Synthetic approaches to Nδ-methylated L-arginine, N ω-hydroxy-L-arginine, L-citrulline, and Nδ- cyano-L-ornithine

Article abstract of DOI:10.1021/jo702150d

(Chemical Equation Presented) N^ω-Methylated arginines such as asymmetric dimefhyl-L-arginine (ADMA) and monomethyl-L-arginine (NMMA) are known as potent physiological inhibitors of nitric oxide synthases (NOSs). To explore a possible physiologi

Full text of DOI:10.1021/jo702150d

Synthetic Approaches to Nδ-Methylated L-Arginine,
Nω-Hydroxy-L-arginine, L-Citrulline, and Nδ-Cyano-L-ornithine
Dennis Schade, Katrin To¨pker-Lehmann, Ju¨rke Kotthaus, and Bernd Clement*
Department of Pharmaceutical Chemistry, Pharmaceutical Institute, Christian-Albrechts-UniVersity of Kiel,
Gutenbergstrasse 76-78, D-24118 Kiel, Germany
bclement@pharmazie.uni-kiel.de
ReceiVed October 3, 2007
Nω-Methylated arginines such as asymmetric dimethyl-L-arginine (ADMA) and monomethyl-L-arginine
(NMMA) are known as potent physiological inhibitors of nitric oxide synthases (NOSs). To explore a
possible physiological and pharmaceutical relevance of Nδ-methylated analogues, a synthetic scheme
had to be developed that would not lead to Nδ-methyl-L-arginine only but also to its presumed metabolites
of NOS catalysis. Two basic synthetic approaches have been pursued to obtain Nδ-methylated derivatives
of L-ornithine, L-citrulline, L-arginine, and Nω-hydroxy-L-arginine. A first attempt utilized conventionally
protected L-ornithine, i.e., the tert-butyl ester and Boc-amine, and led to three end compounds in excellent
yields. Simultaneous protection of the R-amino acid moiety by formation of boroxazolidinones, particularly
by employing 9-borabicyclo[3.3.1]nonane (9-BBN-H), proved to be a convenient option to perform side
chain modifications and led to all of the desired end compounds. Additionally, enantiomeric excess (ee,
%) of crucial synthetic intermediates and end compounds was determined by chiral HPLC.
Introduction
strategies have emerged to provide rational control of NO levels
by taking various metabolic pathways into consideration.
Nevertheless, the predominant enzymes in this regulation
process are nitric oxide synthases (NOSs) and arginases.3
Physiologically, the activity of NOSs can be regulated by
the endogenous, competitive inhibitors asymmetric dimethyl-
L-arginine (ADMA) and monomethyl-L-arginine (NMMA).
These Nω-methylated L-arginines are implicated in the develop-
ment of diseases such as atherosclerosis and endothelial
dysfunction.4 The formation of Nδ-methyl-L-arginine 15 in yeast
cells was described in 1998 and the responsible yeast enzyme
was identified 1 year later.5 However, there has not yet been
reported further evidence of any physiological significance of
15.
Nitric oxide (NO) is a potent vasodilator that regulates
vascular tone and tissue blood flow and inhibits platelet
aggregation and leukocyte adhesion on the endothelial surface.
In addition, it is generated in large quantities during host defense
and immunological reactions and is synthesized in neurons of
the central nervous system, where it acts as a neuromediator
with many physiological functions.1 Accordingly, it is vital that
endogenous NO levels are strictly regulated. As a consequence
it is not surprising that dysregulation leads or contributes to
the development of numerous diseases.2 To date, a number of
* Address correspondence to this author. Phone: +49-431-8801126. Fax:
+49-431-8801352.
(1) (a) Ignarro, L.; Murad, F., Eds. Nitric Oxide: Biochemistry, molecular
biology, and therapeutic implications. In AdVances in Pharmacology;
Academic Press: San Diego, CA, 1995; Vol. 34. (b) Alderton, W. K.;
Cooper, C. E.; Knowles, R. G. Nitric oxide synthases: structures, function
and inhibition. Biochem. J. 2001, 357, 593.
(3) Boucher, J. L.; Moali, C.; Tenu, J. P. Cell Mol. Life Sci. 1999, 55,
1015.
(4) (a) Beltowski, J.; Kedra, A. Pharmacol. Rep. 2006, 58, 159. (b)
Cardounel, A. J.; Cui, H.; Samouilov, A.; Johnson, W.; Kearns, P.; Tsai,
A. L.; Berka, V.; Zweier, J. L. J. Biol. Chem. 2007, 282, 879.
(2) Vallance, P.; Leiper, J. Nat. ReV. Drug DiscoVery 2002, 1, 939.
