoborohydride.16
Our earlier synthesis of (+)-hypusine8 developed a
chiral 4-amino-2-hydroxybutane synthon for accessing
the parent molecule 1 from an L-lysine derivative. In
particular, this fragment made it possible to elaborate
the Nꢀ benzyl group of a protected L-lysine into the N7-
N12 structure of (+)-hypusine. In this report we further
exploit this concept. As shown in Scheme 1, the subse-
quent Nꢀ-alkylation of 4 with (S)-epichlorohydrin at rt
gave the (2S,9S)-chlorohydrin 5 (77%). Displacement of
the chloride in 5 by cyanide ion afforded the protected
(2S,9R)-hypusine skeleton 6 in 70% yield. Simultaneous
debenzylation at N7 and saturation of the terminal nitrile
in 6 was accomplished by hydrogenation using a mixture
a
(a) (BOC)2O, NaHCO3; (b) H2, Pd-C; (c) PhCHO, NaBH3CN;
(d) (S)-(+)-epichlorohydrin, MgSO4; (e) KCN; (f) H2, Pd-C, PtO2;
(g) CBZ-Cl, DIEA; (h) TFA, triethylsilane, CH2Cl2; (i) 3,4-dihydro-
2H-pyran; (j) FMOC-ONSu, Na2CO3.
of 10% Pd-C and PtO2 to give the amino alcohol 7 as a
diacetate (99%). Protection of the amino functions of 7
at N7 and N12 using CBZ-groups provided 8 in 55% yield.
Selective removal of the tert-butyl ester and NR-BOC
protecting groups was accomplished with TFA and tri-
ethylsilane13 to give the di-CBZ-derivative 9 (78%). The
secondary 9-hydroxyl function was protected as tetrahy-
dropyranyl ether17 10 (68%), and subsequent protection
of the remaining NR-amine function with 9-fluorenyl-
methyl N-succinimidyl carbonate (FMOC-ONSu) gave
the hypusine reagent 11 with the desired protecting
groups in 83% yield.18
(9) Stewart, S. M. In The Peptides; Gross, E., Meienhofer, J ., Eds.;
Academic Press: New York, 1981; Vol. 3, p 169.
(10) Tarbell, D. S.; Yamamoto, Y.; Pope, B. M. Proc. Natl. Acad. Sci.
U.S.A. 1972, 69, 730-732.
(11) Pufahl, R. A.; Nanjappan, P. G.; Woodard, R. W.; Marletta, M.
A. Biochemistry 1992, 31, 6822-6828.
(12) Csanady, G.; Medzihradszky, K. Org. Prep. Proced. Int. 1988,
20, 180-184.
Syn t h esis of a H yp u sin e-Con t a in in g P ep t id e.
While the hypusine reagent 11 (Scheme 1) allows for the
(13) Mehta, A.; J aouhari, R.; Benson, T. J .; Douglas, K. T. Tetra-
hedron Lett. 1992, 33, 5441-5444.
(14) Wang, S. S.; Wang, B. S. H.; Hughes, J . L.; Leopold, E. J .; Wu,
C. R.; Tam, J . P. Int. J . Pept. Protein Res. 1992, 40, 344-349.
(15) Bergmann, M.; Zervas, L. Ber. Dtsch. Chem. Ges. 1932, 65,
1192-1201.
(16) Borch, R. F.; Bernstein, M. D.; Durst, H. D. J . Am. Chem. Soc.
1971, 93, 2897-2904.
(17) Bernady, K. F.; Floyd, M. B.; Poletto, J . F.; Weiss, M. J . J . Org.
Chem. 1979, 44, 1438-1447.
(18) Lapatsanis, L.; Milias, G.; Froussios, K.; Kolovos, M. Synthesis
1983, 671-673.

Development of a Hypusine Reagent for Peptide Synthesis
J . Org. Chem., Vol. 62, No. 10, 1997 3287
Sch em e 2.a Hexa p ep tid e Syn th esis
F igu r e 2. Hypusine-containing hexapeptide 12.
assembly of a variety of hypusine-containing peptides,
our target was the hypusine-containing pentapeptide
found in eIF-5A capped at its N-terminus with L-cysteine,
i.e., Cys-Th r -Gly-Hp u -His-Gly (12, Figure 2).6 The
L-cysteine, which is not contained in the natural peptide,
was added to the sequence as a means of covalently
linking this peptidic hapten to a carrier protein for
ultimate use in raising antibodies specific to hypusine-
containing epitopes.19 The solution synthesis, a conver-
gent approach, was carried out by elaborating from the
C to the N terminus, as shown in Scheme 2, and involved
the coupling of three appropriately-protected pieces: Cys-
Thr-Gly, Hpu, and His-Gly.
After several failed attempts with THP and silyl esters,
the carboxamidomethyl (CAM) ester developed by Mar-
tinez20 was employed as a carboxyl protecting group in
generating the Cys-Thr-Gly fragment. This protecting
group is orthogonal to the BOC, CBZ, and FMOC groups.
Thus, the synthesis of the masked Cys-Thr-Gly fragment,
16, began with the N-BOC-Gly-CAM ester 13.20a Re-
moval of the BOC group with trifluoroacetic acid (TFA)
gave the amine salt (60%), which was immediately
coupled with NR-FMOC-L-threonine tert-butyl ether to
give the dipeptide 14 in 85% yield. Treatment of 14 with
10% diethylamine (DEA) in DMF followed by coupling
with N,S-di-CBZ-L-cysteine using BOP and DIEA af-
forded the tripeptide 15 (50%). Removal of the CAM
ester with aqueous Na2CO3 followed by acidification with
aqueous citric acid generated the Cys-Thr-Gly building
block 16 in 77% yield.
