Triurethane-protected guanidines and triflyldiurethane-protected guanidines: New reagents for guanidinylation reactions

Article abstract of DOI:10.1021/jo9814344

New guanidinylation reagents are reported. These reagents consist of N,N',N''-tri-Boc-guanidine (1) and N,N',N''-tri-Cbz-guanidine (2), which allow for the facile conversion of alcohols to substituted guanidines. A series of arginine analogues were synthesized via condensation of a primary or secondary alcohol with the guanidinylation reagents 1 or 2, under Mitsunobu conditions, to produce protected alkylated guanidines. In addition, an extended study of the previously reported reagents N,N'-di-Boc-N''- triflylguanidine (3) and N,N'-di-Cbz-N''-triflylguanidine (4) is presented. The triflyldiurethane-protected guanidine 3 was utilized to guanidinylate primary and secondary amines under mild conditions with high yield in both solution and on solid phase.

Full text of DOI:10.1021/jo9814344

8432
J . Org. Chem. 1998, 63, 8432-8439
Tr iu r eth a n e-P r otected Gu a n id in es a n d
Tr iflyld iu r eth a n e-P r otected Gu a n id in es: New Rea gen ts for
Gu a n id in yla tion Rea ction s
Konrad Feichtinger,† Heather L. Sings, Tracy J . Baker, Kenneth Matthews, and
Murray Goodman*
Department of Chemistry and Biochemistry, University of California, San Diego,
La J olla, California, 92093-0343
Received J uly 22, 1998
New guanidinylation reagents are reported. These reagents consist of N,N′,N′′-tri-Boc-guanidine
(1) and N,N′,N′′-tri-Cbz-guanidine (2), which allow for the facile conversion of alcohols to substituted
guanidines. A series of arginine analogues were synthesized via condensation of a primary or
secondary alcohol with the guanidinylation reagents 1 or 2, under Mitsunobu conditions,1 to produce
protected alkylated guanidines. In addition, an extended study of the previously reported reagents2
N,N′-di-Boc-N′′-triflylguanidine (3) and N,N′-di-Cbz-N′′-triflylguanidine (4) is presented. The
triflyldiurethane-protected guanidine 3 was utilized to guanidinylate primary and secondary amines
under mild conditions with high yield in both solution and on solid phase.
In tr od u ction
agents include derivatives of pyrazole-1-carboxamidine,
S-alkylisothioureas, and protected thiourea derivatives,
the last mostly used in conjunction with mercury salts
or the Mukaiyama reagent.5-14 Despite recent progress,
a completely satisfactory guanidinylating reagent has not
yet been developed. For example, most of the guanidi-
nylating reagents present in the literature are limited
by the availability of the starting amines, though some
examples of the conversion of alcohols to guanidines have
been reported.8 In addition, most of the known guanidi-
nylating reagents are incompatible with solid-phase
synthesis. It is apparent that a completely versatile
guanidinylating reagent must meet several basic crite-
ria: (1) the reagent should not be limited to simple
amines, (2) it should be amenable to solid phase synthe-
sis, (3) it should furnish high yields for a comprehensive
array of substrates in a number of organic and/or aqueous
solvents, and (4) the reagent should be readily available
from inexpensive starting materials.
The guanidine unit of arginine residues plays an
essential role in biologically active proteins, peptides, and
peptidomimetics.3,4 Guanidine itself, the imine of urea,
has a strongly basic character (pKa of guanidinium ion
∼13.5) which renders the side chain of arginine to be fully
protonated under physiological conditions. The positive
charge thus imposed on the molecule forms the basis for
specific interactions between ligand and receptor or
enzyme and substrate, mediated by hydrogen bonds and/
or electrostatic interactions. In addition to simple argi-
nine-containing peptides, a wide array of structurally
diverse molecules that incorporate single or multiple
guanidine units have been isolated from microorganisms,
terrestrial invertebrates, and marine and freshwater
organisms as well as higher plants.3 Many of these novel
metabolites possess unprecedented biological activity
ranging from antimicrobial, antiviral, and antifungal to
neurotoxic. Hence, guanidine-containing bioactive mol-
ecules, particularly the analogues or derivatives of natu-
ral products, are notable targets for drug design and
discovery. Consequently, procedures that allow for the
preparation of guanidines with high yield and under mild
conditions are of great interest in medicinal chemistry,
and much effort has been directed toward developing
efficient synthetic routes for the preparation of these
compounds.
In a recent report, we introduced the diprotected
triflylguanidines, a new class of guanidinylation reagents.
These reagents, N,N′-di-Boc-N′′-triflylguanidine (3) and
N,N′-di-Cbz-N′′-triflylguanidine (4), were utilized to
Typically, the synthesis of guanidine-containing com-
pounds involves treatment of an amine with an electro-
philic amidine species. The most commonly used re-
* To whom correspondence should be addressed. Phone: (619) 534-
4466. Fax: (619) 534-0202. E-mail: mgoodman@ucsd.edu.
† Current address: Cytel Corporation, 3525 J ohn Hopkins Ct., San
Diego, CA.
(1) Mitsunobu, O.; Yamada, M.; Mukaiyama, T. Bull. Chem. Soc.
J pn. 1967, 40, 935.
(2) Feichtinger, K.; Zaph, C.; Sings, H. L.; Goodman, M. J . Org.
Chem. 1998, 63, 3804.
(3) Berlinck, R. G. S. Fortschr. Chem. Org. Naturst. 1995, 66, 119.
(4) Berlinck, R. G. S. Nat. Prod. Rep. 1996, 337.
(5) Poss, M. A.; Iwanowicz, E.; Reid, J . A.; Lin, J .; Gu, Z. Tetrahedron
Lett. 1992, 33, 5933.
(6) Bernatowicz, M. S.; Wu, Y.; Matsueda, G. R. Tetrahedron Lett.
1993, 34, 3389.
(7) Kim, K. S.; Qian, L. Tetrahedron Lett. 1993, 34, 7677.
10.1021/jo9814344 CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/27/1998

New Reagents for Guanidinylation Reactions
J . Org. Chem., Vol. 63, No. 23, 1998 8433
Sch em e 1
Sch em e 2
Sch em e 3
guanidinylate primary and secondary amines under mild
conditions with high yield.2 We have now modified this
class of compounds to include the new guanidinylation
reagents N,N′,N′′-tri-Boc-guanidine (1) and N,N′,N′′-tri-
Cbz-guanidine (2), which allow for the facile conversion
of alcohols to substituted guanidines. The scope and
limitations of N,N′,N′′-tri-Boc-guanidine and N,N′,N′′-tri-
Cbz-guanidine, as well as an extended examination of
the previously described reagents 3 and 4 are reported
herein.
Sch em e 4
Resu lts a n d Discu ssion
Tr iu r eth a n e-P r otected Gu a n id in es: Con ver sion
of Alcoh ols to Gu a n id in es. The triurethane-protected
guanidines, N,N′,N′′-tri-Boc-guanidine (1) and N,N′,N′′-
tri-Cbz-guanidine (2), may be used to convert alcohols
to guanidines in one step. The synthetic routes toward
these symmetrical trisubstituted guanidines are shown
in Scheme 1. The tri-Boc derivative 1 was prepared from
guanidine hydrochloride (5) in a one-pot procedure with
yields up to 83%. Guanidine hydrochloride was added
to a solution of potassium hydroxide and sodium carbon-
ate in DMSO. After the addition of di-tert-butyl dicar-
bonate, the mixture was allowed to stir for 60 h at 40
°C. The product was then precipitated by pouring the
reaction mixture into cold water and purified by recrys-
tallization from acetonitrile.
