Enantioselective syntheses of α-Fmoc-Pbf-[2-13C]-L- arginine and Fmoc-[1,3-13C2]-L-proline and incorporation into the neurotensin receptor 1 ligand, NT8-13

Article abstract of DOI:10.1021/jo9014497

(Chemical Equation Presented) Enantioselective syntheses of selectively labeled, orthogonally protected [2-¹³C]-L-arginine and [1,3- ¹³C₂]-L-proline are described from the commercially available precursors [2-¹³C]bromoacetic acid and potassium [ ¹³C]cyanide. Interestingly the enhanced signal assigned to C-2 in the ¹³C NMR spectrum of α-Fmoc-Pbf-[2-¹³C]-L-arginine was very broad at room temperature. The two Fmoc-labeled amino acids were used to prepare [2-¹³C]-Arg9 and [1,3-¹³C₂]-Pro10 labeled ligand (NT_8-13) by manual Fmoc-SPSS.

Full text of DOI:10.1021/jo9014497

pubs.acs.org/joc
Enantioselective Syntheses of r-Fmoc-Pbf-[2-13C]-L-arginine and
Fmoc-[1,3-13C2]-L-proline and Incorporation into the Neurotensin
Receptor 1 Ligand, NT8-13
Chuanjun Song,† Satita Tapaneeyakorn,‡ Annabel C. Murphy,† Craig Butts,†
Anthony Watts,‡ and Christine L. Willis*,†
†School of Chemistry, University of Bristol, Cantock’s Close, Bristol BS8 1TS, United Kingdom, and
‡Biomembrane Structure Unit, Department of Biochemistry, University of Oxford, Oxford OX1 3QU,
United Kingdom
chris.willis@bristol.ac.uk
Received July 7, 2009
Enantioselective syntheses of selectively labeled, orthogonally protected [2-13C]-L-arginine and
[1,3-13C2]-L-proline are described from the commercially available precursors [2-13C]bromoacetic
acid and potassium [13C]cyanide. Interestingly the enhanced signal assigned to C-2 in the 13C NMR
spectrum of R-Fmoc-Pbf-[2-13C]-L-arginine was very broad at room temperature. The two Fmoc-
labeled amino acids were used to prepare [2-13C]-Arg9 and [1,3-13C2]-Pro10 labeled ligand (NT8-13
by manual Fmoc-SPSS.
)
Introduction
NT acts as a neurotransmitter in the central nervous system
and as a local hormone at the periphery. Only the six
C-terminal amino acids of NT (NT8-13) are required for
ligand binding.3 The membrane receptor, NTS1, belongs to
the family of GPCRs and is a potential target for the
treatment of pain, stress, schizophrenia, Parkinson’s disease,
Alzheimer’s disease, and colon cancer.4 Solving the confor-
mation of such a ligand when bound to its target can be very
useful for the design of new synthetic agonists, antagonists,
and reverse agonists of putative therapeutic use. Further-
more, NMR gives electronic and conformational details of
value in drug design.1,5 To gain further structural informa-
tion, we required site-specific carbon-13 labeling of the
peptide NT8-13 at Arg9 and Pro10 in the [2-13C] and
[1,3-13C2] sites, respectively. For the peptide synthesis a
supply of the analogous protected amino acids was needed.
The enantioselective syntheses of orthogonally protected
[2-13C]-L-arginine and [1,3-13C2]-L-proline are now described
Isotopically labeled amino acids are important for a range
of studies at the chemistry-biology interface. A key applica-
tion is incorporation into ligands, peptides, and proteins for
determining their 3D structures by NMR spectroscopy.1,2
However, often the required selectively labeled amino acid
which would lead to the most structural information is not
commercially available. In contrast, use of uniformly labeled
amino acids which can be purchased may result in spectra
that are too complex to be assigned with confidence due to
unwanted dipolar couplings from neighboring spin sites. A
particular challenge is to resolve the conformation of ligands
on their sites of action in large (Mr . 30K) targets, such as
membrane receptors, including G protein-coupled receptors
(GPCRs). Solid state NMR (SSNMR) can address this
challenge with the prerequisite that site-specific labeling is
required. Thus the development of efficient methods for the
selective labeling of amino acids is an important goal.
A typical example is neurotensin (NT), a tridecapeptide
(Glu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg8-Arg9-Pro10-Tyr-Ile-
Leu13) that is the ligand of neurotensin receptor 1 (NTS1).
(3) Kitabgi, P.; Carraway, R.; Van Rietschoten, J.; Granier, C.; Morgat,
J. L.; Menez, A.; Leeman, S.; Freychet, P. Proc. Natl. Acad. Sci. U.S.A. 1977,
€
74, 1846–1850. Harterich, S.; Koschatzky, S.; Einsiedel, J.; Gmeiner, P.
Bioorg. Med. Chem. 2008, 16, 9359–9368.
(4) Kitabgi, P. Peptides 2006, 27, 2461–2468. Myers, R. M.; Shearman,
J. W.; Kitching, M. O.; Ramos-Montoya, A.; Neal, D. E.; Ley, S. V. ACS
Chem. Biol. 2009, 503–525.