The design of the His-Gly fragment, 18, was predicated
on obtaining efficient coupling between the NR-His group
and reagent 11. Previous work by Yamashiro21 suggested
that tert-butoxycarbonylation of the imidazole side chain
prior to the condensation step substantially increases
coupling yields. For this reason the His-Gly fragment,
18, was assembled in two steps. First, the condensation
of glycine tert-butyl ester and NR-CBZ-L-histidine with
diphenylphosphoryl azide (DPPA),22 afforded 17 (72%).
In the second step, the His side chain was masked by
treatment with di-tert-butyl dicarbonate in THF to give
the dipeptide 18 in 78% yield.
a
(a) TFA; (b) FMOC-Thr(O-t-Bu)-OH, BOP, DIEA; (c) 10% DEA
in DMF; (d) N,S-di-CBZ-cysteine, BOP, DIEA; (e) Na2CO3; (f) citric
acid; (g) DPPA, TEA; (h) (BOC)2O; (i) H2, 10% Pd-C, EtOH, HCl;
(j) Hpu reagent 11, BOP, DIEA; (k) 4-AMP in CHCl3; (l) BOP,
DIEA; (m) TFA, phenol.
with hypusine reagent 11 to give the protected Hpu-His-
Gly tripeptide 19 in 85% yield. The amine generated by
treatment of 19 with 4-(aminomethyl)piperidine23 (68%)
was acylated by the tripeptide acid 16 with BOP to give
the masked conjugate 20 in 81% yield. The final depro-
tection of 20 with TFA and phenol gave the hexapeptide
Cys-Thr-Gly-Hp u -His-Gly as its trifluoroacetic acid salt
12 in 24% yield. The peptide was fully assigned by
DQCOSY, TOCSY, and HMQC NMR (data not shown).
Polymer-bound synthesis of the hexapeptide 12 was
performed on a p-(hydroxymethyl)phenoxymethyl poly-
styrene resin (Wang-resin) using an Applied Biosystems
432A Synthesizer and FMOC chemistry with HBTU as
As shown in Scheme 2, the final hexapeptide 12 was
constructed stepwise from the fragments 11, 16, and 18.
Hydrogenolysis of the NR-CBZ-group of 18 provided the
amine hydrochloride salt (68%), which was condensed
(19) Hashida, S.; Imagawa, M.; Inoue, S.; Ruan, K.-H.; Ishikawa,
E. J . Appl. Biochem. 1984, 6, 56-63.
(20) (a) Martinez, J .; Laur, J .; Castro, B. Tetrahedron 1985, 41, 739-
743. (b) Martinez, J .; Laur, J .; Castro, B. Tetrahedron Lett. 1983, 24,
5219-5222.
(21) Yamashiro, D.; Blake, J .; Li, C. H. J . Am. Chem. Soc. 1972, 94,
2855-2859.
(22) Shioiri, T.; Ninomiya, K.; Yamada, S. J . Am. Chem. Soc. 1972,
94, 6203-6205.
(23) Beyermann, M.; Bienert, M.; Niedrich, H.; Carpino, L. A.; Sadat-
Aalaee, D. J . Org. Chem. 1990, 55, 721-728.

3288 J . Org. Chem., Vol. 62, No. 10, 1997
Bergeron et al.
Sch em e 3.a Dep r otection of th e SP P S
Hexa p ep tid e 21
rected. Samples for amino acid analysis were either hydro-
lyzed with 6 N HCl or with 12 N HCl plus propionic acid for
resin samples and then run on an Applied Biosystems Amino
Acid Analyzer using precolumn PTC-derivatization.
Nr-BOC-NE-CBZ-L-Lysin e ter t-Bu tyl Ester (2). Sodium
hydrogencarbonate (2.81 g, 33.47 mmol) in water (75 mL) was
added to H-Lys(CBZ)-O-t-Bu hydrochloride (12.00 g, 32.18
mmol) in chloroform (100 mL), and the mixture was stirred
at rt for 5 min under an N2 atmosphere. Di-tert-butyl dicar-
bonate (7.02 g, 32.18 mmol) in chloroform (50 mL) was added,
the mixture refluxed for 1.5 h and allowed to cool to rt. The
layers were separated, the aqueous layer was extracted with
chloroform (3 × 100 mL), and the combined organic layers were
dried over magnesium sulfate. Concentration in vacuo fol-
lowed by flash chromatography (3:1 hexane:ethyl acetate) gave
2 (13.82 g, 98%) as a colorless oil. 1H NMR (CDCl3) δ 7.30 (s,
5 H), 5.10 (s, 2 H), 4.82 (m, 1 H), 4.18 (m, 1 H), 3.20 (m, 2 H),
1.90-1.30 (m, 6 H), 1.48 (s, 9 H), 1.46 (s, 9 H). 13C NMR (CD3-
OD) δ 173.8, 158.8, 158.1, 138.4, 129.4, 128.9, 128.7, 82.5, 80.4,
67.3, 55.7, 41.4, 32.4, 30.4, 28.7, 28.3, 24.0. HRMS m/ z calcd
for C23H37N2O6 437.2652, found 437.2643. Anal. Calcd for
C23H36N2O6: C, 63.28; H, 8.31; N, 6.42. Found: C, 63.13; H,
a
(a) HBr/acetic acid in TFA, phenol, pentamethylbenzene,
triisopropylsilane, 1,2-ethanedithiol.
