Practical and efficient synthesis of orthogonally protected constrained 4-guanidinoprolines

Full text of DOI:10.1021/jo005626m

1038
J . Org. Chem. 2001, 66, 1038-1042
P r a ctica l a n d Efficien t Syn th esis of
Or th ogon a lly P r otected Con str a in ed
4-Gu a n id in op r olin es†
Makoto Tamaki, Guoxia Han, and Victor J . Hruby*
Department of Chemistry, University of Arizona,
Tucson, Arizona 85721
F igu r e 1. Structures of guanidino group and designed proline
derivative.
hruby@u.arizona.edu
Received August 27, 2000
In tr od u ction
The design, synthesis, and use of conformationally
constrained amino acids has emerged as a critical strat-
egy for understanding the interaction of peptides and
proteins with their receptors/acceptors. Among the novel
amino acids, 4-substituted prolines have been widely
studied due in part to the readily available starting
material 4-trans-hydroxyproline, which has been used as
a versatile building block for many biologically important
compounds.1 Of the 20 naturally occurring R-amino acids,
proline is the only one with a secondary amino group.
When it is incorporated into a peptide or protein, it can
induce a reverse turn which can provide enhanced
bioactivity. Furthermore, proline residues are present in
many peptide/protein sequences having important bio-
activities, including R-melanotropin (R-MSH, Ac-Ser-Tyr-
Ser -Met -Glu -H is-P h e-Ar g8-Tr p-Gly-Lys-P r o12-Va l-
NH2),2-5 neurotension (NT, Glu-Leu-Tyr-Met-Glu-Asn-
Lys-Pro8-Arg9-Arg10-Pro11-Tyr-Ile-Leu-OH),6-10 and sub-
stance P (SP, Arg1-Pro2-Lys-Pro4-Gln-Gln-Phe-Phe-Gly-
Leu-Met-NH2).11-15 Hence, design and synthesis of con-
formationally constrained proline analogues is an im-
portant tool to study ligand-receptor interactions.
In addition to proline, arginine also plays an important
role in the biological activities of R-MSH, NT, and SP.
The importance of arginine often is due to the strong
basic guanidino group (1), which is almost always pro-
tonated at physiological pH and plays an important role
in molecular recognition either through hydrogen bond-
F igu r e 2. Common protecting groups for the guanidino group
of Arg used in Fmoc chemistry.
ing or electrostatic interactions. For these reasons, we
have developed the synthesis of constrained amino acids
(2) based on proline and arginine (Figure 1).
Retr osyn th etic P a th w a ys. There are several choices
[e.g., Mtr (3), Pmc (4), and Pbf (5)] to orthogonally protect
the guanidino group of Arg for NR-Fmoc chemistry
(Figure 2). Due to side reactions, which can occur during
the cleavage of the Mtr and Pmc groups, these groups
have been replaced, to some extent, by Pbf which has
proven to be a better guanidino protecting group of
Arg.16-20 However, recently we have found that Pbf also
could be problematic during the peptide synthesis of
γ-MSH.21 The tert-butyloxycarbonyl (Boc) group has been
extensively used for the protection of amino groups but
has seldom been used for the protection of the guanidino
group of Arg in the Fmoc chemistry. Recently, Feich-
tinger et al. prepared N,N′-diBoc-N′′-trifluoromethane-
sulfonylguanidine (10)22 as a versatile guanidinating
reagent.23-29 We have investigated the use of this reagent
(16) Carpino, L. A.; Shroff, H.; Triolo, S. A.; Mansour, E. M. E.;
Wenschuh, H.; Albericio, F. Tetrahedron Lett. 1993, 34, 7829-7832.
(17) Nishino, N.; Xu, M.; Mihara, H.; Fujimoto, T.; Ueno, Y.;
Kumagai, H. Tetrahedron Lett. 1992, 33, 1479-1482.
(18) Stierandova, A.; Sepetov, N. F.; Nikiforovich, G. V.; Lebl, M.
Int. J . Pept. Protein Res. 1994, 43, 31-38.
(19) Prochazka, Z.; J ost, K.; Blaha, K. Chem. Listy 1981, 75, 699-
722.
(20) Fields, C. G.; Fields, G. B. Tetrahedron Lett. 1993, 34, 6661-
6664.