The structure of reagent 1 was confirmed by X-ray
analysis. This analysis was necessary to unambiguously
determine that reagent 1 was a symmetric molecule, with
one Boc group on each of the nitrogen atoms (see
Supporting Information). All of the atoms were found
to lie in a single plane with the exception of the tert-butyl
groups. The three carbon-nitrogen bonds of the central
guanidine core all have the same bond length, indicating
a fully conjugated system.
Guanidinylation reagents of the triurethane type react
with primary alcohols under Mitsunobu conditions to
produce protected alkylated guanidines.1,8 This was
exemplified in the synthesis of several orthogonally
protected arginine analogues from suitable precursor
molecules (Schemes 2-4). The precursor alcohols 9,15
10,15 14,16 and 16 were synthesized from an appropriately
protected aspartic or glutamic acid following the proce-
dure of Kokotos.17 For example, commercially available
N-Cbz-Asp-OBn (7) was first converted to the mixed
anhydride derivative with ethyl chloroformate and N-
ethylmorpholine (NEM). The anhydride was then re-
duced with sodium borohydride to afford alcohol 9
(Scheme 2).
Introduction of the fully protected guanidine unit was
accomplished by condensation of 1 mol equiv of the
alcohol with 3.0 equiv of reagent 1 or 2 and 1.5 equiv
each of triphenylphosphine and diethylazodicarboxylate
(DEAD). Reactions with the tri-Boc derivative 1 were
most effectively carried out in refluxing THF or toluene,
and yields of up to 72% were obtained (Schemes 2 and
4). If reagent 2 was used as the guanidinylating species,
the reaction could be carried out at room temperature in
THF (Scheme 3). In addition, yields for reactions with
the tri-Cbz derivative 2 were somewhat higher than those
for comparable reactions with the tri-Boc derivative 1,
ranging from 86% to quantitative yield. In contrast to
the traditional syntheses of guanidine-containing com-
pounds, this new synthesis uses an alcohol as the starting
material, thus opening an additional route to a wide
range of arginine analogues. In addition, the optical
integrity of the R-carbon stereocenter is maintained.
During the initial studies on the conversion of alcohol
10 to the protected arginine analogue 12, a side product
The synthesis of N,N′,N′′-tri-Cbz-guanidine (2) was
likewise facile, with the diprotection of guanidine hydro-
chloride accomplished in one step in good yield (75%).
The diprotected guanidine 6 was then treated with 2
equiv of sodium hydride under anhydrous conditions
followed by acylation of the resulting anion to afford
reagent 2. The crude product was purified by column
chromatography.
(8) Dodd, D. S.; Kozikowski, A. P. Tetrahedron Lett. 1994, 35, 977.
(9) Drake, B.; Patek, M.; Lebl, M. Synthesis 1994, 579.
(10) Katriztky, A. R.; Parris, R. L.; Allin, S. M.; Steel, P. S. Synth.
Commun. 1995, 25, 1173.
(11) Kent, D. R.; Cody, W. L.; Doherty, A. M. Tetrahedron Lett. 1996,
37, 8711.
(12) Levallet, C.; Lerpiniere, J .; Ko, S. Y. Tetrahedron 1997, 53,
5291.
(13) Yong, Y. F.; Kowalski, J . A.; Lipton, M. A. J . Org. Chem. 1997,
62, 1540.
(15) Rosenthal, G. A.; Dahlman, D. L.; Crooks, P. A.; Phuket, S. N.;
Trifonov, L. S. J . Agric. Food Chem. 1995, 43, 2728.
(16) Akimoto, H.; Ootsu, K.; Ito, F. J P Patent 06220060; J apan, p
32.
(14) Bergeron, R. J .; McManis, J . S. J . Org. Chem. 1987, 52, 1700.
(17) Kokotos, G. Synthesis 1990, 299.

8434 J . Org. Chem., Vol. 63, No. 23, 1998
Feichtinger et al.
Sch em e 5
Sch em e 6
Clearly, the outstanding feature of this new class of
guanidinylation reagents is that primary and secondary
alcohols may be converted to a fully protected guanidine
derivative in one step in good to excellent yield.
Tr iflyld iu r eth a n e-P r otected Gu a n id in es: Con -
ver sion of Am in es to Gu a n id in es. In a previous
report, we introduced the triflyldiurethane-protected
guanidines N,N′-di-Boc-N′′-triflylguanidine (3) and N,N′-
di-Cbz-N′′-triflylguanidine (4).2 We have found these
triflylguanidines to be the most reactive among the
reagents synthesized thus far, and initial kinetic studies
propound that they are superior to existing guanidiny-
lating reagents.2 To further demonstrate the utility of
these reagents, we have synthesized a number of differ-
ent arginine and simple amine derivatives in both
solution and on solid phase.
was isolated that resulted from condensation of com-
pound 12 and a second equivalent of alcohol 10. The
overconsumption of compound 10 could be avoided if an
excess (3.0 equiv) of the guanidinylation reagent was
employed. Nevertheless, this observation led to the
examination of the inherent potential of compound 1 to
generate N′-alkylated arginine analogues. Many biologi-
cally interesting guanidines contain two different alkyl
groups in a N,N′-substitution pattern, which are now
easily accessible from reagent 1 through two consecutive
Mitsunobu reactions. An example is given in Scheme 5
with the synthesis of protected ω-methylarginine (20),
an important inhibitor of nitric oxide synthethase. The
tri-Boc reagent 1 was converted to the N-methyl deriva-
tive 19 by reaction of 1 with MeOH, PPh3, and DEAD.
This derivative was then used in a second Mitsunobu
reaction with alcohol 10 to afford compound 20 in good
yield (87%). As an additional example, isopropyl alcohol
was used to produce the alkylated arginine derivative 22
(Scheme 5). The ability to generate dialkylated guanidines
provides an excellent tool for the preparation of novel
alkylated arginine analogues. It should be noted that
some highly reactive alcohols, such as prenyl and benzyl
alcohol, react with the tri-Boc reagent 1 to produce the
dialkylated side product exclusively.
Deprotection of the arginine analogues can be achieved
either through acidolysis or hydrogenolysis. The ability
to use either the Boc derivative 1 or the Cbz derivative
2 allows for greater choice in protection and deprotection
strategies. It should be noted that when N-Cbz-L-
arginine methyl ester (17) was subjected to catalytic
hydrogenation, L-N-Boc-ω,ω′-di-Boc-arginine methyl ester
(18) was isolated in nearly quantitative yield (Scheme
4). Compound 18 appears to result from transfer of the
δ-Boc group of the guanidine side chain to the free amine,
which is generated upon removal of the Cbz group. This
rearrangement may occur in either an intermolecular
reaction or via an intramolecular pseudo-seven-mem-
bered transition state. The positions of the three Boc
groups as shown for compound 18 were confirmed via
total correlation spectroscopy (TOCSY). The TOCSY
spectrum showed correlation between the δ-NH and the
R, â, γ, and δ protons of this amino acid derivative,
confirming that the Boc groups of the guanidine side
chain existed in a ω,ω′-substitution pattern. This side
reaction can be avoided if arginine is not the N-terminal
residue or if no free amine is present in the molecule, as
in the cases described for the conversion of methanol and
isopropyl alcohol to the guanidine derivatives 19 and 21.