an activating agent.24 The cysteine and histidine resi-
dues of the hexapeptide were initially protected with
trityl groups, while the threonine was protected as its
tert-butyl ether. Attempts to release the free hexapeptide
by refluxing in TFA/phenol or with HBr/acetic acid in
TFA with phenol and pentamethylbenzene, respectively,
did not succeed. Use of the more labile 4-methoxytrityl-
(Mmt)25 and 4-methyltrityl-groups (Mtt)26 to protect the
cysteine and histidine derivatives, respectively, produced
the protected hexapeptide 21 (Scheme 3). Final depro-
tection of 21 was then achieved with HBr/acetic acid in
TFA and a “cocktail” of carbocation scavengers (phenol,
pentamethylbenzene, triisopropylsilane, 1,2-ethanedithi-
ol) at rt.14 This method generated very few side products
and provided 12, after purification by HPLC, in 24%
yield. All analytical data were consistent with hexapep-
tide 12 previously prepared by the solution-phase method.
In summary, the hypusine reagent described has been
demonstrated to be a highly useful synthon in accessing
the eIF-5A pentapeptide sequence. While the yields are
generally excellent for these kinds of systems, the most
notable feature is the flexibility that this methodology
offers in synthesizing related eIF-5A mimics. We have
now initiated the development of an antibody for the
pentapeptide-protein coupled system and begun the
synthesis of eIF-5A mimics.
8.28; N, 6.47. [R]27 +5.0° (c 2.00, CHCl3).
D
Nr-BOC-L-Lysin e ter t-Bu tyl Ester Hyd r och lor id e (3).
To a solution of 2 (34.51 g, 79.15 mmol) in 300 mL of ethanol
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 concentrated
and the residue dried in vacuo to give 3 as its hydrochloride
salt (26.59 g, 99%). 1H NMR (CD3OD) δ 3.95 (dd, 1 H, J )
8.8, 5.0 Hz), 2.93 (t, 2 H, J ) 7.7 Hz), 1.84-1.60 (m, 6 H), 1.45
(s, 9 H), 1.43 (s, 9 H). 13C NMR (CD3OD) δ 173.5, 158.2, 82.7,
80.5, 79.5, 55.5, 40.6, 32.1, 28.7, 28.3, 23.9. HRMS m/ z calcd
for C15H31N2O4 303.2284, found 303.2272. [R]26D -10.1° (c 1.00,
CH3OH).
NE-Ben zyl-Nr-BOC-L-Lysin e ter t-Bu tyl Ester (4).
A
solution of 3 (25.97 g, 76.64 mmol) in CHCl3 (300 mL) was
washed with saturated Na2CO3 solution (2 × 100 mL). The
organic layer was dried (MgSO4), filtered, and concentrated.
The resultant oil was combined with benzaldehyde (10.42 g,
98.13 mmol), ethanol (150 mL), and activated 3 Å molecular
sieves (46.0 g) and stirred under N2 for 6 h. Sodium cy-
anoborohydride (2.41 g, 38.4 mmol) was added and the mixture
stirred overnight at rt. The mixture was filtered, and the
filtrate was acidified to pH 2 with 1 N HCl (110 mL). The
solution was concentrated to dryness, dissolved in CHCl3, and
washed with saturated Na2CO3 solution and water. The
organic layer was dried (MgSO4) and concentrated. Flash
column chromatography (10% ethanol/CHCl3) afforded 4 (16.16
g, 54%) as a colorless oil. 1H NMR (CD3OD) δ 7.34-7.20 (m,
5 H), 3.91 (dd, 1 H, J ) 9.0, 5.1 Hz), 3.72 (s, 2 H), 2.58 (t, 2 H,
J ) 7.2 Hz), 1.82-1.30 (m, 6 H), 1.45 (s, 9 H), 1.43 (s, 9 H).
13C NMR (CDCl3) δ 171.9, 155.3, 140.1, 128.3, 128.1, 126.9,
81.6, 79.5, 53.9, 48.9, 32.7, 29.5, 28.3, 27.9, 22.9. HRMS m/ z
calcd for C22H36N2O4 392.2675, found 392.2676. Anal. Calcd
for C22H36N2O4: C, 67.32; H, 9.24; N, 7.14. Found: C, 67.40;
Exp er im en ta l Section
Gen er a l. Reagents were purchased from the Aldrich,
Fluka, or Sigma Chemical Co. and were 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 anhydrous sodium sulfate. THF was distilled from
sodium metal and benzophenone. Acetonitrile was distilled
from NaH. Silica gel 32-60 (40 µm “flash”) from Selecto, Inc.
(Kennesaw, GA) was used for column chromatography. 1H
NMR spectra were recorded at 300 MHz and 13C NMR spectra
at 75 MHz unless otherwise specified. Chemical shifts are
given in parts per million downfield from an internal tetra-
methylsilane or 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid, so-
dium salt, standard. Elemental analyses were performed by
Atlantic Microlabs, Norcross, GA. Melting points are uncor-
H, 9.28; N, 7.16. [R]25 +6.9° (c 1.00, CDCl3).
D
(2S,9S)-7-Ben zyl-2-[(ter t-b u 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 t yl E st er
(5). A mixture of 4 (16.0 g, 40.76 mmol), (S)-(+)-epichlorohy-
drin (4.17 g, 45.0 mmol), and anhydrous MgSO4 (5.33 g, 44.28
mmol) in CH3OH (40 mL) was stirred under N2 for three days.
