Solid-phase functionalization of peptides by an α-hydrazinoacetyl group

Article abstract of DOI:10.1021/jo0343432

A novel procedure for the preparation of α-hydrazinoacetyl peptides is reported on the basis of the solid-phase coupling of partially or fully Boc-protected hydrazinoacetic acid derivatives. The degree of unwanted polymerization of the activated ester dur

Full text of DOI:10.1021/jo0343432

Solid -P h a se F u n ction a liza tion of P ep tid es by a n
r-Hyd r a zin oa cetyl Gr ou p
Dominique Bonnet,*,†,‡ Cyrille Grandjean,‡ Pierre Rousselot-Pailley,† Pascal J oly,†
Line Bourel-Bonnet,† Vale´rie Santraine,‡ He´le`ne Gras-Masse,‡ and Oleg Melnyk*,†
UMR CNRS 8525, Biological Institute of Lille, 1 rue du Pr Calmette, 59021 Lille, France, and
Sedac-Therapeutics, Le Gale´nis, Baˆt. B, 85, rue Nelson Mandela, 59120 Loos, France
dominique.bonnet@sedac-therapeutics.com; oleg.melnyk@ibl.fr
Received March 14, 2003
A novel procedure for the preparation of R-hydrazinoacetyl peptides is reported on the basis of the
solid-phase coupling of partially or fully Boc-protected hydrazino acetic acid derivatives. The degree
of unwanted polymerization of the activated ester during both activation and coupling was found
to be significant for the monoprotected derivative BocNHNHCH2CO2H but could be minimized
with the diprotected derivative BocNHNH(Boc)CH2CO2H and suppressed with the fully protected
acid. Despite the instability of the imidocarbonate group toward acids and bases, a low-cost and
effective route was sought for the preparation of the tris(Boc)-protected derivative. The N,N,N′-
tris(Boc)hydrazinoacetic acid could be introduced on the solid phase after or before peptide elongation
using Fmoc/tert-butyl chemistry. In this latter case, HR MAS NMR analysis of model solid supports
demonstrated the partial loss of one Boc group during the repetitive piperidine treatments. Despite
this slight instability, N,N,N′-tris(Boc)hydrazinoacetic acid was found to be a highly convenient
reagent for the robust and easily scalable preparation of hydrazinopeptides in good yield and high
purity.
In tr od u ction
two one-pot, orthogonal, chemoselective ligation reac-
tions.5
The modification of peptides by an R-hydrazinoacetyl
moiety allows the site-specific derivatization of fully
deprotected peptides owing to the easy discrimination
between the hydrazino group and the other functional-
ities naturally present on peptides. For example, the
large difference in pKa between an R-hydrazinoacetamide
group and ꢀ amino groups was exploited for the chemose-
lective acylation of the former by fatty acid succinimidyl
esters, thus providing a valuable access to complex
lipopeptides.1 The R-hydrazinoacetyl moiety was also
successfully engaged in hydrazone formation by reaction
with R-oxo aldehydes, either for the synthesis of lipopep-
tides,2 the site-specific anchoring of peptides or mannose-
mimetic clusters onto the surface of vesicles,3 the syn-
thesis of oligonucleotide-peptide conjugates,4 or the
synthesis of clustered glycoside-antigen conjugates using
Owing to the usefulness of these peptide derivatives,
we have examined various methods for their solid-phase
synthesis based upon the Fmoc/tert-butyl strategy, since
the R-hydrazinoacetamide group was found to be stable
in the concentrated TFA mixture used for side-chain
deprotection.6 In a first approach, the N-N bond was
elaborated on the solid phase by N-electrophilic amina-
tion of a glycyl residue with N-Boc-3-(4-cyanophenyl)-
oxaziridine (BCPO):1a,b,7 Various hydrazinopeptides were
successfully synthesized using this process, which how-
ever cannot be easily scaled up8 and generalized.5b An
interesting and practical alternative resides in the de-
rivatization of the peptidyl resin by an R-bromoacetyl
group followed by displacement of the bromine atom by
tert-butylcarbazate.9 In search of a robust and easily
(4) Ollivier, N.; Olivier, C.; Gouyette, C.; Huynh-Dinh, T.; Gras-
Masse, H.; Melnyk, O. Tetrahedron Lett. 2002, 43, 997-999.
(5) (a) Grandjean, C.; Rommens, C.; Gras-Masse, H.; Melnyk, O.
Angew. Chem., Int. Ed. 2000, 39, 1068-1072. (b) Grandjean, C.; Gras-
Masse, H.; Melnyk, O. Chem. Eur. J . 2001, 7, 230-239. (c) Grandjean,
C.; Santraine, V.; Fruchart, J .-S.; Melnyk, O.; Gras-Masse, H. Biocon-
jugate Chem. 2002, 13, 887-892.
* To whom correspondence should be addressed. (D.B.) Tel: 33(0)3
20 87 12 16. (O.M) Tel: 33(0)3 20 87 12 14. Fax: 33(0)3 20 87 12 33.
† Biological Institute of Lille.
‡ Sedac-Therapeutics.
(1) (a) Bonnet, D.; Rommens, C.; Gras-Masse, H.; Melnyk, O.
Tetrahedron Lett. 2000, 41, 45-48. (b) Bonnet, D.; Ollivier, N.; Gras-
Masse, H.; Melnyk, O. J . Org. Chem. 2001, 66, 443-449. (c) Melnyk,
O.; Bonnet, D.; Gras-Masse, H. Patent Application FR 99 10626 (08/
19/1999). (d) Bourel-Bonnet, L.; Bonnet, D.; Malingue, F.; Gras-Masse,
H.; Melnyk, O. Bioconjugate Chem. 2003, 14, 494-499.
(6) Bonnet, D.; Samson, F.; Rommens, C.; Gras-Masse, H.; Melnyk,
O. J . Peptide Res. 1999, 54, 270-278.
(7) Klinguer, C.; Melnyk, O.; Loing, E.; Gras-Masse, H. Tetrahedron
Lett. 1996, 37, 7259-7262.
(2) (a) Bonnet, D.; Bourel, L.; Gras-Masse, H.; Melnyk, O. Tetrahe-
dron Lett. 2000, 41, 10003-10007. (b) Melnyk, O.; Bonnet, D.; Bourel,
L.; Gras-Masse, H. Patent Application FR 00 11451 (09/08/2000).
(3) (a) Bourel-Bonnet, L.; Gras-Masse, H.; Melnyk, O. Tetrahedron
Lett. 2001, 42, 6851-6853. (b) Chenevier, P.; Grandjean, C.; Loing,
E.; Malingue, F.; Angyalosi, G.; Gras-Masse, H.; Roux, D.; Melnyk,
O.; Bourel-Bonnet, L. Chem. Commun. 2002, 2446-2447.