(1) Watts, A. Mol. Membr. Biol. 2002, 19, 267–275.
(2) Fielding, L. Curr. Top. Med. Chem. 2003, 3, 39–53.
8980 J. Org. Chem. 2009, 74, 8980–8987
Published on Web 11/02/2009
DOI: 10.1021/jo9014497
r
2009 American Chemical Society

Song et al.
JOCArticle
SCHEME 1. Preparation of R-Fmoc-Pbf-[2-13C]-L-argininea
aReagents and conditions: (a) BnOH, DCC, DMAP, DCM, rt, 4 h, 100%; (b) (1R,2S)-2-amino-1,2-diphenylethanol, Et3N, THF, rt, 2.5 h; (c) Cbz-Cl,
NaHCO3, H2O/DCM, 0 °C, 2.5 h; (d) p-TsOH.H2O, toluene, Dean-Stark, reflux, 60% over the 3 steps; (e) 3-(tert-butyldimethylsilyloxy)-
1-iodopropane, LiHMDS, HMPA, THF, -78 °C to rt, 6 h, 63%; (f) KHSO4, THF/MeOH/H2O, rt, 3 h, 78%; (g) 12, DIAD, PPh3, 80 °C, 16 h;
(h) H2, Pd/C, MeOH/THF, 2 days, 18% over the 2 steps; (i) Fmoc-ONSu, Na2CO3, dioxane/H2O, 0 °C to rt, 2.5 h, then citric acid, 75%.
as well as their incorporation into [2-13C]-Arg9- and
routes are not readily adapted for the incorporation of
carbon-13 at C-2.10 Thus we designed an enantioselective
approach to Fmoc-Pbf-[2-13C]-L-arginine 1 from commer-
cially available [2-13C]bromoacetic acid 2 using Williams’
oxazinone as a chiral glycinate equivalent11 to generate the
required stereogenic center.
[1,3-13C2]-Pro10-labeled NT8-13
.
Results and Discussion
Synthesis of r-Fmoc-Pbf-[2-13C]-L-arginine. The first syn-
thetic target was orthogonally protected [2-13C]-L-arginine.6
Commonly used protecting groups for the highly basic
guanidine function of arginine are arylsulfonyl derivatives,
including PMC (2,2,5,7,8-pentamethylchroman-6-sulfonyl)7
and analogous Pbf (2,2,4,6,7-pentamethyldihydrobenzo-5-
sulfonyl) groups8 which are removed with TFA. Literature
precedents for the synthesis of arginine are rare6,9 and to our
knowledge there are no reports of the preparation of
L-arginine selectively labeled at C-2. Valuable approaches
to unlabeled arginine include the use of aspartic acid and
4-guanidinobutanoic acid as starting materials, but these
First [13C]oxazinone 4 was prepared in 3 steps and 60%
overall yield starting from [2-13C]bromoacetic acid 2
(Scheme 1).11a With use of unlabeled oxazinone, it has been
shown that alkylations may be achieved with a range of
electrophiles to generate anti-R-monosubstituted oxazines in
good yields and excellent stereocontrol (>95% de).11 Thus
we proposed that a direct approach for the synthesis of the
protected arginine 7 (Scheme 1) would be via alkylation of 4
with iodide 9. However, it was found that on treatment of
oxazine 4 with iodide 9 under a variety of conditions
(including with either NaHMDS or LiHMDS in THF/
HMPA commonly used for unactivated alkyl halides11) the
results were disappointing as either no reaction or decom-
position occurred. An alternative strategy to 7 was to
alkylate the oxazine with an electrophile that could be readily
manipulated to give a good leaving group at C-30 for
displacement by a protected guanidine. To this end, pro-
tected guanidine 12 was prepared as shown in Scheme 2.
Initial CBz protection of guanidine hydrochloride 1012 was
(5) Watts, A.; Straus, S. K.; Grange, S.; Kamihira, M.; Lam, Y. H.; Xhao,
Z. In Methods in Molecular Biology-Protein NMR Techniques , Downing,
K., Ed.; Humana Press: Totowa, NJ, 2004; Vol. 278, pp 403-474.
(6) For reviews of the syntheses of amino acids incorporating stable
isotopes, see: (a) Kelly, N. M.; Sutherland, A.; Willis, C. L. Nat. Prod.
Rep. 1997, 14, 205–219. (b) Voges, R. J. Label. Compd. Radiopharm. 2002, 45,
867–897.
(7) (a) Ramage, R.; Green, J. Tetrahedron Lett. 1987, 28, 2287–2290. (b)
Green, J.; Ogunjobi, O. M.; Ramage, R.; Stewart, A. S. J. Tetrahedron Lett.
1988, 29, 4341–4344.
(8) Carpino, L. A.; Shroff, H.; Triolo, S. A.; Mansour, E.-S. M. E.;
Wenschuh, H.; Albericio, F. Tetrahedron Lett. 1993, 34, 7829–7832.
(9) (a) Prabhakaran, P. C.; Woo, N.-T.; Yorgey, P. S.; Gould, S. J.