The guanidinylation agents, N,N′-di-Boc-N′′-triflyl-
guanidine and N,N′-di-Cbz-N′′-triflylguanidine, were ob-
tained in two steps from guanidine hydrochloride (5) as
previously reported (Scheme 6).2 Reaction of compound
5 with Boc-anhydride under strongly alkaline conditions
produced the intermediate N,N′-di-Boc-guanidine (23),8
which was easily converted to the target compound 3 with
triflic anhydride. In a similar sequence of reactions, the
synthesis of reagent 4 commenced with the conversion
of guanidine hydrochloride to N,N′-di-Cbz-guanidine (6).
The diprotected guanidine 6 was then deprotonated with
sodium hydride, followed by treatment of the resulting
anion with triflic anhydride to generate compound 4 in
good yield (75%). Thetriflyldiurethane-protected guanidines
3 and 4 are stable, crystalline substances that have been
stored at room temperature for at least 3 months with
no apparent loss of activity; hence, they should remain
stable indefinitely if refrigerated. The X-ray crystal
structure of reagent 3 (see Supporting Information)
revealed that the triflyl and Boc groups reside on
separate nitrogen atoms and, of the three central carbon-
nitrogen bonds, the one that bears the triflyl group has
the shortest bond length (1.31 Å). Although the remain-
ing two carbon-nitrogen bonds are predicted to have the
same length, the crystal structure showed them to be
significantly different (1.37 vs 1.34 Å). The difference
in bond length might be due to intra- and/or intermo-
lecular hydrogen bonding, both of which were observed
in the packing model of the crystal structure.
In a recent communication,2 a series of structurally
different amines was subjected to reaction with N,N′-di-
Boc-N′′-triflylguanidine (3) or N,N′-di-Cbz-N′′-triflylguani-
dine (4). In a typical reaction, a slight excess of the
amine was added in one portion to a solution of either 3
or 4 and 1 equiv of triethylamine, and the course of the
reaction was monitored by TLC. After the reaction was
complete, triethylamine and the byproduct triflic amide
were removed during a simple aqueous workup proce-

New Reagents for Guanidinylation Reactions
J . Org. Chem., Vol. 63, No. 23, 1998 8435
Ta ble 1. Gu a n id in yla tion of Am in es Usin g
di-Cbz reagent 4 is slightly more acidic than the di-Boc
reagent 3, which may clarify the decreased product
formation observed with reagent 4 and piperidine in
CH2Cl2. Addition of 1 equiv of a tertiary base was found
to accelerate the reaction in most cases; hence, triethyl-
amine is added as a standard procedure. However, even
with the addition of triethylamine, reactions using either
3 or 4 are still slower in protic solvents than in nonpolar
solvents. Therefore, the guanidinylation reactions are
preferably carried out in CH2Cl2 or CHCl3.
N,N′-Di-Boc-N′′-tr iflylgu a n id in e (3)
Ap p lica tion to Solid P h a se Syn th esis. The guanid-
inylation of three separate resin-bound amines was
examined using the general procedure as described for
reagent 3, and these results are summarized in Table 2.
For example, the hexapeptide Boc-Gly-Orn(Mtt)-Gly-Asp-
(tBu)-Ser(tBu)-Pro (entry 1, Table 2) was assembled on
a Rink-amide18 resin by successive coupling of Fmoc-
protected amino acids using Fastmoc chemistry on an
Applied Biosystems peptide synthesizer. The 4-methyl-
trityl (Mtt) protecting group of the Orn residue was
removed with TFA, and the free amino function was
guanidinylated by treatment with a solution of N,N′-di-
Boc-N′′-triflylguanidine (3) and triethylamine in CH2Cl2
for 8 h. A negative Kaiser test19 indicated that the
reaction went to completion, and the peptide was cleaved
from the resin with concomitant removal of the protecting
groups by treatment with TFA. The resin was removed
via filtration, and the combined filtrates were concen-
trated and cooled on an ice bath, and the peptide was
precipitated with cold ether. The peptide was isolated
by centrifugation to afford H-Gly-Arg-Gly-Asp-Ser-Pro-
NH2 as a white powder (77%), which was characterized
by HPLC and HRMS. In addition, the same peptide
sequence was assembled on a phenylacetamidomethyl
resin (PAM)20 using Boc-protected amino acids (entry 2,
Table 2). The free amino functionality of the Orn residue
was guanidinylated as described for the Rink-amide-
bound peptide. The peptide was cleaved from the resin
using HF, and the product, H-Gly-Arg-Gly-Asp-Ser-Pro-
OH, was isolated in quantitative yield by centrifugation
as described above. The peptide was then characterized
by HPLC and HRMS.
a
b
See experimental section for reaction conditions. Isolated
yield after chromatography. c Reference 13. Guanidinylation of
piperidine with reagent 4 resulted in a 15% yield of the corre-
sponding product. e Reference 9.
d
dure. Typically, the crude products obtained in this way
were greater than 95% pure as evidenced by TLC and
1H NMR. To summarize the results of our previous
findings, reagent 3 reacted rapidly with primary and
secondary amines under mild conditions. The reactions
were typically carried out at room temperature and were
usually complete within 1 h. Difficulties were only
observed with highly sterically hindered amines. For
example, tert-butylamine reacted rather sluggishly at
room temperature though a good yield was obtained in
refluxing dichloromethane. Equally impressive were the
results obtained with reagent 4. If a direct comparison
is made between the triflyldiurethane-protected guanidines
3 and 4, little difference is observed for the guandinyla-
tion of benzylamine, though compound 4 gave a slightly
higher yield for aniline.2
Results from further investigation of these reagents
are shown in Tables 1 and 2. For example, reagent 3
may be successfully employed in the guanidinylation of
compounds containing more than one amine in quantita-
tive yield (entry 4, Table 1). In addition, reagent 3
displayed compatibility with functional groups such as
ethers (entry 3, Table 1) and amino alcohols (entry 5,
Table 1). However, the guanidinylation of secondary
amines using di-Cbz-triflylguanidine 4 was less efficient
than that with di-Boc-triflylguanidine 3. Piperidine, for
example, showed minimal product formation using re-
agent 4, though an acceptable yield was obtained with
reagent 3 (entry 2, Table 1) under similar conditions. In
general, N,N′-di-Cbz-N′′-triflylguanidine (4) is an excel-
lent reagent for the guanidinylation of unreactive aro-
matic amines, though for practical purposes, N,N′-di-Boc-
N′′-triflylguanidine (3) is superior overall.
As a final example of the ease and efficiency of using
reagent 3 in the guanidinylation of resin-bound amines,
the octapeptide Boc-Gly-Lys(Mtt)-Ala-Orn(Mtt)-Gly-Asp-
(tBu)-Ser(tBu)-Pro, which contains two potential sites for
guanidinylation, was assembled on a Rink-amide resin
(entry 3, Table 2). The peptide was assembled as
described for H-Gly-Arg-Gly-Asp-Ser-Pro-NH2 and the
product H-Gly-hArg-Ala-Arg-Gly-Asp-Ser-Pro-NH2 was
isolated as an off-white powder (83%).
Con clu sion
In summary, new routes for the guanidinyation of
amines and alcohols have been reported. The prepara-
tion and product isolation for the triurethane-protected
guanidines (1 and 2) and the triflyldiurethane-protected
guanidines (3 and 4) are facile and less demanding than
some known guanidinylation reagents. Efficient routes
To understand the reactivity of the triflyldiurethane-
protected guanidines 3 and 4, one must consider that
they not only behave as electrophiles but also behave as
weak acids, which may explain the variation in reaction
rates in solvents of differing polarity. Polar solvents may
facilitate deprotonation of 3 or 4 by the amine, thus
slowing the guanidinylation process. Furthermore, the
(18) Rink, H. Tetrahedron Lett. 1987, 28, 3787.