(8) BCPO is no longer commercially available. For preparation of
BCPO and other oxaziridines, see: (a) Vidal, J .; Damestoy, S.; Guy,
L.; Hannachi, J .-C.; Aubry, A.; Collet, A. Chem. Eur. J . 1997, 3, 1691-
1709. (b) Vidal, J .; Guy, L.; Ste´rin, S.; Collet, A. J . Org. Chem. 1993,
58, 4791-4793. (c) Vidal, J .; Hannachi, J .-C.; Hourdin, G.; Mulatier,
J .-C.; Collet, A. Tetrahedron Lett. 1998, 39, 8845-8848. (d) Vidal, J .;
Drouin, J .; Collet, A. J . Chem. Soc., Chem. Commun. 1991, 435-437.
10.1021/jo0343432 CCC: $25.00 © 2003 American Chemical Society
Published on Web 08/15/2003
J . Org. Chem. 2003, 68, 7033-7040
7033

Bonnet et al.
transferred to the NR. The use of N-hydroxysuccinimidyl
esters led to diketopiperazine and oligomer formation.
This coupling worked reasonably well provided the
activated ester was formed in situ with hindered hy-
drazino acids. Protection of the NR-position by a benzyl
group solved these problems but required an additional
hydrogenolysis step following peptide assembly.
F IGURE 1. Mono, di and tri-protected R-hydrazinoacetic
derivatives 1-3.
To avoid this additional step following the separation
of the R-hydrazinoacetyl peptide from the solid support,
we have focused our attention on the synthesis and
coupling of R-hydrazinoacetic acid derivatives 1-3, which
were synthesized as depicted in Scheme 1.
Syn th esis of Boc-P r otected R-Hyd r a zin oa cetic
Acid Der iva tives 1-3. Two routes have been devised
for the access to Boc-protected derivatives (Scheme 1).
Path 1 was found suitable for the production of the three
protected acids 1-3 on a laboratory scale and to docu-
ment their ability to be coupled on solid phase. Path 2
was developed as an attractive, low-cost alternative for
the preparation of triprotected derivative 3 on a larger
scale.
scalable methodology for the synthesis of hydrazinopep-
tides using automated solid-phase techniques, we have
envisioned the introduction of the hydrazino moiety
through a classical acid activation using Boc-protected
derivatives of R-hydrazinoacetic acid (Figure 1). We
report in this paper the synthesis of mono-, di-, and
triprotected derivatives 1-3 and the study of their
usefulness for the synthesis of hydrazinopeptides. Some
preliminary results were described before in a short
report.1a Preparation of fully protected derivative 3
proved to be troublesome due to the sensitivity of one of
the Boc groups to both aqueous acids and bases. Coupling
of 1-3 to a model peptidyl resin was examined using
various activation procedures. In particular, the impact
of the activation method upon the extent of polymeriza-
tion of diprotected derivative 2 was documented. The
stability of supported triprotected R-hydrazinoacetyl
moieties toward piperidine, diluted TFA, or acetic anhy-
dride/diisopropylethylamine was also examined using
high-resolution magic angle spinning (HR MAS) NMR
spectroscopy to document its behavior during the Fmoc/
tert-butyl solid-phase peptide synthesis.
P a th 1. This strategy relies on the mono acylation of
commercially available ethyl hydrazinoacetate hydro-
chloride 4 using Boc2O in a H2O/EtOH mixture to give
derivative 6a . Saponification of ester 6a in ethanolic
sodium hydroxide solution yielded Nâ-Boc-protected acid
1, which could be purified by simple precipitation in
t-BuOMe (49% overall yield). Attempts to obtain ester
7a starting directly from ethyl hydrazinoacetate hydro-
chloride 4 failed and gave only mixtures of mono- and
diprotected esters. Thus, synthesis of ester 7a was
performed by treatment of intermediate 6a with an
excess of Boc2O/Et3N in the presence of a catalytic
amount of DMAP. Triprotected ester 7a was next treated
in a NaOH 1 N/EtOH/H2O mixture for 3 h at rt.
Unexpectedly, this experiment yielded exclusively dipro-
Resu lts a n d Discu ssion
During the past decade, several methods allowing the
preparation of either free,10 Nâ-protected or Nâ,NR-
orthogonally bisprotected11 R-hydrazino acids have been
reported. However, little attention has been devoted to
the coupling of these derivatives to peptidyl resins for
the solid-phase synthesis of hydrazinopeptides. Side
reactions arising during the activation and coupling of
Nâ-protected R-hydrazino acids have been partially ad-
dressed only recently by Guy et al.12 Coupling with mixed
anhydrides prepared from ClCO2-i-Bu failed, since once
the anhydride formed, its acyl group was immediately
13
tected acid 2 (66% overall yield) and not the expected
triprotected acid 3. Additional experiments demonstrated
the sensitivity of the Boc group to both aqueous NaOH
and pH 3-5 aqueous media. The electron-withdrawing
effects exerted by the hydrazine substituents in com-
pound 7a may render the imidodicarbonate more sensi-
tive to nucleophiles. A method to selectively cleave off a
Boc-group from a Boc tri-protected hydrazine in acidic
conditions has been previously described by Ma¨eorg et
al.14
These data in hand, access to triprotected acid 3 could
be secured by the careful control of both saponification
and acidification steps in term of time, temperature, and
pH. Saponification of ethyl N,N,N′-tris(Boc)hydrazino-
acetate 7a was performed at 0 °C for only 25 min. The
reaction mixture was acidified to pH 4 with citric acid
and quickly extracted to limit the exposure of 3 to
aqueous acid. Compound 3 could be purified by precipita-
tion from an Et2O/heptane mixture with a 63% overall
yield on a 10 g scale (49% yield on a 50 g scale).
P a th 2. An alternative route to intermediate ester 6a
starting from the cheap starting materials ethyl bro-
(9) See refs 5a,b. A similar approach was exploited for the synthesis
of peptoids: (a) Cheguillaume, A.; Doubli-Bounoua, I.; Baudy-Floc’h,
M.; Le Grel, P. Synlett 2000, 3, 331-334. (b) Kruijtzer, J . A. W.;
Liskamp, R. M. J . Tetrahedron Lett. 1995, 36, 6969-6972. Aminohy-
dantoin: Wilson, L. J .; Li, M.; Portlock, D. E. Tetrahedron Lett. 1998,
39, 5135-5138.
(10) (a) Viret, J .; Gabard, J .; Collet, A. Tetrahedron Lett. 1987, 43,
891-894. (b) Karady, S.; Ly, M. G.; Pines, S. H.; Sletzinger, M. J . Org.
Chem. 1971, 36, 1949-1954.
(11) Nitrosation of amino acids: (a) Achiwa, K.; Yamada, S. Tetra-
hedron Lett. 1975, 31, 2701-2704. Amination of enolates or silyl enol
ethers: (b) Evans, D. A.; Britton, T. C.; Dorow, R. L.; Dellaria, J . F. J .
Am. Chem. Soc. 1986, 108, 6395-6397. (c) Trimble, L. A.; Vederas, J .