Tetrahedron Lett. 1986, 27, 3815–3818. (b) Prabhakaran, P. C.; Woo, N.-T.;
Yorgey, P. S.; Gould, S. J. J. Am. Chem. Soc. 1988, 110, 5785–5791. (c)
Martinkus, K. J.; Tann, C.-H.; Gould, S. J. Tetrahedron 1983, 39, 3493–3505.
(10) (a) Hamilton, D. J.; Sutherland, A. Tetrahedron Lett. 2004, 45, 5739–
5741. (b) Bischoff, R.; Hamilton, D. J.; Jobson, N. K.; Sutherland, A.
J. Label. Compd. Radiopharm. 2007, 50, 322–326. (c) McConnell, R. M.;
Patterson-Goss, C.; Godwin, W.; Stanley, B. J. Org. Chem. 1998, 63, 5648–
5655.
(11) (a) Dastlik, K. A.; Sundermeier, U.; Johns, D. M.; Chen, Y.; Williams, R.
M.; Synlett 2005, 693-696 and references cited therein. (b) Williams, R. M.; Im,
M.-N. J. Am. Chem. Soc. 1991, 113, 9276–9286. (c) Williams, R. M.; Yuan, C.
J. Org. Chem. 1992, 57, 6519–6527. (d) Aoyagi, Y.; Iijima, A. J. Org. Chem.
2001, 66, 8010–8014. (e) Vincent, G.; Williams, R. M. Angew. Chem., Int. Ed.
2007, 46, 1517–1520. (f) For a review, see: Williams, R. M. Aldrichim. Acta
1992, 25, 11–25.
(12) Feichtinger, K.; Sings, H. L.; Baker, T. J.; Matthews, K.; Goodman,
M. J. Org. Chem. 1998, 63, 8432–8439.
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followed by treatment of the diCBz derivative 11 with PbfCl
to give 12.
Following investigations, a successful approach to our
target was developed with 3-(tert-butyldimethylsilyloxy)-
Martin and Liskamp have reported the synthesis of a series
of Fmoc NG-substituted Pbf L-arginine analogues.16 Inter-
estingly the 13C NMR spectra of these acids tended to show a
broadened signal assigned to C-2.
Synthesis of Fmoc-[1,3-13C2]-L-proline. With orthogonally
protected [2-13C]-L-arginine 1 in hand, we next turned our
attention to the synthesis of Fmoc-[1,3-13C2]-L-proline 22 for
which we again used a Williams’ oxazinone as a chiral
glycinate equivalent (Scheme 4). In this case the electrophile
was [1-13C]-3-(tert-butyldimethylsilyloxy)-1-iodopropane
15, which was prepared as shown in Scheme 3. Reaction of
chloroethanol with potassium [13C]cyanide gave the
known17 [13C]hydroxynitrile 13 which, following protection
as the silyl ether, was reduced to give [1-13C]-3-(tert-butyldi-
methylsilyloxy)propan-1-ol 14. Finally the alcohol was con-
verted to iodide 15 with I2, PPh3, and imidazole.
SCHEME 2. Preparation of Protected Guanidine 12a
aReagents and conditions: (i) BnOCOCl, NaOH, DCM/H2O, 0 °C, 19 h,
94%; (b) LiHMDS, THF, 0 °C, 2 h then Pbf-Cl, -78 °C to rt, 20 h, 42%.
1-iodopropane as the electrophile in the key alkylation
step as shown in Scheme 1. First the alkylation condi-
tions were optimized by using unlabeled diphenyloxazinone
4. LiHMDS was added dropwise to a solution of 4
and 3-(tert-butyldimethylsilyloxy)-1-iodopropane in THF/
HMPA (10:1 mixture) at -78 °C giving the novel unlabeled
silyl ether 5 as the sole product. The reaction was repeated
with [13C]-oxazinone 4 giving 5 in 63% yield. Deprotection
of the silyl group of 5 with potassium bisulfate13 gave alcohol
6, the substrate required for introduction of the guanidine
group.
Various approaches were investigated to effect an SN2
displacement of a derivative of alcohol 6 with a good leaving
group (including a tosylate and iodide) at C-30 with protected
guanidine 12. The most reliable method proved to be a
Mitsunobu reaction of 12 with alcohol 6 to generate the
protected arginine 7 (Scheme 1). The crude product was
subjected to a palladium-mediated hydrogenation to give
Pbf-arginine 8. Marfey’s analysis14 of R-amino acid 8
confirmed that the L-enantiomer had been prepared with
excellent stereocontrol.
Boc-protected oxazinone 16, prepared from [1-13C]-
SCHEME 3. Preparation of [1-13C]-3-(tert-Butyldimethylsilyl-
oxy)-1-iodopropanea
aReagents and conditions:(a) K13CN, NaI, H2O/EtOH, 80 °C, 22 h, 90%;
(b) TBSCl, DMAP, Et3N, DCM, rt, 6 h; (c) DIBAL-H, THF, rt, 3 h, then
Rochelle salt, 36% over the 2 steps; (d) NaBH4, EtOH, 0 °C, 0.5 h, then rt,
1 h, 89%; (e) I2, PPh3, imidazole, DCM, 0 °C, 2 h, then rt, 1 h, 76%.