(19) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal.
Biochem. 1970, 34, 595.
(20) Mitchell, A. R.; Erickson, B. W.; Ryabtsev, M. N.; Hodges, R.
S.; Merrifield, R. B. J . Am. Chem. Soc. 1976, 98, 7357.
(21) Data not shown.

8436 J . Org. Chem., Vol. 63, No. 23, 1998
Feichtinger et al.
Ta ble 2. Gu a n id in yla tion of Resin -Bou n d Am in es Usin g N,N′-Di-Boc-N′′-tr iflylgu a n id in e (3)
a
Total yield of peptide after cleavage from resin. HPLC analysis indicated that these molecules were greater than 95% pure.
at a rate such that the reaction mixture was completely
colorless before addition of the next drop. After the addition
was complete, the reaction mixture was heated at reflux for
15-18 h. (Note: reagent 1 is only partially soluble in THF at
room temperature. The solution becomes clear in refluxing
THF). The solution was then cooled to room temperature, and
the precipitate of excess 1 that formed was collected by
filtration and washed with a mixture of THF/hexanes 1:1. The
filtrate was concentrated in vacuo, and the product was
isolated by flash column chromatography on silica gel. Modi-
fications of the molar concentrations described in GPA are
indicated in the experimental text.
for the conversion of both alcohols and amines using
reagents 1-4 have been presented, and we have shown
reagent 3 to be well suited for solid-phase organic
synthesis. Reactions using compound 3 may be carried
out successfully in a wide range of solvents such as
benzene,21 chloroform, dichloromethane, and aceto-
nitrile.21 Remarkably, we have been successful in the
guanidinylation of several aminoglycosides using reagent
3 in aqueous media and these results will be presented
in a future publication. We believe that our new reagents
will find widespread use in the synthesis of both simple
and highly complex guanidine-containing compounds.
Gen er a l P r oced u r e B (GP B) for th e Gu a n id in yla tion
of Am in es a n d /or Am in o Acid s Usin g Rea gen t 3 or 4. In
a typical reaction, the amine (0.5 mmol) was added neat to a
solution of 3 or 4 (0.45 mmol) and triethylamine (0.5 mmol)
in CH2Cl2 (2 mL, filtered over neutral alumina) or CHCl3, and
the mixture was allowed to stir at room temperature until 3
or 4 was consumed as evidenced by TLC. After the reaction
was complete, the mixture was diluted with CH2Cl2 or CHCl3
(3 mL) and washed with 2 M sodium bisulfate, saturated
sodium bicarbonate, and brine. The organic extract was then
dried over sodium sulfate and filtered, and the solvent was
removed under reduced pressure. The crude products were
further purified by flash column chromatography to afford
material suitable for elemental analysis or reversed-phase
analytical HPLC.
Exp er im en ta l Section
Gen er a l. Unless otherwise noted, all materials were
obtained from commercial suppliers and used without further
purification. Analytical thin-layer chromatography was car-
ried out on precoated silica gel plates (Kieselgel 60 F254, E.
Merck & Co, Germany). Flash column chromatography was
performed using silica gel (230-400 mesh) from J . T. Baker.
All NMR spectra were obtained on a 360 MHz spectrometer
assembled at UCSD with a pulse programmer, digitizer, and
an Oxford Instruments superconducting magnet. Chemical
shifts (δ) are in ppm; multiplicities are indicated by s (singlet),
br s (broad singlet), d (doublet), t (triplet), q (quartet), or m
(multiplet). Coupling constants (J ) are reported in hertz.
Mass spectra were obtained at the Scripps Research Institute,
La J olla, CA. Elemental analysis was performed by Desert
Analytics, Tucson, AZ. Independent analysis of purity for
noncrystalline material was achieved using analytical HPLC
(Vydac C18, 25 cm × 0.46 cm, 5 µm) performed on a Millennium
2010 system consisting of a Waters 715 Ultra WISP sample
processor, a Waters TM 996 photodiode array detector, two
Waters 510 pumps, and a NEC Power Mate 486/33I computer.
Solvents used in HPLC analysis were as follows: solvent A,
H2O/0.1%TFA; solvent B, CH3CN/0.1% TFA. Flow rate ) 1.0
mL/min. X-ray crystallography was performed at UCSD.
Gen er a l P r oced u r e A (GP A) for th e Mitsu n obu Rea c-
tion Usin g Rea gen t 1. In a typical reaction, a solution of
the alcohol (1.0 mmol), reagent 1 (3.0 mmol), and PPh3 (1.5
mmol) in anhydrous THF (20 mL) was cooled to -5 °C under
an inert atmosphere. DEAD (1.5 mmol) was added dropwise
N,N′,N′′-Tr i-Boc-gu a n id in e (1). Potassium hydroxide pel-
lets (2.81 g, 50 mmol) and sodium carbonate (5.30 g, 50 mmol)
were finely ground in a mortar and transferred to a three-
necked round-bottomed flask equipped with a magnetic stirrer
and a reflux condenser. DMSO (50 mL) was added, and the
resulting suspension was stirred for 5 min at room tempera-
ture. Guanidine hydrochloride (5, 4.78 g, 50 mmol) was added,
and the mixture was stirred for 5 min. After the addition of
di-tert-butyl dicarbonate (51.7 g, 225 mmol), the mixture was
stirred for 60 h at 40 °C. The white precipitate obtained by
pouring the cold reaction mixture into 1 L of water was
collected by filtration, washed with H2O, and dried overnight
in vacuo. Recrystallization from acetonitrile yielded colorless
needles (14.9 g, 83%). mp 147-150 °C. 1H NMR (CDCl3) δ
1.48 (s, 27H). FABMS m/z (relative intensity) 360 (100, {M +
H}+), 304 (34), 260 (10), 248 (74). Anal. Calcd for

New Reagents for Guanidinylation Reactions
J . Org. Chem., Vol. 63, No. 23, 1998 8437
C16H29N3O6: C, 53.47%; H, 8.13%; N, 11.69%. Found: C,
53.48%; H, 8.34%; N, 11.86%.
mixture was diluted with CH2Cl2 (100 mL), the layers were
separated, and the aqueous layer was extracted with CH2Cl2
(100 mL). The extracts were combined, washed with H2O, and
dried with magnesium sulfate. After filtration and removal
of the solvent under reduced pressure, the crude product was
recrystallized from methanol. Compound 6 (9.85 g, 75%) was
obtained as colorless crystals. mp 149-150 °C. 1H NMR
(DMSO-d6) δ 10.88 (br s, 1H), 8.67 (br s, 2H), 7.40-7.25 (m,
10H), 5.10 (s, 4H); Anal. Calcd for C17H17N3O4: C, 62.38%;
H, 5.23%; N, 12.84%. Found: C, 62.26%; H, 5.01%; N, 12.79%.