(21) Balse, P.; Hruby, V. J . Unpublished results.
(22) Feichtinger, K.; Zapf, C.; Sings, H. L.; Goodman, M. J . Org.
Chem. 1998, 63, 3804-3805. N,N′-Di-Boc-guanidine (9) was synthe-
sized following a slight modification of the literature.22 We were able
to obtain much better yields than those reported by carefully monitor-
ing the reaction by TLC.
* To whom correspondence should be addressed. Tel: 520-621-8617.
Fax: 520-621-8407.
† Part of this work was presented at the 2000 Spring ACS meeting
in San Francisco, CA.
(1) Remuzon, P. Tetrahedron 1996, 52, 13803-13835.
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349.
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104.
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468.
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1319.
Nucleophilic addition to triflic anhydride gave N,N′-di-Boc-N′′-Tf-
guanidine(10, yield: 90%) which was purified by flash chromatography.
We have found that the yield improved if diisopropylethylamine
(DIPEA) is used instead of triethylamine (TEA) as the base. In
addition, the reaction times were much shorter when DIPEA was used
as the base.
(14) Mau, S. E. Dan. Med. Bull. 1999, 46, 35-56.
(15) Walsh, D. A. Rheumatology 1999, 38, 103-112.
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(24) Baker, T. J .; Goodman, M. Synthesis 1999, 1423-1426.
10.1021/jo005626m CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/16/2001

Notes
Sch em e 1. Retr osyn th etic P a th w a y to th e
J . Org. Chem., Vol. 66, No. 3, 2001 1039
Sch em e 3. Retr osyn th etic P a th w a y for
Syn th esis of F m oc P r olin e Der iva tives
4-tr a n s-Su bstitu ted P r olin e Der iva tive
Sch em e 4. Syn th esis of 4-tr a n s-Azid op r olin e
Der iva tive
Sch em e 2. Syn th esis P r otected
4-cis-Gu a n id in op r olin e Der iva tive
SN2 displacement of the tosylate with azide resulted in
the 4-cis-azide 14 with an inverted chiral center at the 4
position.30
Reduction of azide was accomplished under neutral
conditions with PPh3 in THF and water. Other possible
methods were not suitable for this substrate. TLC showed
that the crude product was almost pure and the crude
amine 15 was used directly without purification for
preparing the di-Boc-protected guanidino proline deriva-
tive 16 (Scheme 2).
Guanidino replacement at 4 position was accomplished
by the new guanidilating reagent in a very good yield.
Due to steric hindrance, the displacement reaction pro-
gressed slowly. However, by maintaining the reaction in
an inert atmosphere, no significant side reactions oc-
curred, and after 3 days, the reaction was essentially
complete. After a quick flash column purification, benzyl
NR-Cbz-4-cis-(N,N′-di-Boc)guanidinoprolinate 16 was ob-
tained as a colorless oil (64%).
Hydrogenolysis of the fully protected derivative re-
moved the Cbz and Bn groups giving the desired product,
4-cis-(N, N′-di-Boc)guanidinoprolinate (17, yield: 75%).
The free R-amino group was then protected with Fmoc
using Fmoc-OSu and Na2CO3 in a mixed solvent system.
The conditions presented here gave the best yield (75%)
among several methods tried. In fact, a poor yield was
obtained when TEA (commonly used in Fmoc protection
of proline) was used.
in the synthesis of 4-guanidinoprolines 6 (Scheme 1). To
keep the diBoc protection intact during the synthesis,
proper protection of the R-amino and acidic groups was
essential. For example, groups requiring acid catalysis
during protection and deprotection cannot be applied
during the synthesis, as it would remove the Boc groups.
Moreover, nucleophilic base-sensitive protecting groups
for R-amino and acidic groups could not be used, because
these would interfere with the guanidination step. In
addition, the amino group of the key intermediates, the
4-amino-proline derivatives (7, Scheme 1), could cleave
the protecting groups. Hence, protecting groups which
could be cleaved under neutral condition such as hydro-
genolysis were needed, and we chose the benzyloxycar-
bonyl (Cbz or Z) and benzyl (Bn) protecting groups.