SCHEME 4. Preparation of Fmoc-[1,3-13C2]-L-prolinea
For the peptide synthesis it was necessary to protect the
R-amino group of 8 as the Fmoc carbamate, which pro-
ceeded smoothly15 to furnish the target, protected [2-13C]-
L-arginine 1 in 75% yield. On analysis of the 13C NMR
spectrum of 1 in CDCl3 at room temperature we were
surprised that no significantly enhanced signal due to C-2
was apparent although there was a broad signal at the
expected chemical shift δ 54 ppm. Hence the 1H NMR and
13C NMR spectra of 1 were compared with the analogous
commercial unlabeled R-Fmoc-Pbf-L-arginine. The broad
resonance at δ 54 was confidently assigned to C-2 by analysis
of multiplicity-edited HSQC, DEPT, and COSY spectra. In
the unlabeled material, this crucial resonance (δ 54) could
only be observed in the 13C NMR spectrum following
extended acquisition and with application of a strong apo-
dization function. However, warming the NMR sample to
40 °C caused a sharpening of all of the broad resonances in
the carbon spectrum, confirming a dynamic origin to the
behavior. The 13C NMR spectrum of protected [2-13C]-
L-arginine 1 was run in DMSO at 25 °C and while the
enhanced signal at δ54 was broad, on warming to 80 °C a
significant sharpening of the signal was apparent. Recently
aReagents and conditions: (a) BnOH, DCC, DMAP, DCM, 0 °C, 3 h,
then rt, 1 h, 89%; (b) (1R,2S)-2-amino-1,2-diphenylethanol, Et3N, THF,
rt, 2 h; (c) Boc2O, toluene, reflux, 18.5 h; (d) p-TsOH H2O, toluene,
Dean-Stark, reflux 1 h, 59% over the 3 steps; (e) 15, LiHMDS, HMPA,
THF, -78 °C to rt, 3.5 h, 48%; (f) TBAF, THF, rt, 16.5 h, 57%; (g) TsCl,
DMAP, DCM, rt, 41 h, 67%; (h) TFA, DCM, rt, 2 h, then NaHCO3
(aq), 63%; (i) H2, Pd/C, THF/EtOH, 21.5 h, 78%; (j) Fmoc-Cl, Na2CO3,
H2O/dioxane, 0 °C to rt, 17 h, 85%.
3
bromoacetic acid, was alkylated with iodide 15, using
LiHMDS as the base in a solvent mixture of THF/HMPA,
giving 17 in 48% yield and excellent stereocontrol
(13) Arumugam, P.; Karthikeyan, G.; Perumal, P. T. Chem. Lett. 2004,
33, 1146–1147.
(14) (a) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591–596. (b)
Bushan, R.; Bruckner, H. Amino Acids 2004, 27, 231–247.
(15) Adamson, J. G.; Blaskovich, M. A.; Groenevelt, H.; Lajoie, G. A.
J. Org. Chem. 1991, 56, 3447–3449.
(16) Martin, N. I.; Liskamp, R. M. J. J. Org. Chem. 2008, 73, 7849–7851.
(17) (a) Baxter, R. L.; Hanley, A. B.; Chan, H. W.-S.; Greenwood, S. L.;
Abbot, E. M.; McFalane, I.; Milne, K. J. Chem. Soc., Perkin Trans. 1 1992,
2495–2502. (b) Peng, S.; McGinley, C. M.; van der Donk, W. A. Org. Lett.
2004, 6, 249–352.
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FIGURE 1. (a) HPLC and ESI mass spectrum (insert) of purified [2-13C]-Arg9- and [1,3-13C2]-Pro10-labeled NT8-13. Mass observed: 820.5
Da (M þ H) and 410.8 Da (double positive charge). (b) 13C NMR spectrum of labeled ligand: δC1-Pro10 175.5, 176.2 ppm; δC2-Arg9 54.5
ppm; δC3-Pro10 32.1, 34.4 ppm.
(Scheme 4). Deprotection of silyl ether 17 with TBAF gave
the novel alcohol 18 labeled at the requisite sites for the
synthesis of the target Fmoc-[1,3-13C2]-L-proline 22
(Scheme 4). Activation of the alcohol as the tosylate 19, then
removal of the Boc protecting group with TFA and a base
workup furnished the heterocyclic product 20 in 63% yield.