C. J . Am. Chem. Soc. 1986, 108, 6397-6399. (d) Gennari, C.; Colombo,
L.; Bertolini, G. J . Am. Chem. Soc. 1986, 108, 6394-6395 (e) Oguz,
U.; Guilbeau, G. G.; McLaughlin, M. L. Tetrahedron Lett. 2002, 43,
2873-2875. Nucleophilic substitution of 2-bromo or nosyloxy esters
by hydrazine derivatives (e) Chegillaume, A.; Doubli-Bounoua, I.;
Baudy-Floc’h, M.; Le Grel, P. Synlett 2000, 3, 331-334. (f) Cheguil-
laume, A.; Lehardy, F.; Bouget, K.; Baudy-Floc’h, M.; Le Grel, P. J .
Org. Chem. 1999, 64, 2924-2927. (g) Hoffman, R. V.; Kim, H.-O.
Tetrahedron Lett. 1990, 31, 2953-2956. Other method (e) Cativiela,
C.; Diaz-de-Villegas, M.; Galvez, J . A. Tetrahedron: Asymmetry 1997,
8, 1605-1610.
(13) The diprotected derivative obtained following saponification was
unambiguously identified as corresponding to chemical structure 2
using a heteronuclear multibond coupling (HMBC) NMR experiment.
A clear coupling between the Nâ-H and the carbonyl carbon and no
correlation with the methylene were observed.
(14) Ma¨eorg, U.; Grehn, L.; Ragnarsson, U. Angew. Chem., Int. Ed.
Engl. 1996, 35, 22, 2626-2627.
(12) Guy, L.; Vidal, J .; Collet, A. J . Med. Chem. 1998, 41, 4833-
4843.
7034 J . Org. Chem., Vol. 68, No. 18, 2003

Synthesis of R-Hydrazinoacetylpeptides
SCHEME 1 a
a
Key: (a) overall isolated yield; (i) Boc2O (1.1 equiv), N-methylmorpholine (1.1 equiv), H2O/EtOH (1/1), rt, 2 h; (ii) tert-butyl carbazate
(3 equiv), DMF, 8 h, rt; (iii) glyoxylic acid (4 equiv), Na2HPO4/NaOH, pH 5.2, rt, overnight; (iv) NaOH (2.2 equiv), H2O/EtOH, 0 °C, 30
min, rt, 3 h; (v) Boc2O (3 equiv), DMAP, Et3N, CH2Cl2, rt, 2 h; (vi) NaOH 1 N (1.1 equiv), H2O/EtOH, 0 °C, 25 min; (vii) H2/Pd(OH)2/C,
4°C, 24 h.
SCHEME 2 a
moacetate and tert-butyl carbazate was examined as
shown in Scheme 1 (path 2). In a first approach, the
reaction was carried out using a slight excess of ethyl
bromoacetate since preliminary experiments indicated
that excess of tert-butyl carbazate was difficult to sepa-
rate from ester 6a during workup. The nucleophilic
substitution was found to be faster in DMF than in
CH3CN. However, optimization of the reaction param-
eters could not preclude the formation of a mixture of
the expected compound 6a and a side product arising
from dialkylation of tert-butyl carbazate.
To overcome this problem, we switched to the use of
an excess of tert-butyl carbazate (3 equiv). To avoid
activation of the hydrazino moiety of 6a which could lead
to overalkylation, the reaction was carried out without
a
Key: (a) BOP, DIEA, DMF.
base, the generated HBr being trapped by the excess of
tert-butyl carbazate. Using these experimental conditions,
only traces of the byproduct were detected by TLC.
At this stage of the process, the unreacted tert-butyl
carbazate had to be removed as it was anticipated that
3 and the tetra-Boc hydrazine, inevitably formed during
the next step, would be difficult to separate. To eliminate
the excess by a simple aqueous extraction, we sought to
transform the carbazate into an acidic compound to
facilitate its separation from the intermediate ethyl Boc-
hydrazinoacetate 6a during workup. Indeed, the addition
of glyoxylic acid to the reaction mixture resulted in the
formation of the corresponding hydrazone, which was
readily soluble in water at basic pH.
The monoprotected ester 6a synthesized using this
alternative pathway was further converted into the
triprotected acid 3 as described before with a 45% overall
yield on a 9 g scale.
In a final optimization step, ethyl ester 6a was replaced
by the corresponding benzyl ester to avoid the saponifica-
tion step which can lead to a partial loss of one Nâ-Boc
group. Indeed, removal of the benzyl ester of compound
7b was easily performed by hydrogenolysis in the pres-
ence of palladium hydroxide on carbon (20% Pd). Starting
from benzyl bromoacetate 5b, compound 3 was obtained
via the formation of intermediates 6b and 7b in a 39%
overall yield on a 49 g scale.
equilibrium for each carbamate. In DMF-d7 at 300 K,
the methylene group came out as a singlet for both
compounds 2 and 3, whereas in CD2Cl2 they appeared
as a singlet for diprotected derivative 2 but as two
singlets in a nearly 1/1 ratio for 3.
Solid -P h a se F u n ction a liza tion Stu d ies. With the
three acid compounds 1-3 in hand, we next examined
their coupling onto a solid phase for the functionalization
of peptides by an R-hydrazinoacetyl moiety. A model
tripeptide YLA was assembled on a Wang resin using
the Fmoc/tert-butyl strategy (Scheme 2).
After peptide elongation, the resin was divided into
three samples. The N-terminal Fmoc group was removed
with piperidine, and the peptidyl resins were reacted with
either derivative 1-3 (4 equiv) using an in situ BOP
activation (chosen to minimize the potential risk of
azalactone formation during the activation step). After
30 min at rt, a ninhydrin test15 indicated the completion
of the acylation. Following deprotection and cleavage in
TFA, the peptides were precipitated in cold Et2O and
freeze-dried. The purity of the target peptide 8a was
evaluated by RP-HPLC, CZE and ES-MS. We have found
that only ES-MS allowed detection of the presence of
oligomers (n ) 1-2, Table 1 and Scheme 2) in the
mixture, which could be easily quantified as described
elsewhere.16 Their amount and distribution depended
upon the number of Boc groups on the hydrazino moiety
(Table 1). The use of the monoprotected acid 1 led to the
formation of monomer 8a , dimer 8b and trimer 8c in a
83/14/3 ratio (Table 1, entry 1). These results confirmed
the observations of Guy et al., who reported the formation
of polymeric products during activation of NR-unprotected
NMR Ch a r a cter iza tion . Unlike monoprotected com-
1
pound 1, hydrazino acids 2, and 3 presented complex H
NMR spectra in some deuterated solvents, suggesting a
slow exchange on the NMR time scale between conform-
ers. Indeed, 1H NMR spectra of diprotected acid 2 in
DMSO-d6 at 300 K showed four distinct sets of reso-
nances for Nâ-H proton (relative intensities 53/26/14/7).
At 330 K, the same proton appeared as a broad singlet.