The solids were filtered off and washed with CH3OH. The
filtrate was concentrated at rt to give a yellow oil which was
purified by flash chromatography on silica gel (66% hexane/
1
ethyl acetate) to provide 5 (13.23 g, 77%) as a colorless oil. H
NMR (C6D6) δ 7.18 (m, 5 H), 5.00 (br d, 1 H), 4.40 (m, 1 H),
3.62 (m, 1 H), 3.40-3.10 (m, 4 H), 2.20-2.00 (m, 4 H), 1.63
(m, 1 H), 1.40 (s, 9 H), 1.31 (s, 9 H), 1.20 (m, 2 H). 13C NMR
(C6D6) δ 171.7, 155.2, 138.8, 128.8, 127.9, 127.0, 80.8, 78.8,
67.7, 58.8, 57.2, 53.9, 53.7, 47.3, 32.5, 28.0, 27.5, 26.3, 22.8.
HRMS m/ z calcd for C25H42ClN2O5 485.2782, found 485.2775.
(24) (a) Fields, C. G.; Lloyd, D. H.; Macdonald, R. L.; Otteson, K.
M.; Noble, R. L. Pept. Res. 1991, 4, 95-101. (b) Knorr, R.; Trzeciak,
A.; Bannwarth, W.; Gillessen, D. Tetrahedron Lett. 1989, 30, 1927-
1930.
(25) Barlos, K.; Gatos, D.; Hatzi, O.; Koch, N.; Koutsogianni, S. Int.
J . Pept. Protein Res. 1996, 47, 148-153.
(26) Barlos, K.; Chatzi, O.; Gatos, D.; Stavropoulos, G.; Tsegenidis,
T. Tetrahedron Lett. 1991, 32, 475-478.
[R]25 +5.3° (c 1.00, CHCl3).
D
(2S,9R)-7-Ben zyl-2-[(ter t-b u t oxyca r b on yl)a m in o]-10-
cya n o-9-h yd r oxy-7-a za d eca n oic Acid ter t-Bu tyl Ester (6).

Development of a Hypusine Reagent for Peptide Synthesis
J . Org. Chem., Vol. 62, No. 10, 1997 3289
A mixture of 5 (6.99 g, 14.4 mmol), dry KCN (9.38 g, 144
mmol), and 18-crown-6 (0.76 g, 2.88 mmol) in dry acetonitrile
(275 mL) was stirred at 45 °C for 5 days (it should be noted
that heating to reflux causes significant decomposition). The
reaction mixture was cooled, filtered, and concentrated. The
residue was purified by flash column chromatography on silica
gel (25% ethyl acetate/hexane) to give 6 as a colorless oil (4.82
g, 70%). 1H NMR (CD3OD) δ 7.34-7.18 (m, 5 H), 3.97-3.83
(m, 2 H), 3.67 (dd, 1 H, J ) 13.4, 2.6 Hz), 3.54 (dd, 1 H, J )
13.4, 4.0 Hz), 2.72-2.40 (m, 6 H), 1.80-1.50 (m, 4 H), 1.45 (s,
9 H), 1.44 (s, 9 H), 1.40-1.30 (m, 2 H). 13C NMR (CDCl3) δ
171.8, 155.3, 138.1, 128.8, 128.4, 127.3, 117.1, 81.6, 79.5, 63.6,
58.8, 54.0, 32.6, 28.2, 27.9, 22.1. HRMS m/ z calcd for
C26H42N3O5 476.3124, found 476.3121. Anal. Calcd for
C26H41N3O5: C, 65.66; H, 8.69; N, 8.83. Found: C, 65.71; H,
of 3,4-dihydro-2H-pyran (51 mg each) were added over the next
7 h. The reaction mixture was stirred for an additional 12 h
and concentrated in vacuo. The oil was dissolved in water and
methanol (1:1; 4 mL) and adjusted to pH 7 with saturated
NaHCO3 solution. The solution was concentrated and the
crude oil purified by chromatography on a C-18 column (55%
acetone/water) to give 10 (210 mg, 68%) as a colorless oil and
recovered starting material 9 (20 mg, 8%). 1H NMR (CD3OD)
δ 7.40-7.22 (m, 10 H), 5.10 (m, 2 H), 5.04 (s, 2 H), 4.62-4.32
(m, 1 H), 4.02-3.68 (m, 2 H), 3.50 (m, 1 H), 3.44-3.06 (m, 7
H), 1.98-1.28 (m, 14 H); 13C NMR (CD3OD) δ 174.3, 158.7,
158.1, 138.5, 129.7, 129.6, 129.5, 129.3, 129.0, 128.8, 101.7,
100.1, 74.9, 68.3, 67.4, 65.3, 56.2, 48.4, 38.2, 34.3, 32.6, 32.1,
28.5, 26.4, 23.6, 21.8, 21.2. HRMS m/ z calcd for C31H43N3O8
586.3128, found 586.3118. [R]24 +4.0° (c 0.25, CH3OH).
D
8.67; N, 8.80. [R]25 +4.7° (c 1.00, CHCl3).