It was apparent from the NMR data that a single diaster-
eomer was present with enhanced signals in the 13C NMR
spectrum at δC 172.6 (C-2) and 29.7 (C-9). The optical
rotation, [R]D 135.6 (c 2.0, CH2Cl2), of 20 was in accord with
the literature value18 for the unlabeled opposite enan-
tiomer, [R]D -130.4 (c 2.6, CH2Cl2). Hydrogenolysis of 20
to [1,3-13C2]-L-proline 21, followed by Fmoc protection gave
Fmoc-[1,3-13C2]-L-proline 22 in good yield, ready for synth-
esis of the peptide. The 13C NMR spectrum (CDCl3) of 22
showed the expected enhanced signals with no apparent
broadening. The melting point (115-116 °C) and optical
rotation ([R]D -35.8, c 1.0, DMF) were in accord with
literature values for unlabeled Fmoc L-proline, mp
114-115 °C, [R]D -33.4 (c 1.0, DMF).19
R-Fmoc-Pbf-[2-13C]-L-arginine 1 and Fmoc-[1,3-13C2]-L-
proline 22 were required to assemble the peptide and their
enantioselective syntheses are described from selectively
labeled oxazonines 4 and 16. Readily available [2-13C]-
bromoacetic acid and potassium [13C]cyanide were used as
the sources of isotopic labels. The signal assigned to C-2 in
the 13C NMR spectrum of R-Fmoc-Pbf-[2-13C]-L-arginine
was broad. The two protected labeled amino acids were used
in the synthesis of [2-13C]-Arg9- and [1,3-13C2]-Pro10-la-
beled ligand (NT8-13) by manual Fmoc-SPSS giving the
required product in 27% yield. The synthetic strategies
described herein may be readily adapted for the selective
labeling of further sites in both protected arginine and pro-
line and hence have widespread application both in biologi-
cal NMR and metabolic studies.
Experimental Section
General experimental details are as previously described.20
[3-13C](5S,6R)-4-Benzyloxycarbonyl-5,6-diphenylmorpholin-
2-one, 4. Benzyl [2-13C]bromoacetate 3 (6.54 g, 28.4 mmol) in
THF (10 mL) was added dropwise to a solution of (1R,2S)-2-
amino-1,2-diphenylethanol (6.37 g, 29.9 mmol) and triethyla-
mine (3.45 g, 4.8 mL, 34.1 mmol) in dry THF (100 mL) over 1 h.
After addition, the mixture was stirred at ambient temperature
for 3 h. A precipitate formed, which was filtered, then the filter
cake was rinsed with THF (30 mL). The filtrate was concen-
trated. The solid residue was dissolved in DCM (50 mL) and
washed with water (50 mL). The separated aqueous phase was
extracted with DCM (2 ꢀ 50 mL). Saturated aqueous sodium
hydrogen carbonate (100 mL) was added to the combined
organic extracts. The resulting mixture was cooled in ice. Benzyl
chloroformate (5.33 g, 4.5 mL, 31.3 mmol) was added dropwise
over 20 min to this vigorously stirred mixture. The reaction was
stirred at 0 °C for an additional 2 h, then the layers were
separated. The aqueous layer was extracted with DCM (2 ꢀ
50 mL). The combined organic extracts were washed with brine
(50 mL), then dried (MgSO4), filtered, and evaporated. The
residue was dissolved in toluene (200 mL). p-Toluenesulfonic
acid monohydrate (0.54 g, 2.8 mmol) was added. The resulting
mixture was heated to reflux with a Dean-Stark apparatus until
∼150 mL of toluene was separated. The reaction was then
cooled to ambient temperature. The resulting solid was collected
by filtration and rinsed with toluene-hexane (1:3, 40 mL), then
hexane (20 mL). The mother liquor and washes were combined
and evaporated. The residue was recrystallized from toluene to
Synthesis of the Labeled Neurotensin Receptor 1 Ligand,
NT8-13. The two protected labeled amino acids 1 and 22 were
used to synthesize [2-13C]-Arg9- and [1,3-13C2]-Pro10-la-
beled ligand NT8-13 by manual Fmoc-SPSS. Synthesis was
performed on a 0.1 mmol scale with a Fmoc-Leu-Novasyn
TGA resin with no excess of amino acids and the activating
mixture was HBTU/HOBt/DIEA (1:1:1.5). The Fmoc group
was deprotected with 20% piperidine and the peptide was
cleaved from the resin with trifluoroacetic acid (TFA). As
shown in Figure 1a, purification by preparative RP-HPLC
purification gave the peptide in a purity of >95% and 27%
1
yield. The 13C- NMR spectrum (Figure 1b), MS, and H
NMR spectrum of labeled ligand were used to authenticate
product purity and labeled positions. Detailed NMR studies
with the labeled peptide are in hand and will be reported in
due course.
Conclusions
[2-13C]-Arg9- and [1,3-13C2]-Pro10-labeled NT8-13 have
been designed as the target to facilitate the determination
of the conformation of the ligand bound to the purified
neurotensin receptor 1 by solid state NMR methods.
(18) Williams, R. M.; Im, M.-N. J. Am. Chem. Soc. 1991, 113, 9276–9286.
(19) Chang, C. D.; Waki, M.; Ahmad, M.; Meienhofer, J.; Lundell, E. O.;
Haug, J. D. Int. J. Peptide. Protein Res. 1980, 15, 59–66.
(20) Seden, P. T.; Charmant, J. P. H.; Willis, C. L. Org. Lett. 2008, 10,
1637–1640.