Syn th esis Nr-F m oc-4-tr a n s-(N,N′-d i-Boc)gu a n id i-
n op r olin e. To synthesize the 4-trans-proline isomer 19,
we proposed an SN2 displacement of the 4-cis-halide via
azide (Scheme 3), followed by reduction of the 4-trans
azide to the corresponding trans-amine. Thus, the syn-
thesis of the cis-4-haloproline became our first objective.
To make the synthesis simple and economical, dis-
placement of the trans-4-TsO-proline (13) by iodide was
carried out (Scheme 4). To our surprise, a mixture of
products was obtained. Without separating the crude
mixture, azide displacement was carried out immedi-
ately. However, the desired 4-trans-azide product 21 was
Resu lts a n d Discu ssion
Syn th esis Nr-F m oc-4-cis-(N,N′-d i-Boc)gu a n id in o-
p r olin e. NR-Cbz-4-trans-hydroxyproline (11, Scheme 2)
was first protected as its benzyl ester. The benzyl ester
12 was essentially pure after workup. Tosylation of the
4-hydroxyl group to give 13 was carried out in pyridine.
(25) Gothe, R.; Seyfarth, L.; Schumann, C.; Agricola, I.; Reissmann,
S.; Lifferth, A.; Birr, C.; Filatova, M. P.; Kritsky, A.; Kibirev, V. J .
Prakt. Chem. 1999, 341, 369-377.
(26) Golding, B. T.; Mitchinson, A.; Clegg, W.; Elsegood, M. R. J .;
Griffin, R. J . J . Chem. Soc., Perkin Trans. 1 1999, 349-356.
(27) Watson, S. E.; Markovich, A. Heterocycles 1998, 48, 2149-2155.
(28) J osey, J . A.; Tarlton, C. A.; Payne, C. E. Tetrahedron Lett. 1998,
39, 5899-5902.
(30) It should be noted that, for the tosylation reaction, no significant
difference was observed when the crude benzyl ester was used instead
of the column purified material.
(29) Dubey, I.; Pratviel, G.; Meunier, B. Bioconjugate Chem. 1998,
9, 627-632.

1040 J . Org. Chem., Vol. 66, No. 3, 2001
Notes
deuterated solvents: CDCl3, DMSO-d6, or CD3OD, all of which
were purchased from Cambridge Isotopes or Aldrich.
Sch em e 5. Syn th esis of 4-tr a n s-Gu a n id in op r olin e
Der iva tive
(2S,4S)-Nr-Cbz-4-h yd r oxylp r olin e Ben zyl Ester (12). Tri-
ethylamine (7.7 mL, 55 mmol) was added to the solution of
(2S,4S)-1-(benzyloxycarbonyl)-4-hydroxyl-L-proline (11, 13.25 g,
50 mmol) and benzyl bromide (9.4 g, 55 mmol) in tetrahydro-
furan (50 mL) at 0 °C. After the mixture was stirred for 18 h at
room temperature, the solvent was evaporated in vacuo. The
residue was dissolved in 100 mL of CH2Cl2, washed with HCl
(1 N), H2O, Na2CO3 (5%), and H2O, and then dried over MgSO4
overnight. Evaporation of the solvent under reduced pressure
yielded a light yellow oil, which was almost pure after standing
overnight under high vacuum. It was further purified by flash
chromatoghraphy (eluent, CHCl3/MeOH ) 20:1) to give a color-
less oil (12). Yield: 16.0 g (90%). [R]25 ) -58.0 (c 1.40, CHCl3).
D
1H NMR (CDCl3): 7.33-7.19 (10 H), 5.21-5.00 (4H), 4.58-4.50
(1H), 4.41 (1H), 3.66-3.51 (2H), 2.59 (1H), 2.31-2.22 (1H), 2.07-
2.01 (1H). 13C NMR (cis and trans conformers): 172.51, 172. 31,
155.03, 154.56, 136.35, 136.15, 135.51, 135.30, 128.48, 128.40,
128.34, 128.28, 128.20, 128.05, 128.00, 127.91, 127.77, 127.71,
69.91, 69.18, 67.19, 66.88, 66.75, 58.03, 57.79, 55.15, 54.55, 39.07,
38.26. HR-MS: calcd for C20H22NO5, [M + H]+ )356.1498, found
356.1503. Rf ) 0.24 (hexane/ethyl acetate ) 5:5).
(2S,4S)-Nr-Cbz-4-[(p-Tou len esu lfon yl)oxy]p r olin e Ben z-
yl Ester (13). p-Toluenesulonyl chloride (9.45 g, 50 mmol) was
added portionwise to a solution of alcohol 12 (16.0 g, 45 mmol)
in pyridine (70 mL) at 0 °C. After the mixture was stirred for
12 h at room temperature, the solvent was evaporated in vacuo.