(2S,9R)-11-[(Ben zyloxyca r bon yl)a m in o]-7-(ca r boben -
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 (7). To a solution of 6 (4.80 g, 10.7 mmol) in glacial
acetic acid (100 mL) were added 10% Pd-C (0.50 g) and PtO2
(1.00 g), and H2 gas was introduced. The catalyst was removed
after 6 h by filtration through Celite and washed with acetic
acid. The filtrate was concentrated in vacuo. Azeotropic
removal of acetic acid with toluene provided 7 as a colorless
oil (5.10 g, 99%). 1H NMR (500 MHz) (CD3OD) δ 4.02-3.94
(m, 2 H), 3.14-2.86 (m, 6 H), 1.94 (s, 6 H), 1.87-1.58 (m, 8
H), 1.46 (s, 9 H), 1.44 (s, 9 H). 13C NMR (CD3OD) δ 169.6,
156.54, 85.3, 70.2, 62.6, 56.6, 54.2, 53.9, 34.0, 31.1, 28.2, 26.3,
23.2. HRMS m/ z calcd for C19H40N3O5 390.2968, found
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 (11).
A
solution of 9-fluorenylmethyl N-succinimidyl carbonate (181
mg, 0.53 mmol) in DMF (2.5 mL) was added to a solution of
10 (210 mg, 0.36 mmol) in 9% Na2
CO3 (0.836 mL, 0.72 mmol)
at 0 °C and stirred overnight at rt. The pH was adjusted to 7
with 0.1 N HCl. The mixture was concentrated to an oil and
purified by flash chromatography (90% CHCl3/MeOH) to give
11 (239 mg, 83%) as a colorless oil. 1H NMR (CDCl3) δ 7.78
(m, 2 H), 7.60 (m, 2 H), 7.30 (m, 14 H), 5.72 (m, 2 H), 5.18 (s,
2 H), 5.16 (s, 2 H), 4.60 (m, 1 H), 4.52-4.22 (m, 3 H), 4.20 (m,
1 H), 4.00-3.72 (m, 3 H), 3.50-3.10 (m, 6 H), 2.00-1.22 (m,
14 H);
13C NMR (150 MHz) (CDCl3) δ 174.6, 156.7, 156.4, 143.9,
390.2977. [R]25 +0.6° (c 1.00, CH3OH).
143.8, 141.3, 136.8, 136.5, 128.5, 128.5, 128.1, 128.0, 127.9,
127.7, 127.1, 125.1, 124.9, 120.0, 100.7, 73.5, 67.4, 67.2, 66.6,
53.5, 48.4, 47.2, 32.9, 32.0, 31.5, 30.8, 28.0, 27.3, 25.2, 22.0,
21.1, 19.8. HRMS m/ z calcd for C46H53N3O10Na 830.3629,
found 830.3661. Anal. Calcd for C46H53N3O10: C, 68.38; H,
D
(2S,9R)-11-[(Be n zyloxyca r b on yl)a m in o]-2-[(ter t-b u -
t oxyca r b on yl)a m in o]-9-h yd r oxy-7-(ca r b ob en zyloxy)-7-
a za u n d eca n oic Acid ter t-Bu tyl Ester (8). A solution of 7
(1.17 g, 2.30 mmol) in CHCl3 (100 mL) was washed with
saturated Na2CO3 solution. The aqueous layer was extracted
with CHCl3 (3 × 100 mL), and the combined organic layers
were dried over Na2SO4 and concentrated in vacuo. A solution
of the resultant oil (the free amine, 0.85 g, 2.18 mmol) in CH2-
Cl2 (60 mL) was cooled to 0 °C and treated with diisopropyl-
ethylamine (0.59 g, 4.57 mmol) and benzyl chloroformate (0.79
g, 4.60 mmol). The reaction mixture was stirred overnight at
rt, concentrated to dryness, and purified by flash chromatog-
raphy (50% ethyl acetate/hexane) to give 8 (790 mg, 55%) as
a colorless oil. 1H NMR (CDCl3) δ 7.23 (m, 10 H), 5.45 (m, 1
H), 5.08 (s, 2 H), 5.04 (s, 2 H), 4.10 (m, 1 H), 3.80 (m, 1 H),
3.40 (m, 1 H), 3.23 (m, 5 H), 1.80-1.43 (m, 6 H), 1.41 (s, 18
H), 1.23 (m, 2 H). 13C NMR (CDCl3) δ 171.8, 157.5, 156.9,
155.3, 136.4, 128.4, 128.3, 127.9, 127.7, 81.6, 79.5, 69.2, 67.2,
66.5, 53.7, 48.5, 37.7, 34.8, 32.5, 28.2, 27.9, 22.3. HRMS m/ z
6.61; N, 5.20. Found: C, 68.55; H, 6.63; N, 5.26. [R]26 +3.4°
D
(c 1.00, CHCl3).
FMOC-Th r (O-t-Bu )-Gly Car boxam idom eth yl Ester (14).
A solution of 1320a (1.16 g, 5.0 mmol) in TFA (10 mL) was
stirred for 10 min at 0 °C. The amine TFA salt was precipi-
tated with diethyl ether (130 mL), filtered, dried (0.75 g, 60%),
and dissolved in dry DMF (10 mL). FMOC-Thr(O-t-Bu)-OH
(1.20 g, 3.0 mmol) and BOP (1.33 g, 3.0 mmol) were added.