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provide a second crop of product. The combined solid was
dissolved in DCM (100 mL) and washed with saturated aqueous
sodium bicarbonate (50 mL). The separated aqueous phase was
extracted with DCM (50 mL). The combined organic extracts
were dried (MgSO4), filtered, and evaporated to give [3-13C]-
morpholine carbamate 4 (6.62 g, 60%) as a colorless solid, which
showed a mixture of two rotamers; [R]24D -63.0 (c 1.0, DCM)
{lit.11a [R]D22 -68.7} for the unlabeled (c 1.0, DCM); mp 204 °C
(lit.11a mp 206-207 °C for the unlabeled); νmax/cm-1 1749 (CO)
and 1695 (CO); δH (400 MHz, CDCl3) 7.39-6.65 (15H, m,
Ar-H), 5.88 (1H, m, 6-H), and 5.41-4.18 (5H, m, 5-H, 3-H2
and CH2Ph); δC (100 MHz, CDCl3) 45.3 (C-3, enhanced signal);
m/z (ESI) 411 (Mþ þ Na, 100%) [found (Mþ þ Na) 411.1399,
C2313CH21NNaO4 requires 411.1396].
silyl ether 5 (45 mg, 0.08 mmol) in methanol (10 mL) and water
(2 mL). The resulting mixture was stirred at ambient tempera-
ture for 5 h. The methanol was evaporated and the residue was
partitioned between ethyl acetate (20 mL) and water (20 mL).
The separated aqueous phase was extracted with ethyl acetate
(20 mL). The combined organic extracts were dried (MgSO4),
filtered, and evaporated in vacuo. The residue was purified by
column chromatography (50% ethyl acetate in petroleum ether)
to give unlabeled carbamate 6 (31 mg, 87%) as a colorless solid,
which showed a mixture of two rotamers; [R]24 -40.0 (c 5.3,
D
DCM); mp 160 °C; νmax/cm-1 3389 (OH), 1749 (CO), and 1699
(CO); δH (400 MHz, CDCl3) 7.38-6.53 (15H, m, Ar-H), 5.97
(1H, d, J=2.8 Hz, 6-H), [5.24 (0.3H, d, J=2.8 Hz) and 5.14
(0.7H, d, J = 2.8 Hz), 5-H], [5.21 (0.3H, d, J = 12.3 Hz),
5.04-5.01 (1.7H, m), 4.92 (0.3H, dd, J=10.5 and 4.2 Hz) and
4.87 (0.7H, d, J=12.3 Hz), 3-H and CH2Ph], 3.85-3.60 (2H, m,
30-H2), 2.88 (1H, s, OH), and 2.39-1.69 (4H, m, 10-H2 and 20-
H2); δC (100 MHz, CDCl3) 168.8 and 168.7 (C-2), 154.3 and
153.7 (CO), 135.6, 135.4, 135.3, 134.8, 133.9 (all C), 128.5, 128.4,
128.3, 128.1, 128.0, 127.8, 127.7, 127.4, 127.3, 127.2, 126.3, 126.2
(all CH), 79.1 and 78.9 (C-6), 67.9 and 67.6 (CH2Ph), 61.5 and
61.0 (C-30), 60.8 and 60.6 (C-5), 57.0 and 56.2 (C-3), þ31.9 and
31.0 (C-10), 28.7 and 28.4 (C-20); m/z (ESI) 468 (M þ Na,
100%) [found (Mþ þ Na) 468.1779, C27H27NNaO5 requires
468.1781].
[3-13C]-(3S,5S,6R)-4-Benzyloxycarbonyl-3-[30-(tert-butyldime-
thylsilyloxy)prop-10-yl]-5,6-diphenylmorpholin-2-one, 5. First the
reaction was optimized by using unlabeled material. Lithium
bis(trimethylsilyl)amide (1.0 M solution in THF, 7.7 mL,
7.7 mmol) was added dropwise to a solution of unlabeled mor-
pholine carbamate 4 (2.0 g, 5.2 mmol) and 3-(tert-butyldime-
thylsilyloxy)-1-iodopropane (2.3 g, 7.7 mmol) in HMPA (6 mL)
and dry THF (60 mL) at -78 °C under nitrogen. The resulting
mixture was stirred at -78 °C for 0.5 h, before being allowed to
warm to ambient temperature and left for 5 h. Ethyl acetate (50
mL) was added, and the mixture was washed with saturated
aqueous ammonium chloride (50 mL). The separated aqueous
phase was extracted with ethyl acetate (2 ꢀ 50 mL). The
combined organic extracts were dried (MgSO4), filtered, and