N,N′,N′′-Tr i-Cbz-gu a n id in e (2). Sodium hydride (400 mg,
60% dispersion in mineral oil) was added in small portions to
a suspension of 6 (1.65 g, 5.0 mmol) in anhydrous THF (20
mL) at -45 °C under an argon atmosphere. After the addition
was complete, the mixture was allowed to stir for 1 h at -45
°C. Benzyl chloroformate (0.82 mL, 5.0 mmol) was added, and
the mixture was allowed to warm to room temperature and
stir overnight. The solvent was evaporated in vacuo, and the
residue was dissolved in a mixture of CH2Cl2 (50 mL) and H2O
(25 mL). The phases were separated, and the aqueous layer
was extracted with CH2Cl2 (2 × 50 mL). The extracts were
combined, washed with 1 N HCl and H2O, and dried with
magnesium sulfate. After filtration and evaporation of the
solvent, the crude product was purified by flash column
chromatography on silica gel (eluent, CH2Cl2/ethyl ether 98:
2). N,N′,N′′-Tri-Cbz-guanidine (2, 2.07 g, 90%) was obtained
as a white powder. mp 111-112 °C. 1H NMR (DMSO-d6) δ
10.55 (s, 2H), 7.36 (s, 10H), 5.11 (br s, 6H). FABMS m/z
(relative intensity) 484 (100, {M + Na}+), 462 (24, {M + H}+).
Anal. Calcd for C25H23N3O6: C, 65.07%; H, 5.02%; N, 9.11%.
Found: C, 64.89%; H, 4.74%; N, 8.82%.
N,N′-Di-Boc-N′′-tr iflu or om eth an esu lfon ylgu an idin e (3).
A solution of N,N′-di-Boc-guanidine (23, 0.52 g, 2.0 mmol) and
triethylamine (0.29 mL, 2.1 mmol) in anhydrous CH2Cl2 (10
mL) was cooled to -78 °C under an inert atmosphere. Triflic
anhydride (0.35 mL, 2.1 mmol) was added dropwise at a rate
such that the reaction temperature did not exceed -65 °C.
After the addition was complete, the mixture was allowed to
warm to room temperature within 4 h. The solution was
transferred to a separation funnel, washed with 2 M sodium
bisulfate and H2O, and dried with anhydrous sodium sulfate.
After filtration and removal of the solvent under reduced
pressure, the crude product was purified by flash column
chromatography on silica gel (eluent, CH2Cl2). N,N′-Di-Boc-
N′′-trifluoromethanesulfonylguanidine (3, 686 mg, 88%) was
obtained as pale-yellow crystals. The product was further
purified by recrystallization from hexanes. mp 114-115 °C.
1H NMR (DMSO-d6) δ 11.45 (br s, 2H), 1.45 (s, 18H). FABMS
m/z (relative intensity) 414 (16, {M + Na}+), 392 (13, {M +
H}+), 336 (43), 280 (100), 236 (9). Anal. Calcd for
L-N-Cbz-δ,ω,ω′-t r i-Boc-d esm et h ylen oa r gin in e Ben zyl
Ester (11). To a solution of 1 (1.08 g, 3.0 mmol) and PPh3
(400 mg, 1.5 mmol) in dry toluene (25 mL) was added 9 (340
mg, 1.0 mmol) in toluene (4.1 mL). A solution of DEAD (0.24
mL, 1.5 mmol) in toluene was added dropwise to the above
solution at a rate such that the reaction mixture was com-
pletely colorless before addition of the next drop. After the
addition was complete, the reaction mixture was allowed to
stir at 60 °C for 4 h. The reaction mixture turned clear after
ca. 3 h. Upon cooling the solution to room temperature, a
precipitate of excess N,N′,N′′-tri-Boc-guanidine formed, which
was collected by filtration and washed with toluene. The
filtrate was concentrated in vacuo and the product (11,
colorless oil, 272 mg, 40%) was isolated by flash column
1
chromatography on silica gel (eluent, EtOAc/hexanes 1:4). H
NMR (DMSO-d6) δ 10.62 (s, 1H), 7.38-7.29 (m, 10H), 6.25 (d,
1H, J ) 8.6 Hz), 5.14 (br s, 2H), 5.09 (br s, 2H), 4.44 (m, 1H),
3.85 (q, 2H, J ) 7.2 Hz), 2.19 (m, 2H), 1.45 (s, 9H), 1.42 (s,
18H). HRFABMS calcd for C35H48N4O10 (Mr) 817.2425 (M +
Cs)+, found 817.2445 ∆ ) 2.4 ppm. Analytical HPLC condi-
tions: 220 nm, 30:70 A/B to 10:90 A/B over 30 min, tR ) 27.0
min.
L-N-Cbz-δ,ω,ω′-tr i-Boc-ar gin in e Ben zyl Ester (12). Com-
pound 12 was prepared from (S)-N-Cbz-2-amino-5-hydroxy-
valeric acid benzyl ester (10) according to GPA. The product
12 was isolated as a colorless oil (0.426 g, 61%; eluent,
CH2Cl2/ethyl ether 92:8). 1H NMR (DMSO-d6) δ 10.20 (s, 1H),
7.75 (d, 1H, J ) 7.5 Hz), 7.36-7.29 (m, 10H), 5.04 (d, 2H, J )
12.9 Hz), 4.99 (d, 2H, J ) 12.9 Hz), 4.05 (m, 1H), 3.47 (m, 2H),
1.75-1.53 (m, 4H), 1.39-1.37 (3 s, 27 H). HRFABMS calcd
for C36H50N4O10 (Mr) 721.3425 (M + Na)+, found 721.3445 ∆
) 2.8 ppm. Analytical HPLC conditions: 220 nm, 50:50 A/B
to 5:95 A/B over 30 min, tR ) 24.6 min.
C
12H20F3N3O6S: C, 36.83%; H, 5.15%; N, 10.74%; F, 14.56%;
S, 8.19%. Found: C, 36.93%; H, 5.21%; N, 10.66%; F, 14.80%;
S, 8.33%.
L-N-Boc-δ,ω,ω′-tr i-Cbz-a r gin in e Meth yl Ester (15). To
a solution of (S)-N-Boc-2-amino-5-hydroxy-valeric acid methyl
ester (14, 80 mg, 0.33 mmol) in THF (4 mL) were added 2 (450
mg, 0.98 mmol) and PPh3 (130 mg, 0.49 mmol) under an inert
atmosphere. The mixture was cooled to 0 °C, and DEAD (0.10
mL, 0.49 mmol) was added dropwise at a rate such that the
reaction mixture was completely colorless before addition of
the next drop. After the addition was complete, the reaction
mixture was allowed warm to room temperature and stir
overnight. The reaction was quenched with H2O, and the
solvent was evaporated under reduced pressure. The product
(15, colorless oil, 220 mg, 100%) was isolated by flash column
chromatography on silica gel (eluent, CH2Cl2/ethyl ether 94:
6). 1H NMR (DMSO-d6) δ 10.89 (s, 1H), 7.39-7.30 (m, 15H),
7.20 (d, 1H, J ) 7.8 Hz), 5.09 (br s, 2H), 5.04 (br s, 2H), 4.99
(br s, 2H), 3.90 (m, 1H), 3.52 (br s, 5H), 1.67-1.50 (m, 4H),
1.34 (s, 9H). HRFABMS calcd for C36H42N4O10 (Mr) 691.2979
(M + H)+, found 691.2952 ∆ ) 3.9 ppm. Analytical HPLC
N,N′-Di-Cbz-N′′-tr iflu or om eth an esu lfon ylgu an idin e (4).