10.1021/jo702150d CCC: $40.75 © 2008 American Chemical Society
Published on Web 01/08/2008
J. Org. Chem. 2008, 73, 1025-1030
1025

Schade et al.
SCHEME 1. Synthetic Approach via Conventional
Protecting Group Chemistrya
Attempts to selectively monomethylate the protected L-
ornithine with paraformaldehyde followed by reduction with
sodium borohydride mainly resulted in the dimethylated com-
pound 1. This tertiary amine could then be reacted with
cyanogen bromide (BrCN) in dry dioxane employing the von
Braun degradation reaction.9 The resulting cyanamide 3 was
the key intermediate for the following conversion to three of
the desired amino acids. Deprotection under anhydrous condi-
tions with HCl in dioxane gave the hydrochloride of Nδ-cyano-
Nδ-methyl-L-ornithine 4, whereas partial hydrolysis of 3 under
mild conditions by treatment with dilute alkaline hydrogen
peroxide solution, followed by removal of the protecting groups
with HCl in dioxane, led to the Nδ-methylated L-citrulline 7.
Building up the hydroxyguanidine moiety was achieved by
reacting cyanamide 3 with hydroxylamine base10 in dry dioxane.
Eventually, deprotection yielded Nω-hydroxy-Nδ-methyl-L-argi-
nine as the dihydrochloride 8. Owing to the fact that the oxygen
in hydroxylamine exhibits nucleophilic properties, its reaction
with the cyanamide cannot be ruled out, yielding an unstable
aminoxyformamidine11 derivative that would decompose to the
respective urea. Thus, we performed 15N NMR spectroscopy
experiments with 15N-labeled 8 to show that the reaction
occurred via the nitrogen of hydroxylamine, which was the case
under these reaction conditions, i.e., in aprotic solvent with
hydroxylamine base (Supporting Information).12 Essentially, all
syntheses within this approach could be performed under
conditions which did not significantly lead to racemization
(g98% ee), shown by enantiomeric separation of 7 and 8 on a
Crownpak Cr(+) HPLC column (for details see the Supporting
Information).13 Nevertheless, Nδ-methyl-L-arginine as a pivotal
target compound was not accessible by this route, since
cyanamide 3 could not be converted to the respective guanidine
compound with ammonia under various reaction conditions. We
also tested a direct hydrogenation of hydroxyguanidine 8 using
Pd/charcoal, H2 in methanol (acetic acid 5% v/v) at atmospheric
pressure, which proved difficult. Further attempts are underway
considering successful reductions of other hydroxyguanidines
described in the literature.14
a Reagents and conditions: (a) [CH2O]n, Na2CO3, NaBH4, MeOH, 85%.
(b) BrCN, Na2CO3, dioxane, 94%. (c) HCl, dioxane, 91%. (d) H2O2 (3%),
1 M NaOH, EtOH, 86%. (e) NH2OH, dioxane, 35%. (f) HCl, dioxane, 90%.
(g) HCl, dioxane, 63%.
We aimed to examine a possible physiological role and
pharmaceutical relevance of Nδ-methylated analogues by testing
all enzymes that are involved in regulating endogenous NO
levels, primarily NOSs and arginase.6 The physiological process
of NOS catalysis is a NADPH- and O2-dependent five-electron
oxidation of L-arginine to nitric oxide and L-citrulline via the
intermediate Nω-hydroxy-L-arginine,7 requiring the synthesis of
these Nδ-methyl analogues. The occurrence of an intermediately
formed cyanamide is reported under certain conditions, also
necessitating the synthesis of the respective Nδ-methyl ana-
logue.8
Thus, a second methodology was established that utilized the
simultaneous protection of the R-amino acid moiety of Nδ-
methyl-L-ornithine 10 by intermediate formation of boroxazo-
lidinones.15 This approach was already described for the
preparation of 8 and 15 by Luzzi and Marletta,16b who
demonstrated the usability of B,B-diethyl boroxazolidinones.
First, selective monomethylation of the side chain primary amine
was performed in a four-step synthesis according to literature
Results and Discussion
(9) Braun, V. J Ber. Dtsch. Chem. Ges. 1900, 33, 1438.
(10) Hydroxylamine base was set free from its hydrochloride with sodium
ethanolate. Steudel, R.; Schenk, P. W. In Handbuch der pra¨paratiVen
anorganischen Chemie, 3rd ed.; Brauer, G., Ed.; Ferdinand Enke Verlag:
Stuttgart, Germany, 1975; Vol. 1, p 464.
(11) (a) Zinner, G.; Kleinau, V. Chem.-Z. 1977, 10, 451. (b) Belzecki,
C.; Hintze, B.; Kwiatkowska, S. Bull. Acad. Pol. Sci. Chem. 1970, 18, 379.