evaporated in vacuo. The residue was purified by column
chromatography (10%, then 20% ethyl acetate in petroleum
ether) to give unlabeled carbamate 5 (1.7 g, 58%) as a colorless
solid, which showed a mixture of two rotamers; [R]24D -26.5 (c
12.3, DCM); mp 128-130 °C; νmax/cm-1 1751 (CO) and 1698
(CO); δH (400 MHz, CDCl3) 7.38-6.55 (15H, m, Ar-H), 5.97
(1H, d, J=2.9 Hz, 6-H), [5.27 (0.36H, d, J =2.9 Hz) and 5.15
(0.64H, d, J=2.9 Hz), 5-H], [5.19 (0.36H, d, J=12.3 Hz) and 5.01
(0.64H, d, J=12.3 Hz), HHPh], [5.11 (0.36H, d, J=12.3 Hz) and
4.89 (0.64H, d, J=12.3 Hz), HHPh], [5.05 (0.64H, dd, J=10.0
and 5.3 Hz) and 4.92 (0.36H, dd, J=10.0 and 5.3 Hz), 3-H],
3.78-3.62 (2H, m, 30-H2), 2.35-1.69 (4H, m, 10-H2 and 20-H2),
0.91 and 0.90 (9H, s, 3 ꢀ CH3), 0.09, 0.08, and 0.05 (6H, s, 2 ꢀ
CH3); δC (100 MHz, CDCl3) 168.9 and 168.7 (C-2), 154.3 and
153.9 (CO), 135.7, 135.6, 134.1 (all C), 128.6, 128.4, 128.2, 128.1,
127.9, 127.8, 127.5, 127.4, 126.5, 126.4 (all CH), 79.2 and 78.8
(C-6), 68.0 and 67.6 (CH2Ph), 62.3 and 62.2 (C-30), 61.1 and 60.8
(C-5), 57.5 and 57.2 (C-3), 32.2 and 31.4 (C-10), 29.2 and 29.1 (C-
20), 26.0 and 25.9 (3 ꢀ CH3), 18.3 (C), and -5.3 (2 ꢀ CH3); m/z
(ESI) 582 (Mþ þ Na, 100%) and 560 (Mþ þ H, 15) [found (Mþ
þ Na) 582.2654, C33H41NNaO5Si requires 582.2646].
The above reaction was repeated with [13C]silyl ether 5 (5.36 g,
9.6 mmol) to give [13C]carbamate 6 (3.33 g, 78%) as a colorless
solid, which showed a mixture of two rotamers; δH (400 MHz,
CDCl3) 7.38-6.54 (15H, m, Ar-H), 5.96 (1H, m, 6-H),
5.27-4.73 (4H, m, 5-H, 3-H, and CH2Ph), 3.93-3.63 (2H, m,
30-H2), and 2.39-1.75 (4H, m, 10-H2 and 20-H2); δC (100 MHz,
CDCl3) 57.1 and 56.0 (C-3, enhanced signal); m/z (ESI) 469 (Mþ
þ Na, 100%) [found (Mþ þ Na) 469.1815, C2613CH27NNaO5
requires 469.1815].
N,N0-Dibenzyloxycarbonyl-N00-(2,2,4,6,7-pentamethyldihydro-
benzofuran-5-sulfonyl)guanidine, 12. Lithium bis(trimethylsilyl)-
amide (1.0 M solution in THF, 18.3 mL, 18.3 mmol) was added
to an ice-salt bath cooled solution of dibenzyloxycarbonylgua-
nidine 11 (3.0 g, 9.2 mmol) in dry THF (70 mL). The resulting
mixture was stirred for 2 h, then cooled to -78 °C. 2,2,4,6,7-
Pentamethyldihydrobenzofuran-5-sulfonyl chloride (2.7 g, 9.2
mmol) was added. The resulting mixture was allowed to warm to
ambient temperature and left for 20 h. Water (100 mL) was
added, and the bulk of THF was evaporated. The residue was
extracted with DCM (3 ꢀ 100 mL). The combined organic
extracts were dried (MgSO4), filtered, and evaporated in vacuo.
The residue was purified by column chromatography (DCM) to
give the protected guanidine 12 (2.2 g, 42%) as a colorless solid;
mp 138-140 °C; νmax/cm-1 3237 (NH), 3171 (NH), 1784 (CO),
and 1734 (CO); δH (400 MHz, CDCl3) 10.37 (2H, s, 2 ꢀ NH),
7.33 (10H, s, Ar-H), 5.16 (4H, s, 2 ꢀ CH2Ph), 2.93 (2H, s, 3-H2),
2.61 (3H, s, CH3), 2.53 (3H, s, CH3), 2.09 (3H, s, CH3), and 1.45
(6H, s, 2 ꢀ CH3); δC (100 MHz, CDCl3) 159.7 (CO), 144.6,
139.7, 134.3, 133.4, 129.9 (all C), 128.7, 128.5, 128.4 (all CH),
124.8, 117.7 (both C), 86.6 (C-2), 68.5 (CH2Ph), 42.9 (C-3), 28.4
(2 ꢀ CH3), 19.0, 17.6, and 12.3 (all CH3); m/z (ESI) 602 (Mþ þ
Na, 100%) and 580 (Mþ þ H, 40) [found (Mþ þ Na) 602.1952,
C30H33N3NaO7S requires 602.1931].