Sodium hydride (400 mg, 60% dispersion in mineral oil) was
added to a solution of N,N′-di-Cbz-guanidine (6, 1.65 g, 5.0
mmol) in anhydrous chlorobenzene (50 mL) at 0 °C under an
argon atmosphere. After being stirred for 1 h at 0 °C, the
mixture was cooled to -45 °C. Triflic anhydride (0.84 mL,
5.0 mmol) was added, and the mixture was allowed to warm
to room temperature and stir overnight. The solvent was
removed under reduced pressure, and the residue was dis-
solved in a mixture of ethyl acetate (100 mL) and 2 M sodium
bisulfate (25 mL). The phases were separated, and the organic
layer was washed with H2O and brine and dried with mag-
nesium sulfate. After filtration and removal of the solvent
under reduced pressure, the crude product was purified by
flash column chromatography on silica gel (eluent, CH2Cl2/
ethyl ether 95:5). Compound 4 (1.58 g, 75%) was obtained as
1
a pale-yellow oil that crystallizes in vacuo. mp 74-75 °C. H
conditions: 220 nm, 30:70 A/B to 10:90 A/B over 30 min, tR
7.2 min.
)
NMR (DMSO-d6) 11.55 (br s, 2H), 7.45-7.28 (m, 10H), 5.20
(s, 4H). Electrospray MS m/z (relative intensity) 498 (30, {M
+ K}+), 482 (100, {M + Na}+), 460 (2, {M + H}+). Anal. Calcd
for C18H16F3N3O6S: C, 47.06%; H, 3.51%; N, 9.15%; F, 12.41%;
S, 6.98%. Found: C, 47.37%; H, 3.35%; N, 8.67%; F, 12.79%;
S, 6.92%.
L-N-Cbz-δ,ω,ω′-tr i-Boc-ar gin in e Meth yl Ester (17). Com-
pound 17 was prepared and purified according to GPA with
the following modifications: (S)-N-Cbz-2-amino-5-hydroxyva-
leric acid methyl ester (16, 0.92 g, 3.27 mmol), 1 (5.9 g, 6.4
mmol), PPh3 (1.3 g, 5.9 mmol), THF (50 mL), DEAD (0.93 mL,
5.9 mmol). The product (17) was isolated as a colorless oil
(1.46 g, 72%; eluent, CH2Cl2/ethyl ether 9:1). mp 37-41 °C.
1H NMR (DMSO-d6) δ 10.18 (s, 1H), 7.72 (d, 1H, J ) 7.9 Hz),
7.40-7.26 (m, 5H), 5.01 (s, 2H), 4.03-3.94 (m, 1H), 3.6 (s, 3H),
3.45 (t, 3H, J ) 5.8 Hz), 1.73-1.45 (m, 4H), 1.39-1.37 (3 s,
27H). HRFABMS calcd for C30H46N4O10 (Mr) 645.3112 (M +
N,N′-Di-cbz-gu a n id in e (6). CH2Cl2 (80 mL) was added to
a solution of guanidine hydrochloride (5, 3.82 g, 40 mmol) and
sodium hydroxide (8.0 g, 0.20 mol) in H2O (40 mL), and the
resulting mixture was cooled to 0 °C. Benzyloxycarbonyl
chloride (17.1 mL, 120 mmol) was added dropwise with
vigorous stirring over a period of 45 min. After the addition
was complete, stirring was continued for 20 h at 0 °C. The

8438 J . Org. Chem., Vol. 63, No. 23, 1998
Feichtinger et al.
Na)+, found 645.3125 ∆ ) 2.0 ppm. Analytical HPLC condi-
tions: 220 nm, 60:40 A/B to 20:80 A/B over 30 min, tR) 27.4
min.
found 873.3019 ∆ ) 3.7 ppm. Analytical HPLC conditions:
220 nm, 70:30 A/B to 10:90 A/B over 30 min, tR ) 16.6 min.
N,N′-Bis(ter t-bu toxyca r bon yl)gu a n id in e (23). 1,4-Di-
oxane (50 mL) was added to a solution of guanidine hydro-
chloride (5, 2.39 g, 25 mmol) and sodium hydroxide (4.0 g, 0.1
mol) in H2O (25 mL), and the resulting mixture was cooled to
0 °C. Di-tert-butyl dicarbonate (12.0 g, 55 mmol) was added
in one portion, and the reaction mixture was allowed to warm
to room temperature within 2 h. After being stirred for 20 h,
the mixture was concentrated in vacuo to one-third of its
original volume. The resulting suspension was diluted with
H2O (50 mL) and extracted with ethyl acetate (3 × 50 mL).
The crude product is a mixture of N-Boc-guanidine, N,N′-bis-
(tert-butoxycarbonyl)guanidine (23), and N,N′,N′′-tri-Boc-
guanidine (1). The combined extracts were washed with 10%
citric acid (N-Boc-guanidine remains in the acidic layer and
may be recovered by cooling the acidic layer in an ice bath,
adjusting the pH to 9.0, and extracting with EtOAc), H2O, and
brine and dried with magnesium sulfate. After filtration and
removal of the solvent under reduced pressure, the crude
product was purified by flash column chromatography on silica
gel (eluent, 100% CH2Cl2 to CH2Cl2/MeOH 97:3). N,N′-Bis-
(tert-butoxycarbonyl)guanidine (23, 3.84 g, 59%) was obtained
as a colorless powder. mp 144-145 °C. 1H NMR (DMSO-d6)
10.42 (br s, 1H), 8.47 (br s, 2H), 1.39 (s, 18H). FABMS m/z
(relative intensity) 260 (50, {M + H}+), 204 (48), 148 (100).
Anal. Calcd for C11H21N3O4: C, 50.95%; H, 8.16%; N, 16.21%.
Found: C, 50.83%; H, 8.04%; N, 16.26%.
N,N′-Bis(ter t-b u t yloxyca r b on yl)-N′′-p en t ylgu a n id in e
(E n t r y 1, Ta b le 1). N,N′-Bis(tert-butyloxycarbonyl)-N′′-
pentylguanidine was prepared according to GPB (solvent )
CH2Cl2, reaction time ) 1 h, rt). mp 73-74 °C. 1H NMR
(DMSO-d6) δ 11.48 (s, 1H), 8.26 (br s, 1H), 3.25 (q, 2H, J )
8.5 Hz), 1.52-1.16 (m, 6H), 1.46 (s, 9H), 1.37 (s, 9H), 0.85 (t,
3H, J ) 8.5 Hz). Anal. Calcd for C16H31N3O4: C, 58.34%; H,
9.49%; N, 12.76%. Found: C, 58.36%; H, 9.60%; N, 13.09%.
Di[N,N′-bis-(ter t-bu tyloxycar bon yl)]piper azin e-1,4-car -
boxa m id in e (En tr y 4, Ta ble 1). Di[N,N′-bis-(tert-butyloxy-
carbonyl)]piperazine-1,4-carboxamidine was prepared accord-
ing to GPB (solvent ) CHCl3, reaction time ) 20 h, rt). 1H
NMR (CDCl3) δ 3.64 (br s, 8H), 1.46 (br s, 36H). Anal. Calcd
for C26H46N6O8: C, 54.72%; H, 8.13%; N, 14.73%. Found: C,
54.59%; H, 7.85%; N, 14.64%.