This phenomenon could be the consequence of a cis/trans
(15) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal.
Biochem. 1970, 34, 595-598.
(16) Smart, S. S.; Mason, T. J .; Maeij, N. J .; Geysen, H. M. Int. J .
Peptide Protein Res. 1996, 47, 47-55.
J . Org. Chem, Vol. 68, No. 18, 2003 7035

Bonnet et al.
TABLE 1. ES-MS An a lysis of Hyd r a zin op ep tid e 8
Syn th esized Usin g Der iva tives 1, 2, or 3
TABLE 2. Op tim iza tion of Dip r otected
r-Hyd r a zin oa cetic 2 Cou p lin g
hydrazinopeptidesa,b
hydrazinopeptidesb
entry
1
R-hydrazino acid
8a
8b/8d
8c
entry coupling agents
activation
in situ
8a
8d
1
83 (1.0)
14
3
1
2
BOP (4 equiv)/
DIEA (12 equiv)
PyBOP (4 equiv)/ in situ
DIEA (12 equiv)
92.2
7.8
(0.8)
7.8
(0.5)
0
(0.2)
0
2
2
3
92 (1.2)
100
95.1
4.9
3
0
3
4
DIC(4 equiv)
HOSu (8 equiv)/ 15 min
DIC (4 equiv)
15 min
97.7
98.2
2.3
1.8
a
% estimated by ES-MS as a percentage of the total area (orifice
voltage of 30 V, sample dissolved in H2O/MeCN (4/1) formic acid
0.1%, injection at 5 µL per min). b The standard deviation for three
measurements is indicated in parentheses.
5
6
HOSu (4 equiv)/ overnight at 4 °C
DIC (4 equiv)
82.0
98.0
18.0
2.0
HBTU (4 equiv)/ 1 min
HOBt(4 equiv)
DIEA (12 equiv)a
hydrazino acids. The authors solved this problem by
using Nâ-Boc-NR-Bn-bisprotected hydrazino acids. But in
our hands, the use of diprotected derivative 2 did not
preclude the formation of dimer 8d , which was obtained
at a rate of 8% (Table 1, entry 2). The fact that Nâ in
acid 2 retained some nucleophilic character led us to
examine the use of the fully protected derivative 3.
Indeed, acylation of the peptidyl resin with activated 3
led to the expected R-hydrazino acetyl peptide 8a in a
high purity. Oligomers could not be detected by ES-MS
(Table 1, entry 3).
a
Coupling performed on an automatic peptide syntheziser
b
Applied Biosystems 431 A. % estimated by ES-MS as a percent-
age of the total area (orifice voltage of 30 V, sample dissolved in
H2O/MeCN (4/1) formic acid 0.1%, injection at 5 µL per min).
SCHEME 3 a
Nevertheless, regarding the low rate of dimer formed
with the diprotected acid 2 and the better chemical
stability of this compound compared to the fully protected
one, we undertook the optimization of the coupling step.
Various activating agents such as PyBOP, DIC, DIC/
HOSu, HBTU/HOBt were also investigated as possible
reagents for the solid-phase coupling of the diprotected
acid onto the model peptidyl resin used before. The time
of coupling was fixed at 40 min and the completion of
the reaction was verified using either ninhydrin or TNBS
tests.17 Again, the rate of oligomer formation was quanti-
fied using ES-MS following deprotection and cleavage
from the resin. Regardless of the coupling agent used,
the trimer species 8c were not detected. The amount of
dimer 8d ranged from 18.0% to 1.8% depending on the
nature of the coupling agent (Table 2). Experiments
performed with BOP or PyBOP gave a rate ranging from
4.9% to 7.8% (Table 2, entry 1 and 2). The best results
were obtained using HBTU/HOBt/DIEA, DIC, or DIC/
HOSu (Table 2, entries 3, 4, and 6), but dimer formation
could not be wholly suppressed. As expected, increasing
the time of preactivation from 15 min to overnight
resulted in an increase in the amount of dimer formed
(18%, Table 2, entry 5). Finally, the coupling carried out
on an automated peptide synthesizer with 4 equiv of
compound 2 using a standard HBTU/HOBt/DIEA activa-
tion procedure led to only 2% of dimer formation (Table
2, entry 6).
a
Key: (a) BrCH2CO2H, DIC, NMP; (b) Boc-NHNH2, DIEA,
NMP; (c) HOBt, HBTU, DIEA, NMP.
which can be neglected during a linear synthesis, can be
deleterious during the synthesis of branched dendrimers.
Typically, 2% of dimer formation is expected to generate
about 16% of impurities during the functionalization of
a tetravalent lysinyl tree. We have experienced that
oligomers formed during activation are very difficult to
separate from the target hydrazinopeptide by RP-HPLC.
Thus, we recommend the use of the fully protected
derivative 3 which can be easily synthesized at low cost
using the simple procedure described before.
Solid -P h a se HR-MAS NMR Rea ctivity Stu d ies.
Regarding the partial instability of triprotected acid 3
in both basic or slightly acidic media, we have investi-
gated the behavior of solid supports acylated with 3
during the standard procedures used for the Fmoc/tert-
butyl peptide elongation. To carry out this study, N,N,N′-
tris(Boc)hydrazinoacetyl group was anchored to an Ar-
gogel resin. The modified solid support was treated with
20% piperidine in DMF or 1% TFA in CH2Cl2, i.e., usual
conditions used during peptide elongation for Fmoc or
Mtt removal. The fate of the compound linked onto the
resin was monitored by HR-MAS NMR spectroscopy.
Such a low degree of dimer formation suggest that
derivative 2 could be used for hydrazinopeptide synthesis.
However, the problem is expected to be worse in the case
of a difficult synthesis necessitating multiple couplings19
or when a peptide has to be functionalized by multiple
R-hydrazinoacetyl groups such as for the synthesis of
lysinyl dendrimers. In this latter case, a side reaction,
Mono-, di-, and triprotected hydrazino derivatives were
prepared onto the solid support as described in Scheme
3 and served as references for the chemical shifts. Around
5 mg of each resin were swollen in CD2Cl2 and studied
by HR MAS NMR (Figure 2). The choice of the solvent
for the analysis was found to be crucial to observe a
difference between the three different resins. In DMF-
d7, the CH2 protons appeared as a broad singlet for all
(17) Hancock, W. S.; Battersby, J . E. Anal. Biochem. 1976, 71, 260-
264.
(18) Warrass, R.; Wieruszeski, J . M.; Lippens, G. J . Am. Chem. Soc.
1999, 121, 3787-3788.
7036 J . Org. Chem., Vol. 68, No. 18, 2003

Synthesis of R-Hydrazinoacetylpeptides
SCHEME 4
Syn th esis of a Mod el P ep tid e Usin g Tr ip r otected
Hyd r a zin oa cetic Acid 3. To illustrate the usefulness
of triprotected acid 3 for the efficient preparation of
R-hydrazinopeptides with a good yield and high purity,
model peptide 13 derived from Nef protein encoded by
the Simian Immunodeficiency Virus was synthezised
using the Fmoc/tert-butyl strategy (Scheme 4).