(12) Similar results were obtained for 15Nω-hydroxy-L-arginine: Clement,
B.; Schno¨rwangen, E.; Ka¨mpchen, T.; Mordvintcev, P.; Mu¨lsch, A. Arch.
Pharm. 1994, 327, 793.
From the preparative point of view it was desirable to develop
(a) one common synthetic scheme that leads to all end
compounds and (b) a synthetic sequence with minimum risk
for racemization. Our first strategy started with the methylation
of commercially available NR-Boc-L-ornithine-O-tert-butyl ester
2 (Scheme 1).
(5) (a) Zobel-Thropp, P.; Gary, J. D.; Clarke, S. J. Biol. Chem. 1998,
273, 29283. (b) Niewmierzycka, A.; Clarke, S. J. Biol. Chem. 1999, 274,
814.
(6) Kotthaus, J.; Schade, D.; To¨pker-Lehmann, K.; Beitz, E.; Clement,
B. Bioorg. Med. Chem. 2007, doi: 10.1016/j.bmc.2007.11.066.
(7) Stuehr, D. J.; Kwon, N. S.; Nathan, C. F.; Griffith, O. W.; Feldman,
P. L.; Wiseman, J. J. Biol. Chem. 1991, 266, 6259.
(8) (a) Clague, M. J.; Wishnok, J. S.; Marletta, M. A. Biochemistry 1997,
36, 14465. (b) Rusche, K. M.; Spiering, M. M.; Marletta, M. A. Biochemistry
1998, 37, 15503.
(13) Enantiomeric excess of cyanamide 4 was not determined due to its
hydrolytic instability in the required eluent, i.e. aq HClO4, pH 1.5.
(14) (a) Looper, R. E.; Runnegar, M. T.; Williams, R. M. Tetrahedron
2006, 62, 4549. (b) Nordmann, R.; Loosli, H.-R. HelV. Chim. Acta 1985,
68, 1025.
(15) Nefkens, G. H. L.; Zwanenburg, B. Tetrahedron 1983, 39, 2995.
(16) (a) Diethylboryl complexation procedure under these conditions first
described by Garcia et al. Tetrahedron: Asymmetry 2000, 11, 991. (b) Luzzi,
S. D.; Marletta, M. A. Bioorg. Med. Chem. Lett. 2005, 15, 3934. (c)
Described by Luzzi and Marletta for the diethylboryl complexes.
1026 J. Org. Chem., Vol. 73, No. 3, 2008

Substituted Arginine, Citrulline, and Ornithine
SCHEME 2. Formation of Boroxazolidinone Complexes
Both boron complexes were submitted to the following side
chain manipulations and identical chemical procedures were
employed (Scheme 3). Building up the guanidine group from
11 or 12 was accomplished with N,N′-bis(tert-butyloxycarbo-
nyl)thiourea in DMF with triethylamine as the base.16c The
protected intermediates 13 and 14 could be isolated and
completely characterized.26 Removal of both the Boc-groups
and the boron complexes was carried out under mild conditions
in one pot by treating 13 or 14 with TFA and subsequently
with aqueous 1.5 M HCl, since boroxazolidinones appeared to
be quite stable to TFA.27 Nδ-Methyl-L-arginine was isolated as
the monohydrochloride 15 after cation exchange chromatogra-
phy. Converting boroxazolidinone-protected Nδ-methyl-L-orni-
thine to the corresponding cyanamides 16 and 17 was performed
in dry DME16c with BrCN after unsuccessfully testing a variety
of other etherial solvents to improve yields of this step.
Particularly, the 9-BBN-strategy was expected to significantly
increase solubility in Et2O, THF, or dioxane and thereby
improve this yield-limiting step. However, compound 4 pre-
cipitated as the hydrochloride by flushing gaseous HCl over a
solution of 16 or 17 in dry dioxane. Partial hydrolysis to the
citrulline derivative was realized by treating 16 and 17 with
TFA (60%, aqueous) in acetone, taken the hydrolytic instability
of boron complexes in neutral to alkaline aqueous media into
consideration.27
FIGURE 1. Synthesis and chiral chromatography of Nδ-methylorni-
thine 10 on a Crownpak Cr(+) column using o-PA for postcolumn
derivatization. See the Supporting Information for details.
procedures16b,17-23 with minor modifications, after evaluating
the problematic nature of racemization (Figure 1).24
Several authors reported on the synthesis of 10 but only a
few specified RD-values, leaving open the question of enantio-
meric purity. In particular, the final deprotecting step with
refluxing HBr (47%) turned out to have a major impact on
racemization, which was confirmed by determining two direct
synthetic precursors to be >98% enantiomerically pure (Sup-
porting Information). Reaction times should not exceed 1.5 h
and in addition, HBr should be quickly removed in high vacuum
(<1 mbar) at low temperature, i.e., not exceeding 50-60 °C.