The solution was cooled to 0 °C, and DIEA (1.15 mL, 6.6 mmol)
was added dropwise. The solution was warmed to rt and
stirred overnight. The volatiles were removed in vacuo; the
resulting oil was dissolved in ethyl acetate (200 mL) and
washed with 1 M citric acid, water, 5% aqueous NaHCO3
solution, and water. The organic layer was dried over MgSO4
and concentrated. Flash chromatography (90% ethyl acetate/
hexane) gave 14 as a colorless solid (1.30 g, 85%). 1H NMR
(CDCl3) δ 7.77 (d, 2 H, J ) 7.5 Hz), 7.65 (m, 1 H), 7.60 (d, 2 H,
7.5 Hz), 7.45-7.27 (m, 4 H), 6.60 (s br, 1 H), 5.87 (d br, 1 H),
5.54 (s br, 1 H), 4.67 (m, 2 H), 4.51-4.35 (m, 2 H), 4.28-4.12
(m, 4 H), 4.05 (m, 1 H), 1.29 (s, 9 H), 1.08 (d, 3 H, J ) 6.4 Hz).
calcd for C35H52N3O9 658.3703, found 658.3774. [R]24 +4.6°
D
(c 0.50, CHCl3).
(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 (9). The
ester 8 (500 mg, 0.76 mmol) was added to a mixture of
trifluoroacetic acid (1.12 g, 9.90 mmol), CH2Cl2 (2.05 g, 24.0
mmol), and triethylsilane (220 mg, 1.9 mmol) and stirred at
rt for 20 h. The reaction mixure was concentrated to dryness
and stirred again in the mixture as described above for an
additional 6 h. The reaction mixture was concentrated and
the resultant oil dissolved in 1.0 mL water and adjusted to
pH 8 with saturated NaHCO3 solution. The solution was
concentrated and the residue purified by chromatography on
a C-18 column (55% acetone/water) to give 9 (300 mg, 78%)
as a colorless oil. 1H NMR (CD3OD) δ 7.40-7.25 (m, 10 H),
5.11 (s, 2 H), 5.06 (s, 2 H), 3.82 (m, 1 H), 3.55 (m, 1 H), 3.40-
3.10 (m, 6 H), 1.95-1.30 (m, 8 H). HRMS m/ z calcd for
Anal. Calcd for C27
H33N3O7: C, 63.39; H, 6.50; N, 8.21.
Found: C, 63.14; H, 6.60; N, 8.10. [R]22
-0.8° (c 1.00, CH3-
D
OH).
CBZ-Cys(CBZ)-Th r (O-t-Bu )-Gly Ca r boxa m id om et h yl
Ester (15). A solution of 14 (0.46 g, 0.90 mmol) and dieth-
ylamine (0.8 mL) in dry DMF (8 mL) was stirred overnight at
rt. The volatiles were removed in vacuo, and the residue was
dissolved in dry DMF (15 mL). N,S-Di-CBZ-L-cysteine (0.35
g, 0.90 mmol) and BOP (0.40 g, 0.90 mmol) were added; the
solution was cooled to 0 °C. DIEA (0.25g, 1.89 mmol) was
added, and the solution was stirred at rt for 2 days. The
reaction mixture was concentrated in vacuo, diluted with ethyl
acetate (100 mL), and washed with 10% citric acid, water, 5%
aqueous NaHCO3 solution, and water. The organic layer was
dried over MgSO4 and concentrated in vacuo. The resulting
oil was purified by flash column chromatography (80% ethyl
acetate/10% hexane/10% CHCl3) to obtain 15 (0.29 g, 50%).
Mp 95-97 °C. 1H NMR (CDCl3) δ 7.70 (m, 1 H), 7.28 (s, 10
H), 6.78 (s, 1 H), 6.00 (s, 1 H), 5.50 (s, 1 H), 5.23 (s, 2 H), 5.17
(s, 2 H), 4.62 (m, 2 H), 4.41 (m, 1 H), 4.30 (m, 1 H), 4.25 (m, 1
H), 4.15 (m, 1 H), 3.94 (m, 1 H), 3.30 (m, 2 H), 1.65 (s, 1 H),
1.20 (s, 9 H), 1.07 (d, 3 H). Anal. Calcd for C31H40N4O10S: C,
C26H36N3O7 502.2553, found 502.2531. [R]24 +4.2° (c 1.00,
D
CH3OH).
(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 (10). Trifluoroacetic acid (115 mg, 1.01 mmol)
was added to a solution of 9 (265 mg, 0.53 mmol) in CHCl3 (5
mL). The solution was concentrated in vacuo. The resultant
oil was dissolved in CH2Cl2 (15 mL), and 3,4-dihydro-2H-pyran
(51 mg, 55 µL, 0.61 mmol) was added at rt. The reaction
progress was monitored by TLC, and three additional portions

3290 J . Org. Chem., Vol. 62, No. 10, 1997
Bergeron et al.
56.35; H, 6.10; N, 8.48. Found: C, 56.62; H, 6.14; N, 8.38.
CHCl3 (10 mL) was stirred for 2 h at rt. An additional portion
of 4-(aminomethyl)piperidine (1.0 mL) was added, and stirring
was continued for another 40 min. The reaction mixture was
taken up in CHCl3 (50 mL) and extracted with phosphate
buffer (pH 5.5; 3 × 75 mL). The organic layer was dried over
Na2SO4 and concentrated; the residue was purified by flash
chromatography (10% methanol/CHCl3) to give H-Hpu(N7,N12-
di-CBZ,O9-THP)-His(BOC)-Gly-O-t-Bu as a colorless oil (88
mg, 68%). A portion of this oil (10 mg, 11 µmol) and 16 (7 mg,
11 µmol) in dry DMF (2 mL) was cooled to 0 °C. BOP (6 mg,
12 µmol) was added, and the solution was stirred for 30 min.