Fmoc-Lys(Mtt)-OH was first coupled onto a Pal-PEG-
PS resin using HBTU/HOBt activation. Following Mtt
deprotection with 1% TFA in CH2Cl2,20 N,N,N′-tris(Boc)-
hydrazinoacetic acid 3 was incorporated on ꢀ-NH2 group
using a PyBOP in situ activation to furnish resin 12. The
peptide was elaborated on the solid phase using standard
Fmoc/tert-butyl protocols, and then deprotected and
cleaved from the resin with TFA/EDT/H2O. Hydrazi-
nopeptide 13 was isolated with a 27% overall yield
following RP-HPLC purification. Peptide homogeneity
was verified by ES-MS analysis and no byproducts
arising from polymerization were detected.
It is worth noting that the organic solvent used for the
RP-HPLC purification was found to be crucial. Indeed,
we have observed a rapid decomposition of R-hydrazi-
nopeptides when stored in the lyophilized form following
RP-HPLC purification with acetonitrile as the mobile
phase. This behavior was unexpected, since 6-hydrazi-
nonorleucine-containing peptides were found to be stable
for months when purified under the same conditions.6
The decomposition occurred irrespective of the nature of
the counterion (chloride, trifluoroacetate). Finally, R-hy-
drazinopeptides proved to be fully stable when acetoni-
trile was replaced by 2-propanol.21
F IGURE 2. HR MAS NMR spectra in CD2Cl2 at 600 MHz
recorded with the LED diffusion sequence: (A) resin 10; (B)
resin 11; (C) resin 11 treated with piperidine 20% in DMF
during 3 h at rt.
the three derivatives, whereas in CD2Cl2 the CH2 protons
were observed as a singlet for resin 10 but as two singlets
for resin 11 (Figure 2A,B). 1H NMR spectra were recorded
on a 600 MHz using the Longitudinal Eddy current Delay
(LED) sequence to eliminate residual signals arising from
protonated species diffusing freely in solution.18 This
sequence allowed us to perform the analyses in the
presence of the reagents used in the stability study.
These studies allow us to propose an efficient route for
the solid-phase incorporation of R-hydrazinoacetyl moiety
either on a C-terminal or N-terminal part of a peptide.
The fully N,N,N′-triprotected acid 3 was found to be a
valuable compound to suppress any oligomerization and
gave access to highly pure hydrazinopeptides. As an
alternative, the more stable N,N′-diprotected acid 2 can
First, the potential removal of an Mtt group while
keeping the chemical integrity of tri-protected hydrazi-
noacetyl group was examined by treating resin 11 with
1% TFA in CH2Cl2 for 30 min and 1, 3, or 5 h at rt. Within
this time, the Boc group of the imidodicarbonate dis-
played excellent stability. The stability of the same Boc
group toward 20% piperidine in DMF was then exam-
ined. After 30 min at rt, a sample of this resin was
washed and loaded in a rotor. Based on the integration
of the CH2 signals, we found a conversion of 5% of the
triprotected form into the diprotected one. An analysis
after 3 h indicated about 17% of deprotection (Figure 2C).
The resin was then washed and treated with Ac2O 10%/
diisopropylethylamine 5% in CH2Cl2 for 10 min to
determine the propensity of the Nâ-deprotected group to
undergo acetylation. No product of acetylation could be
(19) However, we have shown the propensity of the Nâ of the bis-
Boc hydrazino acetic acid anchored onto a resin to be acylated using a
BOP in situ activation. Indeed, following a first coupling of the
diprotected acid, driven to completion as indicated by a negative Kaiser
test, a second and a third coupling in the same conditions were
performed on solid phase. Following the number of coupling, the
proportion of dimer increased from 7.8% (1 coupling) to 13.4% (2
couplings) and 18.0% (3 couplings).
(20) Bourel, L.; Carion, O.; Gras-Masse, H.; Melnyk, O. J . Peptide
Sci. 2000, 6, 264-270.
(21) Bonnet, D.; Gras-Masse, H.; Melnyk, O. Manuscript in prepara-
tion.
1
detected in the H HR MAS NMR spectrum.19
J . Org. Chem, Vol. 68, No. 18, 2003 7037

Bonnet et al.
performed at 298 K on a 600 MHz spectrometer equipped with
a 4 mm HR MAS probe using a 6 kHz spinning rate. 1H NMR
spectra were recorded with 16 scans and diffusion filtered
experiment (LED) with 64 scans. Gradient parameters for the
LED based sequence were as previously described.18
be used using an HBTU/HOBt activation and as long as
a 2% dimer contamination is acceptable.
Con clu sion
Syn th esis of N-ter t-Bu tyloxyca r bon yl Hyd r a zin oa ce-
tic Acid 1. Commercially available ethyl hydrazinoacetate
(20.1 g, 130 mmol) and di-tert-butyl dicarbonate (31.3 g, 144
mmol) were dissolved in 130 mL of a mixture H2O/EtOH (1/
1). N-Methylmorpholine (15.8 mL, 144 mmol) were added
dropwise, and the resultant mixture was stirred for 2 h in a
water bath. NaOH (11.5 g, 287 mmol) was then added in five
aliquots, and the solution was stirred for further 2 h at rt.
Following dilution with a saturated solution of NaCl, the
mixture was first extracted with diethyl ether (1 100 mL). The
aqueous layer was then acidified to pH 3 with a solution of
citric acid and extracted with diethyl ether (2 × 100 mL). The
combined extracts were washed with water (50 mL), dried over
Na2SO4, filtered, concentrated under reduced pressure, and
dried under vacuum. The resultant solid was precipitated after
dilution in methyl tert-butyl ether to afford 1 (12.2 g, 49%
overall yield) as a white solid: mp 124-125 °C; HRMS [M +
H]+ calcd for C7H15O4N2 191.1032, found 191.1028 (-2.2 ppm);
MALDI-TOF [M + Na]+ calcd 213.085, found 213.087; 1H NMR
(MeOH-d4, 300 MHz) δ 4.07 (s, 2H), 1.45 (s, 9H); 13C NMR
(MeOH-d4, 75 MHz) δ 173.3 and 172.2, 81.4, 53.0, 52.3, 51.8,
27.6, 27.5. Anal. Calcd for C7H14N2O4: C, 44.20; H, 7.42; N,
14.73. Found: C, 44.16; H, 7.59; N, 14.32.
As part of our program on the solid-phase synthesis of
hydrazinopeptides, we have developed a new strategy to
prepare R-hydrazinoacetyl peptides in an efficient and
readily scalable way. The approach reported in this work
relies on the coupling of Boc-protected derivatives of
R-hydrazinoacetic acid directly onto the peptide. A low
cost route to a fully Boc-protected derivative is described.