This way 98% ee of the L-enantiomer could be prepared in good
yields (84% for the last deprotecting step). Figure 1 also
demonstrates the consequence of longer reaction times (2 h)
and distillation at higher temperatures (80-90 °C), which was
a 71% ee Nδ-methylornithine batch.
Nω-Hydroxy-Nδ-methyl-L-arginine 8 was obtained directly by
reacting hydroxylamine hydrochloride with 16 or 17 in dry
methanol.16c Thereby, the boroxazolidinones are cleaved, but
building up the hydroxyguanidine moiety apparently proceeds
faster. Under these conditions it cannot be circumvented that
the respective citrulline derivative 7 originates as a side product
(1-1.5%) via the mechanism discussed above. This contamina-
tion increases with prolonged reaction times, and 2.5 h was
found to be the ideal compromise between yield and side
product. Attempts to avoid this side reaction were undertaken
by changing to aprotic solvents but with concomitant, major
losses in yield again. Chromatographic workup was then
performed on a cellulose column and afforded Nω-hydroxy-Nδ-
methyl-L-arginine as the free amino acid that was subsequently
isolated as the dihydrochloride 8 by flushing gaseous HCl over
a suspension in dioxane.28
Compound 10 was converted to the diethylboryl complex 11
by refluxing with triethylborane (1 M solution in THF) in
DME16a,b for 72 h (Scheme 2). However, rather long reaction
times, the fact that 11 could not be isolated in high purity, and
difficulties in subsequent reactions prompted us to choose
9-borabicyclo[3.3.1]nonane (9-BBN-H) as an alternate com-
plexing reagent. 9-BBN-H was easily and efficiently introduced
by refluxing it with 10 in methanol for 1.5 h according to the
protocol of Dent et al.25
(17) Rudinger, J.; Gut, V. Tetrahedron 1968, 24, 6351.
(18) Erlanger, B. F.; Sachs, H.; Brand, E. J. Am. Chem. Soc. 1954, 76,
1806.
(19) Yamamoto, H.; Yang, J. T. Biopolymers 1974, 13, 1093.
(20) Benoiton, L. Can. J. Chem. 1964, 42, 2043.
(21) Thomas, K.; Kapfhammer, J.; Flaschentra¨ger, B. Hoppe-Seyler’s
Z. Physiol. Chem. 1923, 125, 75.
(22) Klein, C.; Schulz, G.; Steglich, W. Liebigs Ann. Chem. 1983, 1623.
(23) Paik, W. K.; Paik, K. P.; Sangduk, K. Anal. Biochem. 1980, 104,
343.
(24) Protocols of different authors for synthetic intermediates leading to
10 and the final deprotecting step were considered and evaluated. Detailed
synthetic protocols are provided in the Supporting Information along with
the references. Note: So far reported characterization data were in most
cases incomplete and are summarized in the Supporting Information.
(25) (a) Dent, W. H., III; Erickson, W. R.; Fields, S. C.; Parker, M. H.;
Tromiczak, E. G. Org. Lett. 2002, 4, 1249. (b) Further articles reporting
about similar application of 9-BBN-H: Cudic, M.; Mari, F.; Fields, G. B.
J. Org. Chem. 2007, 72, 5581. Syed, B. M.; Gustafsson T.; Kihlberg, J.
Tetrahedron 2004, 60, 5571. Walker, W. H., IV; Rokita, S. E. J. Org. Chem.
2003, 68, 1563.
(26) A complete spectroscopic characterization of the herein described
complexed intermediates is given in the Supporting Information, including
2D NMR experiments with 9-BBN complexes.
(27) 9-BBN complex 12 was examined in terms of pH stability (HPTLC
experiments, data not shown). It withstands even concentrated TFA over
several hours, making it suitable in orthogonal sets, but hydrolyzes rapidly
in dilute HCl as well as at pH >6. Maximum stability was observed at pH
2-4.
(28) Hydroxyguanidines are known to be most stable as salts of strong
acids. Literature: (a) hydroxyguanidine: Walker, J. B.; Walker, M. S. J.
Biol. Chem. 1959, 234, 1481. (b) Nω-hydroxy-L-arginine: Fukuto, J. M.
Meth. Enzymol. 1996, 268, 365.
J. Org. Chem, Vol. 73, No. 3, 2008 1027

Schade et al.
SCHEME 3. Synthetic Approach via Boron Complexing Strategya
a Reagents and conditions: (a) N,N′-Bis(tert-butyloxycarbonyl)thiourea, HgCl2, TEA, DMF, 41%. (b) Concentrated TFA, 1.5 N HCl, 85%. (c) BrCN,
TEA, DME, 29%. (d) HCl(g), dioxane, 75-85%. (e) 60% TFA(aq), acetone; HCl(g), dioxane, 60-80%. (f) NH2OH‚HCl, TEA, MeOH; HCl(g), dioxane, 80%.