The residue was poured into an ice bath (50 g), and the mixture
was extracted with ethyl acetate (100 mL × 3). The extract was
washed with HCl (0.5 mL, 0.5 M), H2O, Na2CO3 (10%), H2O,
and then dried over Na2SO4. Concentration of the solvent in
vacuo gave an oily residue, which was purified by column
chromatography (hexanes-ethyl acetate 7:3) to give as a color-
obtained in a low yield, ∼30%. The reaction was repeated,
using bromide. Unfortunately, slow rates and low yields
of the desired trans-product led us to abandon this
approach.
An alternative method to invert a chiral center of an
alcohol is the Mitsunobu reaction (Scheme 5). When
trans-4-hydroxy proline derivative 12 was subjected to a
Mitsunobu reaction, the corresponding cis-bromoproline
22 was obtained in a high yield (90%) after purification.
Azide displacement of Br with NaN3 in DMF afforded
the 4-trans-azide proline derivative (21, 94% after puri-
fication). Reduction of azide by PPh3 in THF and water
gave the amine 23 which was used directly without
purification. Nucleophilic addition of 4-trans-aminopro-
line using the guadinino reagent 10 was much faster than
with the cis counterpart. The reaction time was 1 day
with an improved yield of almost 80% after purification.
Hydrogenolysis of the fully protected guanidino com-
pound 24 removed the Cbz and Bn protecting groups and
afforded the free proline derivative with free R-amino and
acidic groups 25. Fmoc protection (same protocol as
discussed above) provided fully orthogonally protected
NR-Fmoc 4-trans-(N,N′-di-Boc)guanidinoproline (26) in an
almost quantitative yield (96%).
In summary, we have successfully synthesized enan-
tiomerically pure proline analogues with 4-guanidino
substitutions. These analogues are fully protected for
Fmoc chemistry for peptide and protein synthesis. There
are a number of advantages over the analogues made for
Boc chemistry.31 Applications of these compounds in the
design of novel bioactive ligands are underway.
less oil (13). Yield: 19.4 g (86%), [R]30 ) -35.3 (c 1.18, CHCl3).
D
1H NMR (CDCl3): 7.79-7.76 and 7.40-7.20 (14H), 5.23-5.50
(4H), 4.68-4.49 (1H), 3.78-3.65 (2H), 2.61-2.45 (1H and 3H),
2,23-2.12 (1H). 13C NMR: 171.68, 171.49, 154.42, 153.85,
145.33, 136.12, 136.02, 135.30, 135.08, 133.38, 133.30, 130.09,
130.05, 128.60, 128.51, 128.45, 128.39, 128.20, 128.12, 127.90,
127.74, 78.67, 78.03, 67.43, 67.20, 67.08, 57.62, 57.33, 52.43,
52.11, 37.25, 36.03, 21.66. HR-MS: calcd for C27H28NO7S [M +
H]+ ) 510.1586, found 510.1592. Rf ) 0.25 (hexane/ethyl acetate
) 7:3).
(2S,4S)-Nr-Cbz-4-a zid op r olin e Ben zyl Ester (14). Sodium
azide (3.30 g, 51 mmol) was added to a solution of tosylate 13
(6.46 g, 12.7 mmol) in DMF (30 mL). The mixture was heated
at 65-70 °C for 8 h. The reaction mixture was diluted with
water, and the mixture was extracted with ethyl acetate. The
extract was washed with brine and then dried over Na2SO4
overnight. Concentration of the solvent in vacuo gave an oily
residue, which was purified by column chromatography (hex-
anes-ethyl acetate 8:2) to give 14 as a colorless oil, which
solidified as a white powder from an ether-hexane solution.