We have shown the need to fully protect the hydrazino
moiety to avoid polymerization of the activated ester
during the coupling. The process has been fully auto-
mated allowing the synthesis of various hydrazinopep-
tides in a good yield and high purity. In our laboratory,
R-hydrazinoacetyl peptides have proved to be valuable
partners in the hydrazone chemical ligation with R-oxo
aldehyde derivatives giving a straightforward access to
synthetic peptide conjugates and high molecular weight
constructs.
Exp er im en ta l Section
1
Gen er a l Con sid er a tion s. H and 13C NMR spectra were
Syn th esis of Eth yl [N-(ter t-Bu tyloxyca r bon yl)h yd r a zi-
n o]a ceta te 6a . P a th 1. Commercially available ethyl hy-
drazinoacetate (14.9 g, 96.4 mmol) and di-tert-butyl dicarbon-
ate (26.1 g, 120 mmol) were dissolved in 70 mL of a mixture
H2O/EtOH (1/1). Following dissolution, the mixture was cooled
to 0 °C, and 13.2 mL (120 mmol) of N-methylmorpholine was
added dropwise. The resultant mixture was stirred for 15 min
at 0 °C and 2 h at rt. The solution was then diluted with water
saturated with KH2PO4 and extracted with diethyl ether (2 ×
70 mL) and petroleum ether (2 × 70 mL). The combined
organic layers were dried over Na2SO4, filtered, and concen-
trated under reduced pressure to give crude 6a (19.8 g) as an
orange oil which was used without any further purification
for the next step: 1H NMR (CDCl3, 300 MHz, 323 K) δ 4.19
(q, 2H, J ) 7.1 Hz), 4.11 (s, 2H), 1.45 (m, 9H), 1.26 (t, 3H, J )
7.1 Hz); 13C NMR (CDCl3, 75 MHz, 323 K) δ 175.5, 174.3,
161.7, 160.8, 85.8, 85.2, 65.5, 65.4, 57.41, 32.8, 32.7, 32.6, 18.7.
Anal. Calcd for C9H18N2O4: C, 49.53; H, 8.31; N, 12.84; O,
29.32. Found: C, 49.78; H, 8.36; N, 12.33; O, 29.27.
recorded at 300 or 75.5 MHz. Chemical shifts are given in ppm
and referenced to internal TMS. For the assignment of signals
1
1H, 13C, and H-13C heteronuclear single quantum correlation
(HSQC) spectroscopy experiments were used. Thin-layer chro-
matography was performed using E. Merck plates of silica gel
60 with fluorescent indicator. Visualization was effected by
spraying plates with 5% phosphomolybdic acid in EtOH
followed by heating at 120-140 °C. All reagents were used
directly as obtained commercially.
N,N′,N′-Tris(tert-butyloxycarbonyl)hydrazino]acetic acid is
now commercially available from NOVABIOCHEM.
Solid-phase peptide syntheses were performed using stan-
dard Fmoc/t-Bu chemistry on Perseptive Pioneer peptide
synthesizers. The amino acids were activated using HBTU/
HOBt/diisopropylethylamine (AA/HBTU/HOBt/DIEA: 4 equiv/4
equiv/4 equiv/8 equiv) in DMF. Side chain protections were
as follows: Tyr(t-Bu), Lys(Boc) or Lys(Mtt), Ser(t-Bu), Trp-
(Boc), Thr(t-Bu), Asp(O-t-Bu), Glu(O-t-Bu). Fmoc amino acids
were purchased from Senn Chemicals.
P a th 2. Ethyl bromoacetate 5a (5.6 mL, 50.4 mmol, 1 equiv)
diluted in DMF (10 mL) was added by means of a dropping
funnel to a stirred solution of tert-butylcarbazate (19.8 g, 151
mmol) in DMF (40 mL) at rt. The mixture was further stirred
for 8 h. A solution of glyoxylic acid was added to the reaction
vessel. The solution was prepared as follows: solid glyoxylic
acid (18.6 g, 200 mmol) was dissolved in 0.4 M aqueous
Na2HPO4 (50 mL), and the pH adjusted to 5.2 by addition of
5 N aqueous NaOH. Following overnight stirring, the crude
mixture was poured into saturated aqueous NaHCO3 (300 mL)
and extracted by diethyl ether (5 × 100 mL). The combined
extracts were washed with brine (2 × 50 mL), dried over
Na2SO4, filtered, concentrated under reduced pressure, and
dried under vacuum to give crude ethyl [N-(tert-butyloxycar-
bonyl)hydrazino]acetate 6a (7.56 g) as a yellow oil which was
directly engaged in the next step: Rf ) 0.48 (heptane/AcOEt
1:1).
Ch a r a cter iza tion of P u r ified Hyd r a zin op ep tid es. The
RP-HPLC analyses were performed on a C18 Vydac (4.6 × 250
mm) column using water/2-propanol linear gradient (0-100%
B in 60 min, 1 mL/min, 50 °C, 215 nm). The following buffers
were used: (eluent A) water containing 0.05% TFA by volume;
(eluent B) 2-propanol/water 2/3 by volume containing 0.05%
TFA by volume.
Capillary zone electrophoresis (CZE) was performed on an
Applied Biosystems 270A-HT apparatus in a 75 µm × 50 cm
fused silica capillary, with 40 mÅ current and a 30 kV field.
The analyses were undertaken in a pH 3.0 sodium citrate
buffer.
For electrospray mass spectrometry (ES-MS) studies, the
m/z range 200-2100 was scanned using an ionspray voltage
of 4500 V. The orifice plate voltage was ranged between 30
and 60 V. The temperature in the ionization chamber was 60
°C.
Amino acid compositions were determined using a Beckman
amino acid analyzer model 7300 with ninhydrin detection,
following hydrolysis in evacuated sealed tubes with 6 N HCl/
phenol:10/1 (v/v) at 110 °C for 24 h.
MAS NMR Exp er im en ts. Resin was washed twice with
CH2Cl2 and filtered. Then, 5 mg of this resin were loaded in a
4 mm rotor and swollen with CD2Cl2. Tetramethylsilane (TMS)
was added as a internal reference. All NMR experiments were
Syn t h esis of Ben zyl [N-(ter t-Bu t yloxyca r b on yl)h y-
d r a zin o]a ceta te 6b via P a th 2. Benzyl bromoacetate (51.1
mL, 321 mmol) diluted in DMF (64 mL) was added over 10
min by means of a dropping funnel to a stirred solution of tert-
butylcarbazate (128 g, 968 mmol) in DMF (256 mL) at rt. The
mixture was further stirred for 18 h. To the reaction vessel
was then added a solution of sodium glyoxylate prepared as
follows: solid glyoxylic acid (119.0 g, 1285 mmol) was dissolved
7038 J . Org. Chem., Vol. 68, No. 18, 2003

Synthesis of R-Hydrazinoacetylpeptides
in H2O (192 mL) and then carefully added to a 10 N solution
of NaOH (51.20 g, 1280 mmol) in H2O (128 mL) at 0 °C to
give a yellow solution (pH ∼4-5, paper). The reaction mixture,
from which a precipitate appeared, was stirred for further 24
h at rt and then poured into a saturated aqueous NaHCO3
solution (1.5 L) and extracted by diethyl ether (4 × 300 mL).