N ,N ′-B i s -(t er t -b u t y lo x y c a r b o n y l)-N ′′-2-h y d r o x y -
et h ylgu a n id in e (E n t r y 5, Ta b le 1). N,N′-Bis-(tert-butyl-
oxycarbonyl)-N′′-2-hydroxyethylguanidine was prepared ac-
cording to GPB (solvent ) CH2Cl2, reaction time ) 2 h, rt). 1H
NMR (DMSO-d6) δ 11.48 (s, 1H), 8.42 (br s, 1H), 4.88 (t, 1H,
J ) 5.3 Hz), 3.47 (q, 2H, J ) 5.1 Hz), 3.33 (m, 2H), 1.46 (s,
9H), 1.37 (s, 9H). Anal. Calcd for C13H25N3O5: C, 51.48%; H,
8.31%; N, 13.86%. Found: C, 51.23%; H, 8.33%; N, 13.60%.
H-Gly-Ar g-Gly-Asp -Ser -P r o-NH2 (En tr y 1, Ta ble 2).
The hexapeptide Boc-Gly-Orn(Mtt)-Gly-Asp(tBu)-Ser(tBu)-Pro
was assembled on a Rink-amide18 resin (179 mg, 0.1 mmol)
by successive coupling of Fmoc-protected amino acids using
Fastmoc chemistry on an Applied Biosystems automated
peptide synthesizer. After coupling of Boc-Gly-OH in the last
step, the synthesis was completed manually as described
below. The resin was washed with CH2Cl2 (5 × 10 mL) and
with a solution of 1% TFA and 2.5% triisopropylsilane (TIS)
in CH2Cl2 (2 × 10 mL). The Mtt group was removed by
shaking the resin for 2 h in a solution of 1% TFA and 2.5%
TIS in CH2Cl2. The resin was washed with 10% DIEA in
CH2Cl2 (3 × 10 mL) and with CH2Cl2 (5 × 10 mL). The free
amino function was guanidinylated by treatment with a
solution of 3 (180 mg, 0.5 mmol) and triethylamine (70 µL,
0.5 mmol) in CH2Cl2 (2 mL) for 8 h. A negative Kaiser test19
indicated that the reaction had gone to completion. The resin
was washed with CH2Cl2 (5 × 10 mL), methanol, and CH2Cl2
(3 × 10 mL) and dried in vacuo over KOH. The peptide was
cleaved from the resin by treatment with a solution of 2.5%
TIS and 2.5% H2O in TFA for 1 h. The resin was removed by
filtration and washed with TFA (3 × 2 mL). The filtrates were
combined and concentrated under reduced pressure to a small
volume (ca. 0.5 mL). The solution was cooled in an ice bath,
L-N-Boc-ω,ω′-d i-Boc-a r gin in e Meth yl Ester (18). Com-
pound 17 (1.46 g, 2.3 mmol) was dissolved in MeOH (20 mL).
After 10% Pd/C (150 mg) was added, the solution was allowed
to stir under an atmosphere of H2 for 4 h. The product was
filtered over Celite, and the solvent was evaporated to give
an oil, which was precipitated with ether to give 18 as a white
foam (1.05 g, 99%). 1H NMR (CDCl3) δ 11.39 (s, 1H), 8.88 (br
s, 1H), 4.85 (m, 2H), 3.73 (s, 3H), 3.15 (m, 2H), 1.82 (m, 2H),
1.49 (m, 2H), 1.49-1.41 (3 s, 27H). HRFABMS calcd for
C
22H40N4O8 (Mr) 511.2744 (M + Na)+, found 511.2754 ∆ ) 2.0
ppm. Analytical HPLC conditions: 220 nm, 40:60 A/B to 20:
80 A/B over 30 min, tR ) 8.7 min.
N-Meth yl-N,N′,N′′-tr i-Boc-gu a n id in e (19). Compound 19
was prepared according to GPA with the following modifica-
tions: anhydrous methanol (0.04 mL, 1.0 mmol), 1 (539 mg,
1.5 mmol), PPh3 (393 mg, 1.5 mmol), DEAD (0.24 mL, 1.5
mmol). The mixture was allowed to cool to room temperature,
and the solvent was evaporated under reduced pressure. The
crude product was suspended in MeOH (10 mL), and the
precipitate of excess N,N′,N′′-tri-Boc-guanidine that formed
was collected by filtration and washed with MeOH. The
filtrate was concentrated in vacuo, and the product (19) was
isolated as a colorless oil (182 mg, 49%; eluent, CH2Cl2/ethyl
ether 98:2). FABMS m/z (relative intensity) 396 (100, {M +
Na}+), 374 (13, {M + H}+); 1H NMR (DMSO-d6) δ 10.17 (s,
1H), 2.94 (s, 3H), 1.43-1.36 (3 s, 27H). HRFABMS calcd for
C
17H31N3O6 (Mr) 374.2291 (M + H)+, found 374.2298 ∆ ) 1.9
ppm. Analytical HPLC conditions: 220 nm, 50:50 A/B to 5:95
A/B over 30 min, tR ) 14.8 min.
L-N-Cbz-ω-m eth yl-δ,ω,ω′-tr i-Boc-a r gin in e Ben zyl Ester
(20). Compound 20 was prepared according to GPA with the
following modifications: (S)-N-Cbz-2-amino-5-hydroxyvaleric
acid benzyl ester (10, 97 mg, 0.273 mmol), 19 (66 mg, 0.177
mmol), PPh3 (0.11 g, 0.408 mmol), anhydrous THF (3.5 mL),
DEAD (84 µL, 0.408 mmol). The reaction mixture was allowed
to cool to room temperature and the solvent was evaporated
in vacuo. The product (20) was isolated as a colorless oil (113
mg, 87%; eluent, ethyl acetate/hexanes 1:3). 1H NMR (DMSO-
d6) δ 7.80 (d, 1H, J ) 7.9 Hz), 7.39-7.28 (m, 10H), 5.10 (s,
2H), 5.06-4.94 (m, 2H), 4.11-4.00 (m, 1H), 3.53-3.44 (m, 2H),
2.89 (s, 3H), 1.75-1.50 (m, 4H), 1.40-1.34 (3 s, 27H). HR-
FABMS calcd for C37H52N4O10 (Mr) 845.2738 (M + Cs)+, found
845.2709 ∆ ) 3.4 ppm. Analytical HPLC conditions: 220 nm,
30:70 A/B to 10:90 A/B over 30 min, tR ) 12.2 min.
N-P r op yl-N,N′,N′′-tr i-Boc-gu a n id in e (21). Compound 20
was prepared according to GPA with the following modifica-
tions: isopropyl alcohol (0.07 mL, 0.92 mmol), 1 (0.50 g, 1.4
mmol), PPh3 (0.365 g, 1.4 mmol), DEAD (0.22 mL, 1.4 mmol).
The filtrate was concentrated in vacuo, and the product (21)
was isolated as a colorless oil (249 mg, 67%; eluent, EtOAc/
hexanes 1:4). 1H NMR (CDCl3) δ 1.50-1.44 (2 s, 27H), 1.48
(m, 1H, beneath tert-butyl groups), 1.34 (d, 6H, J ) 6.7 Hz).
HRFABMS calcd for C19H35N3O6 (Mr) 402.2604 (M + H)+,
found 402.2613 ∆ ) 2.2 ppm. Analytical HPLC conditions:
220 nm, 50:50 A/B to 5:95 A/B over 30 min, tR ) 18.6 min.