Yield: 4.42 g (87%). Mp: 66-68 °C. [R]25 ) -47.3 (c 0.96,
D
CHCl3). 1H NMR (CDCl3): 7.39-7.38 (10H), 5.29-5.06 (4H),
4.60-4.49 (1H), 4.21 (1H), 3.85-3.76 (1H), 3.64-3.56 (1H), 2.52-
2.42 (1H), 2.28-2.24 (1H). 13C NMR: 171.11, 170.85, 154.10,
136.30, 136.22, 135.49, 135.33, 128.56, 128.45, 128.39, 128.30,
128.25, 128.14, 128.06, 128.00, 127.91, 67.40, 67.33, 67.14, 59.26,
58.32, 57.93, 57.68, 51.51, 51.21, 36.14, 35.12. HR-MS: calcd
for C20H21N4O4 [M + H]+ ) 381.1563, found 381.1573. Rf ) 0.21
(hexane/ethyl acetate ) 7:3).
(2S,4S)-Nr-Cbz-4-a m in op r olin e Ben zyl Ester (15). A solu-
tion of azide 14 (838 mg, 2.2 mmol), tetrahydrofuran (10 mL),
triphenylphosphine (1.15 g, 4.4 mmol), and water (0.08 mL, 4.4
mmol) was refluxed for 6 h with stirring and then concentrated
in vacuo. The residue was dissolved in diethyl ether (30 mL)
and HCl (0.1 N, 20 mL). The aqueous layer was extracted with
diethyl ether (30 mL × 2) and was subsequently neutralized with
Na2CO3 (10%), and then the mixture was extracted with dichlo-
romethane (30 mL × 3). The combined CH2Cl2 layers were dried
over MgSO4 overnight, filtered, and concentrated in vacuo to
afford amine 15 as a colorless oil, which was used to the next
reaction without further purification. A small portion of the
crude product was further purified by flash chromatography for
analysis. 1H NMR (CDCl3): 7.45-7.22 (10H), 5.25-5.00 (4H),
Exp er im en ta l P r oced u r es
All reagents, unless otherwise noted, were purchased from
Aldrich Chemical Co. Flash chromatography was performed
using Merck silica gel 60 (70-230 mesh). TLC was performed
on Whatman silica gel 60A MK6F plates and was developed with
ninhydrin in ethanol followed by heating. Optical rotation values
were obtained using an automatic polarimeter. High-resolution
mass spectra were obtained using the FAB method. NMR
spectra were obtained on a 500 MHz instrument equipped with
gradient shimming. Solvents used were one of the following
(31) Webb, T. R.; Eigenbrot, C. J . Org. Chem. 1991, 56, 3009-3016.

Notes
J . Org. Chem., Vol. 66, No. 3, 2001 1041
4.44-4.35 (1H), 3.77-3.68 (1H), 3.60-3.51 (1H), 3.34-3.28 (1H),
2.47-2.38 (1H), 1.87-1.80 (1H). 13C NMR: 172.86, 172.73,
154.88, 154.25, 141.20, 136.54, 136.40, 135.59, 135.38, 132.11,
132.04, 131.95, 131.93, 128.61, 128.55, 128.52, 128.49, 128.47,
128.41, 128.36, 128.30, 128.27, 128.23, 128.16, 128.06, 128.01,
127.96, 127.88, 127.83, 127.45, 126.92, 67.32, 67.28, 67.16, 67.14,
67.01, 66.93, 65.08, 65.06, 65.05, 58.44, 58.15, 55.55, 55.11, 51.07,
(eluent, hexane/ethyl acetate ) 8:2) afforded product 22 as a
white solid. Yield: 4.52 g (90%). Mp: 82-83 °C. [R]25 ) -42.5
D
1
(c 1.12, CHCl3). H NMR (CDCl3): 7.40-7.28 (10H), 5.30-5.04
(4H), 4.58-4.47 (1H), 4.36-4.31 (1H), 4.21-4.11 (1H), 3.88-
3.80 (1H), 2.90-2.82 (1H), 2.55-2.46 (1H). 13C NMR: 171.09,
170.83, 154.28, 153.80, 136.20, 136.14, 135.40, 135.23, 128.54,
128.45, 128.40, 128.32, 128.27, 128.17, 128.08, 128.04, 127.93,
67.48, 67.40, 67.27, 67.20, 58.42, 58.20, 55.90, 55.54, 41.99, 41.30,
50.22, 39.46, 38.66. HR-MS: calcd for C20H23N2O4 [M + H]+
355.1658, found 355.1654. Rf ) 0.40 (CHCl3/CH3OH ) 9:1).