The combined extracts were washed with brine (2 × 50 mL),
dried over Na2SO4, filtered, concentrated under reduced pres-
sure, and dried under vacuum to give crude benzyl [N-(tert-
butyloxycarbonyl)hydrazino]acetate 6b (83.0 g) as a yellow
syrup which was directly used in the next step: Rf ) 0.53
mixture was stirred for 25 min at 0 °C and then neutralized
by slow addition of an aqueous citric acid solution (634
mg‚mL-1) to pH 4 (paper). The crude mixture was then diluted
with H2O (50 mL) and extracted with diethyl ether (2 × 80
mL) and CH2Cl2 (2 × 80 mL). The combined extracts were
washed with brine (2 × 40 mL), dried over Na2SO4, filtered,
and concentrated under reduced pressure. The oily residue was
precipitated after dilution with a cooled mixture of diethyl
ether/heptane to furnish pure [N,N′-tris(tert-butyloxycarbonyl)-
hydrazino]acetic acid (9.6 g, 63% overall yield): Rf ) 0.24 (1:1
heptane/AcOEt); mp 119-120 °C; 1H NMR (DMSO-d6, 300
MHz) 4.04 (s, 2H), 1.42 (m, 27H); 13C NMR (DMSO-d6, 75
MHz)169.3, 153.5, 149.8, 82.9, 81.7, 81.1, 53.1, 51.2, 27.8. Anal.
Calcd for C17H30N2O8: C, 52.30; H, 7.74; N, 7.17; O, 32.78.
Found: C, 52.63; H, 7.73; N, 7.21; O, 32.81.
1
(heptane/AcOEt 1:1); H NMR (CDCl3, 300 MHz) δ 1.44 (s, 3
× 3H), 3.74 (s, 2H), 5.17 (s, 2H), 7.35 (brs, 5H); 13C NMR
(CDCl3, 75 MHz) δ 28.2, 52.6, 67.1, 81.5, 125.2, 128.4, 128.6,
128.7, 135.1, 156.3, 170.5; TOF-PDMS MW calcd 280.1, m/z
found 224.7 [M + H - tBu]+, 180.1 [M + H - Boc]+.
Syn th esis of [N,N,N′-Tr is(ter t-bu tyloxyca r bon yl)h y-
d r a zin o]a cetic Acid 3. Crude benzyl [N-(tert-butyloxycar-
bonyl)hydrazino]acetate 6b (80 g, 285 mmol) was dissolved in
CH2Cl2 (51 mL) containing Et3N (125 mL, 891 mmol) and then
cooled to 0 °C. This mixture was then added by means of a
dropping funnel over 2 h to a solution of di-tert-butyl dicar-
bonate (194 g, 889 mmol) and DMAP (1.09 g, 0.89 mmol) in
CH2Cl2 (64 mL) under N2 at 0 °C. The reaction mixture was
warmed to rt with stirring for 24 h. As the reaction was not
completed (presence of a mixture of di- and tri-Boc-protected
derivatives as monitored by TLC), additional di-tert-butyl
dicarbonate (194 g, 889 mmol) was introduced to the reac-
tion vessel. The mixture was stirred for 48 h and then diluted
with CH2Cl2 (160 mL) and washed with saturated aqueous
KH2PO4 (3 × 255 mL). The organic phase was dried over
Na2SO4, filtered, and concentrated under reduced pres-
sure to give an orange-brown oil which was further passed
through a short column of silica gel (40-60 µm, 1.4 kg)
(eluent: CH2Cl2/AcOEt 97:3). The filtrate was concentrated
under reduced pressure and dried overnight over P2O5 to give
the fully protected derivative 7b as an orange oil which was
used directly in the next step: Rf ) 0.53 (heptane/AcOEt 7:3).
To a solution of crude benzyl [N,N,N′-tris(tert-butyloxycarbo-
nyl)hydrazino]acetate 7b (145 g, 302 mmol) in EtOH (1000
mL) cooled at 0 °C was added palladium hydroxide on carbon
(20% Pd/C, 5.8 g, 4% w/w). The mixture was then hydrogeno-
lyzed at atmospheric pressure of hydrogen for 24 h at 4 °C.
The crude mixture was filtered through a pad of Celite which
was washed with CH2Cl2. The solvent was concentrated under
reduced pressure to give an orange oil. Following precipitation
in a cooled mixture of diethyl ether/cyclohexane, pure [N,N,N′-
tris(tert-butyloxycarbonyl)hydrazino]acetic acid (49 g, 39%
overall yield) was obtained as a white solid: Rf ) 0.24
(heptane/AcOEt 1:1); HRMS calcd for C17H31N208 391.2080,
found 391.2082; MALDI-TOF [M + Na]+ calcd 413.190, found
413.1919. Anal. Calcd for C17H30N2O8: C, 52.30; H, 7.74; N,
7.17; O, 32.78. Found: C, 52.26; H, 7.77; N, 7.22; O, 32.63.
Syn th esis of Eth yl [N,N,N′-Tr is(ter t-bu tyloxyca r bon -
yl)h yd r a zin o]a ceta te 7a via P a th 1. Crude ethyl [N-(tert-
butyloxycarbonyl)hydrazino]acetate 6a (19.8 g, 91.1 mmol) was
dissolved in CH2Cl2 (16 mL) containing Et3N (38.5 mL, 276
mmol) at 0 °C under N2. This mixture was then added by
means of a dropping funnel to a solution of di-tert-butyl
dicarbonate (60.2 g, 276 mmol) and DMAP (3.37 g, 27.6
mmol) in CH2Cl2 (20 mL) under N2 at 0 °C. The reaction
mixture was warmed to rt while stirring. After 2 h, the crude
mixture was then diluted with CH2Cl2 (50 mL) and washed
with saturated aqueous KH2PO4 (3 × 75 mL). The organic
layer was dried over Na2SO4, filtered, and concentrated under
reduced pressure to give an orange oil which was further
passed through a short column of silica gel (40-60 µm, 160 g,
eluent: CH2Cl2/AcOEt 97/3 by volume). The filtrate was
concentrated under reduced pressure and dried overnight over
P2O5 to give the fully protected derivative 7a which was
directly used in the next step (yellow oil, 36.9 g): Rf ) 0.68
(heptane/AcOEt 1:1); MALDI-TOF MW calcd 418.2, m/z found
441.4 [M + Na]+, 457.4 [M + K]+; 1H NMR (CDCl3, 300 MHz)
δ 4.16 (s, 2H), 3.71 (q, 2H, J ) 7.2 Hz), 1.46 (m, 27H), 1.23 (t,
3H, J ) 7.2 Hz); 13C NMR (CDCl3, 75 MHz) δ 167.74, 150.4,
150.2, 83.6, 82.4, 82.0, 60.9, 58.4, 53.3, 51.5, 28.0, 18.4, 14.2.