L-N-Cbz-ω-p r op yl-δ,ω,ω′-tr i-Boc-a r gin in e Ben zyl Ester
(22). Compound 22 was prepared according to GPA with the
following modifications. To a solution of PPh3 (0.11 g, 0.402
mmol) and (S)-N-Cbz-2-amino-5-hydroxyvaleric acid benzyl
ester (10, 96.0 mg, 0.268 mmol) in THF (4 mL) was added 21
(0.243 g, 0.604 mmol) via cannulation. After the addition of
DEAD (84 µL, 0.402 mmol) was complete, the reaction mixture
was heated at reflux for 15 h. The mixture was allowed to
cool to room temperature, and the solvent was evaporated
under reduced pressure. The filtrate was concentrated in
vacuo, and the product (22) was isolated as a colorless oil (126
mg, 64%; eluent, EtOAc/hexanes 1:4). 1H NMR (CDCl3) δ
7.36-7.26 (m, 10H), 5.73 (br s, 1H), 5.16 (br s, 2H), 5.09 (br d,
2H, J ) 2.9 Hz), 4.06 (m, 1H), 3.61 (m, 2H), 1.86 (m, 1H), 1.74-
1.68 (m, 4H), 1.45-1.41 (3 s, 27H), 1.23 (d, 6H, J ) 6.5 Hz).
HRFABMS calcd for C39H56N4O10 (Mr) 873.3051 (M + Cs)+,

New Reagents for Guanidinylation Reactions
J . Org. Chem., Vol. 63, No. 23, 1998 8439
and the peptide was precipitated by adding ice cold ether (10
mL). The precipitate was isolated by centrifugation and
washed with ethyl ether (3 × 10 mL). After drying in vacuo,
the product H-Gly-Arg-Gly-Asp-Ser-Pro-NH2 (entry 5, Table
2; 54 mg, 77%) was obtained as an off-white powder. HR-
FABMS calcd for C22H38N10O9 (Mr) 719.1878 (M + Cs)+, found
719.1899 ∆ )2.9 ppm. Analytical HPLC conditions: 220 nm,
100:0 A/B to 85:15 A/B over 15 min, tR ) 12.6 min.
reaction vessel was flushed with nitrogen and cooled to -78
°C. HF (ca. 5 mL) was condensed into the vessel, and the
mixture was stirred for 1 h at 0 °C. The HF was evaporated
by flushing with nitrogen, and the vessel containing the resin
was dried in vacuo over KOH. The resin was washed with
TFA (3 × 2 mL) and filtered. The combined filtrates were
concentrated in vacuo to ca. 0.5 mL and cooled to 0 °C. Ice
cold ether (10 mL) was added, and the precipitate was isolated
by centrifugation and washed with ether (3 × 10 mL). After
drying in vacuo the product H-Gly-Arg-Gly-Asp-Ser-Pro-OH
(entry 6, Table 2; 59 mg, 100%) was obtained as an off-white
powder. HRFABMS calcd for C22H37N9O10 (Mr) 720.1718 (M
+ Cs)+, found 720.1743 ∆ )3.5 ppm. Analytical HPLC
H-Gly-Ar g-Gly-Asp -Ser -P r o-OH (en tr y 2, Ta ble 2). Boc-
Pro-PAM resin (114 mg, 0.1 mequiv) was washed with CH2Cl2
(5 × 10 mL), and the resin was treated with TFA/anisole/
CH2Cl2 (25:5:70) for 2 min. The solution was filtered, and the
Boc group was removed by treatment with TFA/anisole/CH2Cl2
(25:5:70) for 30 min. The resin was washed with CH2Cl2 (3 ×
10 mL), 2-propanol, CH2Cl2, 2-propanol, and CH2Cl2 (3 × 10
mL). The resin was treated with 10% DIEA in CH2Cl2 (3 ×
10 mL) for 2 min. The solution was filtered, and the resin
was washed with CH2Cl2 (3 × 10 mL) and DMF (3 × 10 mL).
A solution of Boc-Ser(Bn)-OH (148 mg, 0.5 mmol), 1-hydroxy-
benzotriazole (0.5 mmol), and 2-dimethylaminoisopropyl chlo-
ride (0.5 mmol) in DMF (2 mL) was added, and the mixture
was allowed to stir for 4 h. The solution was filtered, and the
resin was washed with DMF (3 × 10 mL), CH2Cl2 (3 × 10 mL),
2-propanol, and CH2Cl2 (3 × 10 mL). The above cycle was
repeated with Boc-Asp(cHex)-OH (156 mg, 0.5 mmol), Boc-Gly-
OH (88 mg, 0.5 mmol), Boc-Orn(Fmoc)-OH (227 mg, 0.5 mmol),
and Boc-Gly-OH (88 mg, 0.5 mmol). After completion of the
sequence, the resin was washed with DMF (3 × 10 mL) and
with a 20% solution of piperidine in DMF. The Fmoc group
was removed by shaking the resin for 30 min in a 20% solution
of piperidine in DMF. The resin was then washed with DMF
(3 × 10 mL) and CH2Cl2 (3 × 10 mL). The free amino function
was guanidinylated by treatment with a solution of 3 (180 mg,
0.5 mmol) and triethylamine (70 µL, 0.5 mmol) in CH2Cl2 (2
mL) for 8 h. A negative Kaiser test19 was obtained, indicating
the reaction had gone to completion. The resin was washed
with CH2Cl2 (3 × 10 mL) followed by TFA/anisole/CH2Cl2 (25:
5:70) for 2 min. The solution was filtered, and the Boc group
was removed by treatment with TFA/anisole/CH2Cl2 (25:5:70)
for 30 min. The resin was washed with CH2Cl2 (3 × 10 mL),
methanol (3 × 10 mL), and CH2Cl2 (3 × 10 mL), and dried in
vacuo over KOH. The resin was placed in a Teflon reaction
vessel, and anisole (0.2 mL) was added as a scavenger. The
conditions: 214 nm, 100:0 A/B to 80:20 A/B over 20 min, tR
)
11.6 min.
H -Gly-h Ar g-Ala -Ar g-Gly-Asp -Ser -P r o-NH 2 (E n t r y 3,
Ta ble 2). The hexapeptide Boc-Gly-Lys(Mtt)-Ala-Orn(Mtt)-
Gly-Asp(tBu)-Ser(tBu)-Pro was assembled on a Rink-amide18
resin (0.1 mmol, 179 mg) as described for H-Gly-Arg-Gly-Asp-
Ser-Pro-NH2. The free amino functions were guanidinylated
by treatment with a solution of 3 (359 mg, 1.0 mmol) and
triethylamine (140 µL, 1.0 mmol) in CH2Cl2 (2 mL) for 16 h.
The peptide was cleaved from the resin as described for H-Gly-
Arg-Gly-Asp-Ser-Pro-NH2. After being dried in vacuo, the
product H-Gly-hArg-Ala-Arg-Gly-Asp-Ser-Pro-NH2 (entry 3,
Table 2; 88 mg, 83%) was obtained as an off-white powder.
HRFABMS calcd for C32H57N15O11 (Mr) 828.4440 (M + H)+,
found 828.4468 ∆ ) 3.4 ppm. Analytical HPLC conditions:
220 nm, 100:0 A/B to 80:20 A/B over 20 min, tR ) 15.2 min).
Ackn owledgm en t. We thank NIGU-Chemie GmBH,
Germany, for financial support.
Su p p or tin g In for m a tion Ava ila ble: ORTEP diagrams,
tables of crystallographic data, bond lengths and angles,
atomic coordinates, and anisotropic thermal parameters are
available for compounds 1 and 3 (7 pages). This material is
contained in libraries on microfiche, immediately follows this
article in the microfilm version of the journal, and can be
ordered from the ACS; see any current masthead page for
ordering information.
J O9814344