)
40.93, 39.90. HR-MS: calcd for C20H21BrN11O4 [M + H]+
)
(2S,4S)-Nr-Cbz-4-N,N′-d i-Boc-gu a n id in op r olin e Ben zyl
Ester (16). Compound 15 (2.2 mmol, crude product was used
directly) in CH2Cl2 (5 mL) was added in one portion to a solution
of the guanidination reagent 7 (860 mg, 2.2 mmol) and triethyl-
amine (0.31 mL, 2.2 mmol) in CH2Cl2 (15 mL) at 0 °C, stirred
for 3 days at room temperature, and then concentrated in vacuo.
The residue was purified by flash column chromatography on
silica gel (eluent, hexane/ethyl acetate ) 7:3) to give a colorless
420.0652, found 420.0636. Rf ) 0.43 (hexane/ethyl acetate ) 7:3).
(2S,4R)-Nr-Cbz-4-a zid op r olin e Ben zyl Ester (21). The SN2
reaction between 22 (1.25 g, 3 mmol) and sodium azide (1.32 g,
20 mmol) was performed by the same method as used to prepare
14. This reaction was performed at room temperature for 1 day.
The purification of 21 was performed by column chromatography
(hexane/ethyl acetate ) 8:2) to give a colorless oil. Yield: 2.15 g
1
(94%). [R]25 ) -51.7 (c 1.63, CHCl3). H NMR (CDCl3): 7.38-
D
oil (16). Yield: 850 mg (65% from compound 14, two steps). [R]25
7.19 (10H), 5.24-5.00 (4H), 4.55-4.45 (1H), 4.19-4.18 (1H),
3.78-3.73 (1H), 3.68-3.54 (1H), 2.38-2.30 (1H), 2.21-2.14 (1H).
13C NMR: 171.88, 171.70, 154.56, 154.00, 136.24, 136.13, 135.38,
135.17, 128.63, 128.61, 128.60, 128.55, 128.53, 128.49, 128.45,
128.40, 128.22, 128.15, 128.08, 127.95, 127.89, 67.46, 67.39,
67.19, 67.07, 59.21, 58.64, 57.95, 57.66, 51.81, 51.32, 36.30, 35.26.
HR-MS: calcd for C20H21N4O4 [M + H]+ ) 381.1563, found
381.1574. Rf ) 0.58 (hexane/ethyl acetate ) 7:3).
D
) -24.0 (c 1.06, CHCl3). 1H NMR (CDCl3): 8.60-8.55 (1H, NH),
7.32-7.18 (10H), 5.32-4.84 (4H), 4.80-4.77 (1H), 4.48-4.39
(1H), 3.94-3.87 (1H), 3.52-3.43 (1H), 2.62-2.53 (1H), 2.08-
1.99 (1H), 1.51-1.45 (18H). 13C NMR: 172.02, 171.91, 163.10,
155.48, 154.61, 154.05, 152.73, 136.09, 135.97, 135.42, 135.28,
128.40, 128.28, 128.16, 128.06, 127.98, 127.92, 127.87, 127.82,
127.63, 83.27, 79.45, 67.30, 67.20, 66.98, 66.90, 58.11, 57.83,
52.39, 52.00, 49.19, 48.41, 36.54, 35.55, 28.10, 27.87, 27.64. HR-
MS: calcd for C31H41N4O8 [M + H]+ ) 597.2924, found 597.2913.
Rf ) 0.24 (hexane/ethyl acetate ) 7:3).
(2S,4R)-Nr-Cb z-4-a m in op r olin e Ben zyl E st er (23). The
selective hydrogenation of azide in 21 (1.52 g, 4 mmol) was
performed by the same method as used to prepare 15. Compound
23, obtained as a colorless oil, was used to the next reaction
without further purification. A small portion of the crude product
was further purified by flash chromatography. 1H NMR
(CDCl3): 7.64-7.18 (10H), 5.21-4.92 (4H), 4.55-4.48 (1H),
3.79-3.73 (1H), 3.70-3.65 (1H), 3.30-3.17 (1H), 2.17-2.11 (1H),
2.08-1.96 (1H), 1.64 (2H). HR-MS: calcd for C20H23N2O4 [M +
H]+ ) 355.1658, found 355.1660. Rf ) 0.42 (CHCl3/CH3OH )
9:1).