TABLE 1. Overall Yields (%) for End Compounds
1 M HCl to 7-8. MeOH was removed under reduced pressure,
the water phase was adjusted to pH 4 with acetic acid, and unreacted
starting material was extracted with CH2Cl2. The product was
obtained by another extraction with CH2Cl2 at pH 8. The organic
phases were dried with Na2SO4 and evaporated in a vacuum. Further
purification was realized by chromatography on silica gel (CH2-
Cl2/MeOH, 8.5:1.5). The title compound crystallized from the
collected fractions in the refrigerator. Yield 941 mg (85%). Mp
entry
route Aa
route Bb
4
7
8
72.7 (30.8)
61.8 (26.2)
17.6 (7.5)
N.A.
8.9
8.4
8.4
15
12.6
a Conventional protecting strategy starting from commercially available
NR-Boc-L-ornithine-O-tert-butyl ester 2. It is to be noted that 2 is prepared
in a four-step synthesis from L-ornithine in a 42.4% overall yield.28b,29,30
Thus, for comparibility reasons, % values in parentheses represent yields
from L-ornithine. b Boron complexing strategy (9-BBN-H approach).
1
46 °C. H NMR (DMSO-d6, 300 MHz) δ 1.38 (s, 9H), 1.39 (s,
3
9H), 1.46-1.53 (m, 4H), 2.28 (s, 6H), 2.44 (t, 2H, J ) 6.5 Hz),
3
3
3.76 (t, 1H, J ) 6.0 Hz), 7.18 (d, 1H, J ) 7.5 Hz); 13C NMR
(CDCl3, 75 MHz) δ 23.3, 28.0, 28.4, 30.7, 45.2, 53.9, 59.2, 79.2,
81.5, 155.5, 172.0.
In conclusion, the presented boron complexing methodology
proved to be suitable for the herein described side chain
modifications and therefore underlines its specific applicability
in amino acid chemistry. 9-BBN-H as the complexing reagent
was superior to the respective diethylboryl complexes in terms
of ease of introduction, purity of intermediates, stability, and
yield in places. Importantly, no significant racemization took
place, either after obtaining optically pure Nδ-methyl-L-ornithine
10 or starting from 2. This fact is vital for the accurate
determination of Ki values for NOS inhibition and testings with
all other enzymes involved in the nitric oxide pathway. Besides
overall yields, the conventional protecting scheme might be
preferred with respect to building up a hydroxyguanidine moiety,
considering its stability toward hydroxylamine and the discussed
formation of the unwanted urea derivative.
NR-(tert-Butyloxycarbonyl)-Nδ-cyano-Nδ-methyl-L-ornithine
tert-Butyl Ester (3). 1 (500 mg, 1.58 mmol) was dissolved in 3
mL of dry 1,4-dioxane and the solution was added dropwise to a
solution of 270 mg of Na2CO3 and 178 mg (1.75 mmol) of
cyanogen bromide in 3 mL of 1,4-dioxane under nitrogen atmo-
sphere. After 20 h of stirring Na2CO3 was filtered off and the
reaction mixture was concentrated in a vacuum to give an oily
residue. The crude product was purified by flash chromatography
(EtOAc/n-pentane, 10:7). Yield 486 mg (94%). 1H NMR (DMSO-
d6, 300 MHz) δ 1.39 (s, 9H), 1.40 (s, 9H), 1.57-1.75 (m, 4H),
2.78 (s, 3H), 2.95 (t, 2H, 3J ) 6.5 Hz), 3.79 (m, 1H), 7.15 (d, 1H,
3J ) 7.9 Hz); 13C NMR (CDCl3, 75 MHz) δ 23.0, 27.9, 28.2, 29.8,
38.8, 52.4, 53.2, 79.7, 82.2, 118.2, 155.3, 171.3.
Nδ-Cyano-Nδ-methyl-L-ornithine Hydrochloride (4). From 3:
A 291 mg (0.889 mmol) sample of the protected cyanamide 3 was
dissolved in 7.5 mL of 4 M HCl in dry 1,4-dioxane and the solution
was allowed to stand for 12 h under nitrogen atmosphere. The
precipitated hydrochloride was filtered under a nitrogen stream,
washed with dry EtOAc, and stored under nitrogen at -20 °C due
to its high hygroscopicity and hydrolytic instability. Yield 168 mg
(91%). From 16, 17: A 0.288 mmol sample of thoroughly dried
16 or 17 was dissolved in 14 mL of dry 1,4-dioxane and a stream
of HCl was flushed over the liquid. After 24 h in the refrigerator
the desired compounds precipitated. For complete precipitation, dry
Et2O was added and 4 filtered under a nitrogen stream. Yield 44.8-
50.8 mg (75-85%). 1H NMR (DMSO-d6, 300 MHz) δ 1.64-1.86
Experimental Section
NR-(tert-Butyloxycarbonyl)-Nδ-dimethyl-L-ornithine tert-Butyl
Ester (1) NR-(tert-Butyloxycarbonyl)-L-ornithine tert-butyl ester
hydrochloride (1.14 g, 3.5 mmol) was dissolved in CH2Cl2 and the
corresponding base was set free by treatment with gaseous NH3.