DIEA (4.5 µL, 26 µmol) was added dropwise. The solution was
warmed to rt and stirred overnight. The reaction mixture was
concentrated in vacuo. The residue was dissolved in ethyl
acetate (15 mL) and extracted with 5% aqueous NaHCO3
solution (10 mL) and water (10 mL). The organic layer was
concentrated under reduced pressure and the residue purified
by flash chromatography (10% ethanol/CHCl3) to give 20 as a
colorless oil (13 mg, 81%). 1H NMR (600 MHz) (CD3OD) δ 7.99
(m, 1 H), 7.28-7.14 (m, 21 H), 5.17-5.07 (m, 2 H), 5.06-4.88
(m, 6 H), 4.58 (dd, 1 H, J ) 9.0, 4.2 Hz), 4.51-4.32 (m, 2 H),
4.26 (m, 1 H), 4.14 (m, 1 H), 4.10 (m, 1 H), 4.08-3.98 (m, 2
H), 3.86-3.58 (m, 6 H), 3.40-2.94 (m, 8 H), 2.88 (m, 1 H),
1.70-0.94 (m, 17 H), 1.48 (s, 9 H), 1.34 (s, 9 H), 1.08 (s, 9 H).
HRMS m/ z calcd for C77H105N10O20S 1521.7227, found 1521.736.
[R]25 -14.0° (c 1.10, CH3OH).
D
CBZ-Cys(CBZ)-Th r (O-t-Bu )-Gly-OH (16). To a solution
of 15 (0.90 g, 1.36 mmol) in DMF (14 mL) was added aqueous
Na2CO3 solution (2.72 mmol, 8 mL) at rt. The solution warmed
on addition, and water was added until the solution was clear.
After 5 min, the pH was adjusted to 6 with citric acid (0.5 N,
9 mL). The reaction mixture was concentrated in vacuo; the
residue was dissolved in ethyl acetate and washed with 0.5 N
citric acid. The organic layer was dried over Na2SO4 and
concentrated in vacuo. The residue was purified by column
chromatography using LH-20 (lipophilic Sephadex; 50 g; 1%
ethanol/toluene) to give 16 as a white solid (0.63 g, 77%). Mp
124-125 °C. 1H NMR (CD3OD) δ 7.39-7.25 (m, 10 H), 5.24
(s, 2 H), 5.12 (s, 2 H), 4.46 (dd, 1 H, J ) 8.6, 5.0 Hz), 4.33 (d,
1 H, J ) 3.0 Hz), 4.16 (m, 1 H), 3.92 (m, 2 H), 3.45 (dd, 1 H,
J ) 14.3, 5.0 Hz), 3.16 (dd, 1 H, J ) 14.3, 8.6 Hz), 1.20 (s, 9
H), 1.09 (d, 3 H, J ) 6.4 Hz). Anal. Calcd for C29H37N3O9S:
C, 57.70; H, 6.18; N, 6.96. Found: C, 57.76; H, 6.24; N, 7.02.
[R]22 +8.6° (c 0.50, CHCl3).
D
CBZ-His-Gly-O-t-Bu (17). A solution of glycine t-Bu ester
hydrochloride (2.20 g, 13.0 mmol) and NR-CBZ-L-histidine
(3.44 g, 11.9 mmol) in dry DMF (40 mL) was cooled to 0 °C
under an N2 atmosphere, and diphenylphosphoryl azide
(DPPA, 3.58 g, 13.0 mmol) was added dropwise over 5 min.
The solution was stirred for 10 min, and triethylamine (2.63
g, 26.0 mmol) was added. The solution was stirred at rt
overnight and concentrated in vacuo, and the residue purified
by flash chromatography (8% ethanol/CHCl3) to give 17 as a
white solid (3.45 g, 72%). Mp 64-66 °C. 1H NMR (CD3OD) δ
7.56 (d, 1 H, J ) 1.1 Hz), 7.30 (m, 5 H), 6.88 (s, 1 H), 5.04 (m,
2 H), 4.42 (m, 1 H), 3.80 (m, 2 H), 3.12 (m, 1 H), 2.91 (m, 1 H),
1.46 (s, 9 H). Anal. Calcd for C20H26N4O5: C, 59.69; H, 6.51;
N, 13.92. Found: C, 59.43; H, 6.46; N, 13.81.
[R]26 -1.2° (c 1.00, CHCl3).