(2S,4S)-4-N,N′-Di-Boc-gu a n id in op r olin e (17). Compound
16 (800 mg, 1.34 mmol) was hydrogenated over palladium on
carbon (5%, 80 mg) in methanol (40 mL) for 7 h at room
temperature under H2 atmosphere (3 atm). The catalyst was
filtered off, and the filtrate was concentrated in vacuo to give a
residue which was purified by the recrystallization from methanol/
ether/hexane. Yield: 374 mg (75%). Mp: >300 °C (dec, getting
dark starting at 285 °C). [R]25 ) -32.5 (c 0.31, CH3OH). 1H
D
NMR (CD3OD): 4.61-4.59 (1H), 4.01-3.98 (1H), 3.53-3.50 (1H),
3.39-3.36 (1H), 2.73-2.67 (1H), 2.14-2.09 (1H), 1.50 (9H), 1.45
(9H). 13C NMR (CD3OD): 171.95, 162.61, 155.58, 152.38, 83.33,
79.24, 60.80, 49.88, 49.83, 34.56, 30.88, 27.06, 26.77. HR-MS:
calcd for C16H29N4O6 [M + H]+ ) 373.2089, found 373.2083. Rf
) 0.43 (n-BuOH/AcOH/H2O ) 4:1:1).
(2S,4R)-NrCbz-4-N,N′-d i-Boc-gu a n id in op r olin e (24). The
coupling between 23 (4 mmol) and guanidination reagent 7 (1.56
g, 4 mmol) was performed by the same method as used to
prepare 16. DIPEA (517 mg, 4 mmol) was used as a base instead
of triethylamine. This reaction was performed for 1 day at room
temperature. The purification of 24 was performed by flash
column chromatography and recrystallization from ether/hexane.
(2S,4S)-Nr-F m oc-4-N,N′-d i-Boc-gu a n id in op r olin e (18).
Compound 17 (200 mg, 0.54 mmol) and sodium carbonate (113
mg, 1.08 mmol) were suspended in water (10 mL) and cooled in
an ice bath. A solution of Fmoc-OSu (268 mg, 0.8 mmol) in
dimethylformamide (10 mL) was added in one portion at 0 °C
and stirred at room temperature for 15 min. The mixture was
diluted with water (20 mL) and acidified to pH ) 2 with HCl (3
M in water). The mixture was extracted with ethyl acetate (3 ×
50 mL). The extract was washed with water, dried with Na2-
SO4, and evaporated. The crude product was purified by flash
chromatography on silica gel (eluent, hexane/ethyl acetate ) 7:3
and chloroform/methanol ) 20:1) and recrystallization from
ether/hexane. Yield: 247 mg (77%). Mp: >300 °C (dec, getting
dark starting at 220 °C). [R]25D ) -25.2 (c 0.50, CHCl3). 1H NMR
(DMSO-d6): 8.43-8.39 (1H), 7.87-7.28 (8H), 4.61-4.60 (1H),
4.38-4.12 (1H and 3H), 3.79-3.74 (1H), 3.37-3.32 (1H), 2.64-
1.92 (2H), 1.44-1.38 (18H). 13C NMR: 174.52, 174.08, 163.33,
155.21, 154.19, 152.12, 144.13, 144.05, 141.17, 141.10, 128.18,
128.13, 127.61, 125.65, 120.60, 83.50, 78.89, 67.67, 65.36, 58.16,
57.74, 52.86, 52.26, 49.88, 48.98, 47.11, 46.99, 36.21, 35.07, 28.42,
28.04. HR-MS: calcd for C31H39N4O8 [M + H]+ ) 595.2768,
found: 595.2771. Rf ) 0.43 (CHCl3/CH3OH ) 9:1).