The suspension was filtered and the organic solvent removed in
vacuo. The resulting yellow oil was dissolved in 30 mL of dry
MeOH and 1.8 g of Na2CO3 and 440 mg of paraformaldehyde (13.9
mmol) was added. This suspension was stirred for 2 h before adding
a 3-fold excess of NaBH4 (364 mg, 10.4 mmol). After 24 h the
reaction was quenched by adding water and adjusting the pH with
3
(m, 4H), 2.81 (s, 3H), 3.01 (t, 2H, J ) 6.9 Hz), 3.90 (m, 1H),
8.58 (s, 3H), 8.97 (br s, 1H); 13C NMR (DMSO-d6, 75 MHz) δ
1028 J. Org. Chem., Vol. 73, No. 3, 2008

Substituted Arginine, Citrulline, and Ornithine
22.4, 26.8, 38.1, 51.4, 51.6, 118.4, 170.6; HRMS m/z calcd for
C7H14N3O2 (MH+) 172.10805, found 172.10805.
98.1% (Crownpak Cr(+)). 1H NMR (DMSO-d6, 300 MHz) δ 1.62-
1.79 (m, 4H), 2.92 (s, 3H), 3.32 (m, 2H), 3.91 (m, 1H), 7.90 (s,
1H), 7.71 (s, 2H), 8.47 (s, 3H), 9.98 (s, 1H), 10.53 (s, 1H); 13C
NMR (DMSO-d6, 75 MHz) δ 22.2, 26.6, 35.7, 48.5, 51.5, 157.9,
NR-(tert-Butyloxycarbonyl)-Nδ-methyl-L-citrulline tert-Butyl
Ester (5). A 196 mg (0.6 mmol) sample of the protected cyanamide
3 were dissolved in 17 mL of EtOH. After adding 58 mL of a
dilute (3%) aqueous H2O2 solution and 1.15 mL of 1 M NaOH the
reaction mixture was stirred for 4 h. The title compound was
extracted twice with CH2Cl2, then the combined organic phases
were dried with Na2SO4 and evaporated under reduced pressure.
The resulting oil was subjected to flash chromatography (CH2Cl2/
MeOH, 7:1) and the purified product crystallized upon drying in
vacuo. Yield 178 mg (86%). Mp 40 °C. 1H NMR (DMSO-d6, 300
MHz) δ 1.38 (s, 9H), 1.39 (s, 9H), 1.46-1.59 (m, 4H), 2.73 (s,
3H), 3.10 (t, 2H, 3J ) 6.4 Hz), 3.76 (m, 1H), 5.71 (s, 2H), 7.11 (d,
1H, 3J ) 7.6 Hz); 13C NMR (CDCl3, 75 MHz) δ 23.4, 28.0, 28.3,
29.7, 34.5, 48.5, 53.2, 79.8, 82.1, 155.7, 158.9, 171.8.
170.6; 15N NMR (DMSO-d6, 15N-labeled compound, measuring time
1
15
1
5 h, INEPT 3 h) δ 136.6 (d, J( N- H) ) 97.5 Hz, NHOH); HRMS
m/z calcd for C7H17N4O3 (MH+) 205.12952, found 205.12956.
Preparation of Nδ-Methyl-L-ornithine (10): Summary. First,
the preparation of Nδ-(p-toluenesulfonyl)-L-ornithine 10a was
accomplished according to the literature17 but using Na2EDTA for
the removal of Cu(II) instead of H2S. Yield 71%. R20D +21.0 (2%,
6 N HCl) [lit.18,19 R23 +20.8 (2%, 6 N HCl)]. Subsequent
D
conversion to NR-benzoyl-Nδ-(p-toluenesulfonyl)- L-ornithine 10b
was performed with benzoylchloride in 1 M NaOH.16b,20 Yield 79%.