D
Cys-Th r -Gly-Hp u -His-Gly, Tr iflu or oa cetic Acid Sa lt
(12). Method a (Scheme 2): A solution of 20 (13 mg, 7.2 µmol)
and phenol (5 mg, 50 µmol) was heated to reflux for 90 min in
degassed TFA (5.0 mL) under an argon atmosphere. The
reaction mixture was concentrated in vacuo and the residue
applied to a C-18 plug (Supelco; water/acetonitrile ) 88/22 +
0.1% TFA). Further purification was performed by preparative
HPLC (solvent systems A, aqueous 0.1% TFA; and B, 0.1%
TFA in CH3CN; linear gradient of 0-20% B in 50 min; flow
rate 4.0 mL/min; detection at 214 nm; retention time ) 8.4
CBZ-His(BOC)-Gly-O-t-Bu (18). A solution of di-tert-butyl
dicarbonate (1.33 g, 6.0 mmol) in THF (3 mL) was added
dropwise at 0 °C to a solution of 17 (2.0 g, 5.0 mmol) in THF
(15 mL). The reaction mixture was stirred overnight at rt and
concentrated in vacuo. The residue was purified by flash
chromatography (60% ethyl acetate/hexane) to give 18 (1.96
g, 78%). 1H NMR (CDCl3) δ 8.00 (s, 1 H), 7.30 (m, 6 H), 6.60
(m, 1 H), 5.10 (s, 2 H), 4.58 (m, 1 H), 3.85 (m, 2 H), 3.03 (m, 2
H), 2.38 (s, 1 H), 1.60 (s, 9 H), 1.40 (s, 9 H). Anal. Calcd for
C20H26N4O5: C, 59.75; H, 6.82; N, 11.15. Found: C, 59.69; H,
min) using a C-18 reverse phase column (Dynamax 300 A C18
)
to give 12 as a colorless oil (2 mg, 24%). 1H NMR (D2O) (600
MHz) δ 8.69 (d, 1 H, J ) 1.2 Hz), 7.38 (s, 1 H), 4.80 (m, 1 H),
4.49 (d, 1 H, J ) 4.9 Hz), 4.41 (t, 1 H, J ) 5.6 Hz), 4.36 (dd, 1
H, J ) 8.5, 6.0 Hz), 4.29 (m, 1 H), 4.11 (m, 1 H), 4.09-4.00
(m, 4 H), 3.37 (dd, 1 H, J ) 15.5, 7.2 Hz), 3.29-3.06 (m, 9 H),
1.99 (m, 1 H), 1.86 (m, 2 H), 1.76 (m, 3 H), 1.45 (m, 2 H), 1.32
(d, 3 H, J ) 6.4 Hz). MS (MALDI-Tof) m/z calcd for
C27H48N10O9S 688.33 (M+), found 688.96.
6.87; N, 11.09. [R]22 -4.0° (c 1.00, CH3OH).
D
F MOC-Hp u (N7,N12-d i-CBZ,O9-THP )-His(BOC)-Gly-O-
t-Bu (19). To a solution of 18 (185 mg, 0.37 mmol) and 1 N
HCl (370 µL) in ethanol (15 mL) was added 10% Pd-C (18
mg), and the reaction mixture was hydrogenated for 2.5 h at
rt. The reaction mixture was filtered through Celite, concen-
trated in vacuo, and purified by flash chromatography (9:1
CHCl3:ethanol) to give H-His(BOC)-Gly-O-t-Bu hydrochloride
(101 mg, 68%). A portion of this ammonium salt (69 mg, 0.17
mmol) and 11 (134 mg, 0.17 mmol) were dissolved in dry DMF
(12 mL). The solution was cooled to 0 °C. BOP (87 mg, 0.20
mmol) was added and stirred for 20 min. DIEA (47.5 mg, 0.37
mmol) was added dropwise at 0 °C, and the solution was
warmed to rt and stirred overnight. The volatiles were
removed under reduced pressure, and the residue was dis-
solved in ethyl acetate and washed with 5% aqueous NaHCO3
solution and water. The organic layer was dried over MgSO4
and concentrated in vacuo and the residue purified by flash
chromatography (4% ethanol/CHCl3) to give 19 as a colorless
oil (165 mg, 85%). 1H NMR (600 MHz) (CD3OD) δ 7.86 (m, 1
H), 7.70 (d, 2 H, J ) 7.5 Hz), 7.58 (m, 2 H), 7.31-7.11 (m, 15
H), 5.00 (m, 2 H), 4.92 (s, 2 H), 4.60 (m, 1 H), 4.50-4.32 (m, 1
H), 4.30-4.26 (m, 2 H), 4.08 (t, 1 H, J ) 6.8 Hz), 3.90 (m, 1
H), 3.86-3.52 (m, 5 H), 3.38-2.76 (m, 8 H), 1.70-1.12 (m, 14
H), 1.40 (s, 9 H), 1.28 (s, 9 H). HRMS m/ z calcd for
C63H80N7O14 1158.5763, found 1158.5739. [R]26D -1.5° (c 1.00,
CHCl3).
Meth od b (Sch em e 3): The polymer-bound peptide 21 was
synthesized using an Applied Biosystems 432A Synthesizer.
Amino acid analysis for 21: Gly 2.09, His 1.03, Thr 0.88. An
aliquot of 21 (49 mg, 19.3 µmol), phenol (250 mg), and
pentamethylbenzene (250 mg) were dissolved in degassed TFA
(5.0 mL) at 0 °C. Saturated HBr in acetic acid solution (0.2
mL), triisopropylsilane (0.1 mL), and 1,2-ethanedithiol (0.1
mL) were added under an argon atmosphere. The solution
was stirred at rt 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 by preparative HPLC as in method a using a C-18
reverse phase column (Dynamax 300 A C18) to give 12 as a
colorless oil (5.2 mg, 24%). 1H NMR and MS analytical data
were identical with those for 12 prepared by method a. [R]26
D
-16.7° (c 0.30, H2O). Amino acid analysis: Gly 1.94, His 1.02,
Thr 1.04.
Ack n ow led gm en t. Financial support was provided
by SunPharm Corp., J acksonville, FL. The authors
thank A.Y.K. Chung of the Dept. of Biochemistry and
Protein Core ICBR, University of Florida, Gainesville,
FL, for performing the SPPS and HPLC purifications;
H. D. Plant and J . R. Rocca of the Center for Structural
BiologysBrain Institute, University of Florida, Gaines-
ville, FL, for high-field and 2D NMR spectral analyses.
CBZ-Cys(CBZ)-Th r (O-t-Bu )-Gly-Hpu (N7,N12-di-CBZ,O9-
THP )-His(BOC)-Gly-O-t-Bu (20). A solution of 4-(amino-
methyl)piperidine (1.0 mL) and 19 (156 mg, 0.14 mmol) in
J O970119Z