Yield: 1.87 g (78% from compound 21). Mp: 118-119 °C. [R]25
D
) -27.7 (c 0.50, CHCl3). 1H NMR (CDCl3): 11.42 (1H), 8.48-
8.47 (1H), 7.34-7.20 (10H), 5.22-5.00 (4H), 4.78-4.76 (1H),
4.56-4.46 (1H), 3.91-3.88 (1H), 3.58-3.43 (1H), 2.32-2.26 (2H),
1.48-147 (18H). 13C NMR: 171.78, 171.53, 163.22, 155.63,
154.78, 154.16, 136.35, 136.22, 135.44, 135.25, 128.56, 128.46,
128.40, 128.27, 128.16, 128.10, 128.00, 127.82, 83.54, 83.48,
79.54, 67.27, 67.10, 66.97, 57.94, 57.67, 52.60, 52.15, 49.38, 48.75,
36.80, 35.79, 28.22, 28.01. HR-MS: calcd for C31H41N4O8: [M +
H]+ ) 597.2924, found 597.2925. Rf ) 0.43 (hexane/ethyl acetate
) 7:3).
(2S,4R)-4-N,N′-Di-Boc-gu a n id in op r olin e (25). The hydro-
genation of 24 (1.50 g, 2.5 mmol) was performed by the same
method as used to prepare 17. Yield: 840 mg (91%). Mp: >300
°C (dec, getting dark starting at 290 °C). [R]25 ) -7.31 (c )
D
0.56, CH3OH). 1H NMR (DMSO-d6): 8.24-8.23 (1H), 4.45-4.44
(1H), 3.77-3.74 (1H), 3.43-3.40 (1H), 3.04-3.00 (1H), 2.24-
2.06 (2H), 1.45-1.37 (18H). HR-MS: calcd for C16H29N4O6 [M
+ H]+) 373.2087, found 373.2085. Rf ) 0.5 (n-BuOH/AcOH/H2O
) 4:1:1).
(2S,4R)-Nr-F m oc-4-N,N′-d i-Boc-gu a n id in op r olin e (26).
The coupling between 25 (500 mg, 1.34 mmol) and Fmoc-OSu
(670 mg, 2 mmol) was performed by the same method as used
(2S,4R)-NR-Cbz-4-br om op r olin e Ben zyl Ester (22). Di-
ethyl azodicarboxylate (10.3 g, 59 mmol) was added dropwise
with stirring to a solution of triphenylphosphine (15.7 g, 60
mmol) in anhydrous tetrahydrofuran (100 mL) under argon.
After 20 min, lithium bromide (10.4 g, 120 mmol) was added to
the nearly colorless solution followed by the alcohol 12 (4.27 g,
12 mmol) in anhydrous tetrahydrofuran (50 mL). The mixture
was stirred at room temperature for 20 h. After evaporation of
the solvent, the residue was poured into water and extracted
twice with diethyl ether. The combined ether layer was washed
with brine and dried over anhydrous Na2SO4. After removal of
the solvent under reduced pressure, a solid residue was obtained.
Purification by flash column chromatography on silica gel
to prepare 18. Yield: 770 mg (96%). Mp: 87-89 °C. [R]30
)
D
-17.0 (c 0.55, CHCl3). 1H NMR (DMSO-d6): 8.26-8.23 (1H),
7.92-7.27 (8H), 4.55-4.54 (1H), 4.42-4.14 (1H and 3H), 3.73-
3.69 (1H), 3.33-3.28 (1H), 2.45-2.13 (2H), 1.44-1.38 (18H). 13
C
NMR: 173.95, 173.55, 163.32, 162.73, 155.57, 155.49, 154.32,
154.12, 152.43, 144.19, 144.11, 144.04, 141.17, 141.09, 128.16,
127.61, 127.51, 125.72, 125.67, 125.53, 120.56, 83.63, 79.02,
67.59, 67.34, 58.04, 57.80, 51.83, 51.35, 49.43, 48.50, 47.08, 46.99,
36.21, 35.74, 34.66, 31.40, 31.20, 28.40, 18.10, 22.50. HR-MS:
calcd for C31H39N4O8 [M + H]+ ) 595.2768, found: 595.2773. Rf
) 0.28 (CHCl3/CH3OH ) 9:1).

1042 J . Org. Chem., Vol. 66, No. 3, 2001
Notes
Ack n ow led gm en t. We thank Dr. Doyle for the use
of the polarimeter in his laboratory. This research was
supported by grants from the US Public Health Service,
Grant Nos. DA06284 and DK17420. The views ex-
pressed are those of authors and not necessarily the
reflection of those of the USPHS. Mass Spectrometer
and NMR facilities in the Department of Chemistry at
the University of Arizona were partially funded by
grants from the NSF. The help of Dr. M. Kavanara with
the manuscript is appreciated.
J O005626M