Ee 99.2% ((R,R)-Whelk O1); R20 -5.0 (2%, DMF) [lit.20 R23
D
D
-3.0 (2%, DMF)]. Methylation to NR-benzoyl-Nδ-methyl-Nδ-(p-
toluenesulfonyl)- L-ornithine 10c was realized using dimethyl sulfate
in 2 equiv of NaOH.21,22 Yield 90%. Ee 98.8% ((R,R)-Whelk O1);
R20 -4.2 (2%, DMF) [lit.20 R23 -3.0 (2%, DMF)]. Nδ-Methyl-
NR-(tert-Butyloxycarbonyl)-Nω-hydroxy-Nδ-methyl-L-argin-
ine tert-Butyl Ester (6). 3 (373 mg, 1.14 mmol) was dissolved in
4 mL of dry 1,4-dioxane and the solution was added dropwise to
a solution of 57 mg (1.7 mmol) of hydroxylamine in 2 mL of 1,4-
dioxane. The solution was then stirred for 12 h. The solvent was
evaporated under reduced pressure and the crude product was further
purified by flash chromatography (CH2Cl2/MeOH/AcOH, 8.5:1.5:
0.05). Pooled fractions were concentrated on a rotary evaporator.
To remove remaining acetic acid, toluene was added and evaporated
again. The purified compound was obtained as a yellow oil. Yield
D
D
L-ornithine was obtained by deprotection in refluxing hydrobromic
acid (47%).16b,23 In terms of racemization it is of importance to
terminate the reaction after 1.5 h and to remove HBr in a high
vacuum (<1 mbar), not above 50-60 °C. The product was
recrystallized from ethanol. Yield 84%. Mp 243 °C; Ee 98.2%
(Crownpak Cr(+)); R20 +23.2 (2%, 6N HCl) [lit.20 R23 +19.7
D
D
(2%, 6 N HCl); lit.31 R20 +25.5 (2%, 6 N HCl)].
D
9-Borabicyclo[3.3.1]non-9-yl[Nδ-methyl-L-ornithinato-O,N]-
boron Hydrochloride (12). 10 (2.0 g, 11 mmol) was added to a
stirred, hot solution of 1.61 g of crystalline, dimeric 9-borabicyclo-
[3.3.1]nonane (13.2 mmol monomer) in 24 mL of MeOH under
nitrogen atmosphere and refluxed for 1.5 h. The suspension was
filtered and the residue was washed two times with n-hexane and
1
144 mg (35%). H NMR (DMSO-d6, 300 MHz) δ 1.37 (s, 18H),
1.51-1.59 (m, 4H), 2.88 (s, 3H), 3.24 (t, 2H), 3.78 (m, 1H), 7.11
(d, 1H, 3J ) 7.5 Hz), 7.71 (s, 2H), 9.83 (s, 1H); 13C NMR (CDCl3,
75 MHz) δ 23.0, 27.9, 28.0, 28.4, 36.3, 50.4, 53.7, 80.0, 82.3, 156.2,
158.1, 172.0.
Nδ-Methyl-L-citrulline Hydrochloride (7). From 5: The pro-
tected compound 5 (311 mg, 0.9 mmol) was dissolved in 9 mL of
4 M HCl in dry 1,4-dioxane and the solution was allowed to stand
for 20 h under nitrogen atmosphere. For complete precipitation,
dry Et2O was added and the highly hygroscopic title compound
was obtained by filtration under a nitrogen stream. The product
was washed with dry EtOAc and stored at -20 °C under nitrogen
due to its high hygroscopicity. Yield 183 mg (90%). From 16, 17:
16 or 17 (0.144 mmol) was dissolved in 3 mL of acetone and 1
mL of TFA (60%) was added. The solution was stirred for 2-3 h
at room temperature and concentrated in vacuo. Completion of the
reaction was monitored via TLC (isopropanol/water/AcOH, 6:3:1,
Rf 0.68). HCl(aq) (10 mL, 1.5 M) was added and the mixture was
stirred for another 2 h at room temperature. The product was
purified by column chromatography on microgranular cellulose
(ACN/water, 5:3). After removal of the solvent the oily product
was dispersed in 1,4-dioxane and gaseous HCl was flushed over
the liquid to obtain the hydrochloride 5. Yield 122-163 mg (60-
1
Et2O. Yield 2.83 g (85%). Mp 275 °C dec. H NMR (DMSO-d6/
CDCl3 1:1, 300 MHz) δ 0.51 (s, 1H), 0.56 (s, 1H), 1.43 (m, 2H),
1.58 (m, 2H), 1.62-2.07 (m, 12H), 2.59 (s, 3H), 2.94 (m, 2H),
3.57 (m, 1H), 5.87 (br t, 1H), 6.45 (dd, 1H), 9.02 (s, 2H); 13C NMR
(DMSO-d6/CDCl3 1:1, 75 MHz) δ 22.3, 22.5, 23.6, 23.9, 24.3, 27.4,
30.97, 31.04, 31.2, 31.3, 32.6, 48.0, 53.9, 173.7; LRMS (ESI) 267
(MH+).