Anal. Calcd for C19H34N2O8: C, 54.53; H, 8.19; N, 6.69.
Found: C, 54.81; H, 8.25; N, 6.71.
Syn th esis of [N,N′-Bis(ter t-bu tyloxyca r bon yl)h yd r a zi-
n o]a cetic Acid 2. To a solution of crude ethyl [N,N,N′-tris-
(tert-butyloxycarbonyl)hydrazino]acetate 7a (19.2 g, 88.3 mmol)
in EtOH (135 mL) cooled at 0 °C was added cooled aqueous 1
N NaOH (135 mL, 135 mmol) by means of a dropping funnel.
Following 30 min of stirring at 0 °C and a further 3 h at rt,
the crude mixture was diluted with H2O (110 mL) and
extracted with diethyl ether (2 × 80 mL). The aqueous layer
was then acidified to pH 2 by means of a solution 1 N HCl
and extracted with CH2Cl2 (2 × 80 mL) and Et2O (2 × 80 mL).
The combined extracts were washed with brine (2 × 40 mL),
dried over Na2SO4, filtered, and concentrated under reduced
pressure. The resultant oily residue was precipitated after
dilution with a cooled mixture of diethyl ether/heptane to
afford pure [N,N′-di-(tert-butyloxycarbonyl)hydrazino]acetic
acid (17.9 g, 66% overall yield): Rf ) 0.24 (heptane/AcOEt 1:1);
mp 113-114 °C; HRMS: calcd for C12H23O6N2 291.1556, found
291.1551 (-1.7 ppm); MALDI-TOF [M + Na]+ calcd 313.137,
found 313.141; 1H NMR (DMSO-d6, 300 MHz) δ 9.23 (s, 0.53H)
9,16 (s, 0.26H), 8.84 (s, 0.14H), 8.74 (s, 0.07H), 3.97 (s, 2H),
1.39 (m, 18H); 13C NMR (DMSO-d6, 75 MHz, 300 K) 170.1,
155.0, 154.1, 80.1, 79.5, 54.6, 53.1, 27.9; HMBC δH NH 9.23
and 9.16, coupling to δC 155.0; δH CH2 3.97, coupling to δC
170.1 and 155.0. Anal. Calcd for C12H22N2O6: C, 49.65; H, 7.64;
N, 9.65; O, 33.07. Found: C, 50.09; H, 7.84; N, 9.57; O, 32.64.
Syn t h esis of H -SKWDDP WGE VLAWKF DP TLAYTY-
EAK(COCH2NHNH2)-NH2. Peptide 13 was elaborated start-
ing from 3 mmol of Fmoc-Pal-PEG-PS resin (0.16 mmol/g,
Applied Biosystems, Foster City, CA). Fmoc-L-Lys(Mtt)-OH
(7.5 g, 12 mmol) was coupled using HBTU (4.55 g, 12 mmol)/
HOBt (1.84 g, 12 mmol)/DIEA (3.38 mL, 48 mmol). The Mtt-
protecting group was removed by 1% TFA in CH2Cl2, and the
deprotection was monitored by RP-HPLC as previously re-
ported.20 Following washing with CH2Cl2 and DMF, (Boc)2N-
N(Boc)CH2COOH (1.41 g, 3.6 mmol) was coupled using PyBop
(1.87 g, 3.6 mmol) and DIEA (1.25 mL, 7.2 mmol) activation
(30 min at rt). The completion of the reaction was monitored
using a TNBS test. Resin was then successively rinsed with
DMF, CH2Cl2, and diethyl ether and dried under vacuum, and
0.1 mmol of resin was loaded in a reactor vessel for SPPS in
a Pioneer Perseptive Biosystems syntheziser. Peptide 13 was
then elaborated using a Fmoc/tert-butyl strategy. Each coup-
ling step was followed by treatment with Ac2O/DIEA/DMF (3/
0.3/96.7) by volume. Cleavage and deprotection steps were
performed using 10 mL of TFA/EDT/H2O (95/2.5/2.5) by
Syn th esis of [N,N,N′-Tr is(ter t-bu tyloxyca r bon yl)h y-
d r a zin o]a cetic Acid 3 F ollow in g Sa p on ifica tion . To a
solution of crude ethyl [N,N,N′-tris(tert-butyloxycarbonyl)-
hydrazino]acetate 7a (15.05 g, 36.0 mmol) in EtOH (40 mL)
cooled at 0 °C was added ice-cooled aqueous 1 N NaOH (39.6
mL, 39.6 mmol) by means of a dropping funnel. The reaction
J . Org. Chem, Vol. 68, No. 18, 2003 7039

Bonnet et al.
volume mixture during 2 h at rt. The crude peptide was
precipitated in cold diethyl ether, redissolved in water/acetic
acid 5/1 by volume, and lyophilized. Purification was performed
on a C3 Zorbax column (15 × 500 mm) using buffers A and B
(20-50% B in 30 min, 50% for 25 min, 50-70% in 20 min, 3
mL‚min-1, detection at 215 nm). Following a freeze-drying
step, peptide 13 was obtained as a white powder (98.4 mg,
27%): ES-MS [M + H]+ calcd 3188.8, found 3187.0; RP-HPLC
purity (215 nm) one peak >95%.
Abbr eviation s: BOP, benzotriazol-1-yloxytris(dimeth-
ylamino)phosphonium hexafluorophosphate; CZE, cap-
illary zone electrophoresis; DIC, diisopropylcarbodi-
imide; DIEA, diisopropylethylamine; EDT, ethanedithiol;
ES-MS, electrospray-mass spectroscopy; HBTU, N-[(1H-
benzotriazol-1-yl)(dimethylamino)methylene]-N-methyl-
methanaminium hexafluorophosphate N-oxide; HOBt,
N-hydroxybenzotriazole; HOSu, N-Hydroxysuccinimide;
PyBOP, benzotriazole-1-yl-oxytrispyrrolidinophosphon-
ium hexafluorophosphate.
Ack n ow led gm en t. We thank Bernadette Coddeville
and Guy Ricart for the ES-MS studies (Universite´ des
Sciences et Technologies de Lille), Ge´rard Montagne for
performing the NMR experiments, and Dr. David Hill
for proofreading the manuscript. We gratefully acknowl-
edge financial support from Institut Pasteur de Lille,
University of Lille 2, Agence Nationale de Recherche
sur le SIDA (ANRS) and Centre National de la Recher-
che Scientifique (CNRS).
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra for
compounds 1-3, 6a ,b, and 7a ,b, 13C NMR spectra for com-
pounds 1-3 and 6a ,b, MS spectra for compounds 1-3 and 13,
and HPLC for peptide 13. This material is available free of
charge via the Internet at http://pubs.acs.org.
J O0343432
7040 J . Org. Chem., Vol. 68, No. 18, 2003