9-Borabicyclo[3.3.1]non-9-yl[Nω,Nω′-bis(tert-butyloxycarbo-
nyl)-Nδ-methyl-L-argininato-O,N]boron (14). Guanylation of 12
was performed according to ref 15b. Yield 302 mg (41%). Mp
110 °C (foaming; 270 °C dec). 1H NMR (DMSO-d6, 300 MHz) δ
0.48 (s, 1H), 0.52 (s, 1H), 1.27-1.50 (m, 20H), 1.50-1.90 (m,
14H), 2.90 (s, 3H), 3.33 (m, 2H), 3.53 (m, 1H), 5.81 (br dd, 1H),
6.38 (br dd, 1H), 9.52 (s, 1H); 13C NMR (DMSO-d6, 75 MHz) δ
22.2, 23.4, 23.5, 23.8, 24.2, 27.4, 27.9, 30.7, 31.1, 31.2, 35.6, 48.9,
54.2, 151.1, 152.0, 159.9, 173.3; LRMS (ESI) 457 (MH+).
9-Borabicyclo[3.3.1]non-9-yl[Nδ-cyano-Nδ-methyl-L-ornithi-
nato-O,N]boron (17). Reaction of 12 with cyanogen bromide was
performed according to ref 15b. Yield 135 mg (29%). Mp 235 °C.
1H NMR (DMSO-d6) δ 0.49 (s, 1H), 0.53 (s, 1H), 1.42 (m, 2H),
1.50-1.97 (m, 14H), 2.59 (s, 3H), 3.00 (qt, 2H), 3.54 (m, 1H),
5.84 (br t, 1H), 6.43 (br dd, 1H); 13C NMR (DMSO-d6, 75 MHz)
δ 22.2, 23.4, 23.6, 23.8, 24.1, 27.3, 30.7, 31.1, 31.2, 38.2, 51.7,
54.0, 118.5, 173.3; LRMS (ESI) 292 (MH+).
1
80%). Ee 98.1% (Crownpak Cr(+)). H NMR (DMSO-d6, 300
3
MHz) δ 1.51-1.76 (m, 4H), 2.76 (s, 3H), 3.18 (t, 2H, J ) 6.7
Hz), 3.90 (m, 1H), 5.18 (s, 2H), 8.38 (s, 3H), 13.68 (s, 1H); 13C
NMR (DMSO-d6, 75 MHz) δ 22.8, 27.0, 34.3, 47.3, 51.7, 159.0,
170.8; HRMS m/z calcd for C7H16N3O3 (MH+) 190.11862, found
190.11849.
Nω-Hydroxy-Nδ-methyl-L-arginine Dihydrochloride (8). From
6: The protected L-arginine derivative 6 (319 mg, 0.885 mmol)
was dissolved in 6.4 mL of 4 M HCl in dry 1,4-dioxane and left
for 18 h under nitrogen. Complete precipitation was achieved by
adding dry Et2O. The highly hygroscopic compound was filtered
under a nitrogen stream and washed several times with dry EtOAc.
It was best stored under nitrogen at -20 °C. Yield 147 mg (63%).
From 16, 17: Synthesis from 16 and 17 was performed according
to ref 15b with the following alterations: reaction times were 2.5
h at room temperature. After purification by column chromatog-
raphy on microgranular cellulose (ACN/0.1% TFA(aq), 5:3) the oil
was dispersed in 1,4-dioxane and gaseous HCl was flushed over
the liquid. The product crystallized after standing in the refrigerator
and was isolated after 24 h by filtration. Yield 76.4 mg (80%). Ee
Acknowledgment. We thank Dr. Ulrich Girreser for per-
forming NMR and MS experiments and his helpful discussion
of analytical issues. We also thank Mrs. Melissa Zietz and Mr.
Sven Wichmann for excellent technical assistance.
Supporting Information Available: General experimental
methods, reagents, and material; complete synthetic protocols and
spectral data for compounds 1-17; elemental analyses and HPLC
(29) Harris, J. I.; Work, T. S. Tetrahedron Lett. 2006, 47, 5159.
(30) Feldman, P. L. Tetrahedron Lett. 1991, 32, 875.
(31) Belyaev, A. A.; Krasko, E. V. Synthesis 1991, 5, 417.
J. Org. Chem, Vol. 73, No. 3, 2008 1029

Schade et al.
chromatograms for purity assessment; chiral chromatography on
This material is available free of charge via the Internet at
http://pubs.acs.org.
1
Crownpak Cr(+) and (R,R)-Whelk O1; 1D H, 13C NMR spectra;
2D NMR spectra of 12 and 14; IR spectra of boron-complexed
cyanamides 16 and 17; and 15N NMR spectrum of 15N-labeled 8.
JO702150D
1030 J. Org. Chem., Vol. 73, No. 3, 2008