The above reaction was repeated with [13C]morpholine car-
bamate 4 (5.91 g, 15.2 mmol) giving [13C]carbamate 5 (5.37 g,
63%) as a colorless solid, which showed a mixture of two
rotamers; δH (400 MHz, CDCl3) 7.40-6.56 (15H, m, Ar-H),
5.99 (1H, d, J=2.9 Hz, 6-H), [5.28 (0.35H, d, J=2.9 Hz) and 5.17
(0.65H, d, J=2.9 Hz), 5-H], [5.21 (0.35H, d, J=12.2 Hz) and 5.02
(0.65H, d, J=12.2 Hz), HHPh], [5.13 (0.35H, d, J=12.2 Hz) and
4.92 (0.65H, d, J=12.2 Hz), HHPh], [5.06 (0.65H, ddd, J=148.8,
9.6, and 5.5 Hz) and 4.93 (0.35H, ddd, J=148.8, 9.6, and 5.5 Hz),
3-H], 3.78-3.62 (2H, m, 30-H2), 2.37-1.70 (4H, m, 10-H2 and 20-
H2), 0.93 and 0.91 (9H, s, 3 ꢀ CH3), 0.10, 0.09, and 0.07 (6H, s,
2 ꢀ CH3); δC (100 MHz, CDCl3) 57.4 and 57.2 (C-3, enhanced
signal); m/z (ESI) 583 (Mþ þ Na, 100%), 561 (Mþ þ H, 10), and
455 (40) [found (Mþ þ Na) 583.2702, C3213CH41NNaO5Si
requires 583.2680].
[3-13C]-(3S,5S,6R)-4-Benzyloxycarbonyl-3-{30-[N,N0-dibenzylox-
ycarbonyl-N00-(200,200,400, 600,700-pentamethyldihydrobenzofuran-
500-sulfonyl)guanidino]prop-10-yl}-5,6-diphenylmorpholin-2-one,7.
First the reaction was investigated with use of unlabeled ma-
terial. Diisopropyl azodicarboxylate (81 mg, 0.08 mL, 0.40
mmol) was added dropwise to a solution of the unlabeled
alcohol 6 (100 mg, 0.22 mmol), protected guanidine 12 (261
mg, 0.45 mmol), and triphenylphosphine (105 mg, 0.40 mmol) in
dry THF (20 mL) at 0 °C under nitrogen. After addition, the
resulting mixture was heated to 80 °C for 29 h and cooled. Water
[3-13C]-(3S,5S,6R)-4-Benzyloxycarbonyl-5,6-diphenyl-3-(30-
hydroxyprop-10-yl)morpholin-2-one, 6. First the reaction was
optimized with use of unlabeled material. Potassium hydrogen-
sulfate (11 mg, 0.08 mmol) was added to a mixture of unlabeled
8984 J. Org. Chem. Vol. 74, No. 23, 2009

Song et al.
JOCArticle
(20 mL) was added and the separated aqueous phase was
extracted with ethyl acetate (2 ꢀ 50 mL). The combined organic
extracts were dried (MgSO4), filtered, and evaporated in vacuo.
The residue was purified by column chromatography (20%,
then 30% ethyl acetate in petroleum ether) to give unlabeled
masked arginine 7 (80 mg, 36%) as a sticky oil, which showed a
mixture of rotamers; [R]D23 -4.0 (c 1.5, DCM); νmax/cm-1 1759
(CO) and 1707 (CO); δH (400 MHz, CDCl3) 7.37-6.51 (25H, m,
Ar-H), 5.99 (1H, m, 6-H), 5.21-4.73 (8H, m, 3-H, 5-H, and 3 ꢀ
CH2Ph), 3.98-3.73 (2H, m, 30-H2), 2.97-2.86 (2H, m, 300-H2),
2.53 and 2.49 (3H, s, CH3), 2.46 and 2.43 (3H, s, CH3),
2.31-1.74 (4H, m, 10-H2 and 20-H2), 2.07 and 2.06 (3H, s,
CH3), 1.46 and 1.45 (6H, s, 2 ꢀ CH3); δC (100 MHz, CDCl3)
168.4 and 168.3, 160.1, 154.1 and 153.7, 150.1 and 149.7, 139.6
and 139.5, 135.8, 135.6, 135.0, 134.7 and 134.6, 134.4, 134.3,
133.4, 129.0 (all C), 128.6, 128.5, 128.4, 128.2, 128.0, 127.8,
127.7, 127.6, 127.5, 127.3, 126.6, 126.5 (all CH), 125.3 and 125.2
(C), 118.1 (C), 87.0 (C-200), 78.9 and 78.6 (C-6), 69.1 and 69.0
(CH2Ph), 68.4 and 68.3 (CH2Ph), 67.8 and 67.4 (CH2Ph), 61.0
and 60.7 (C-5), 57.1 and 56.9 (C-3), 48.4 and 48.3 (C-30), 42.9 (C-
300), 32.4 and 31.3 (C-10), 28.5 (2 ꢀ CH3), 25.3 and 25.1 (C-20),
19.1, 17.9, and 12.4 (all CH3); m/z (ESI) 1029 (Mþ þ Na, 100%)
and 1007 (Mþ þ H, 40) [found (Mþ þ Na) 1029.3743,
C57H58N4NaO11S requires 1029.3715].
colorless solid; [R]24D -2.4 (c 0.7, DCM) and [R]D -6.0 (c 1.0,
DMF), [R]D -5.5 (c 1.0, DMF) for an unlabeled commercial
sample, mp 134-136 °C (lit.21 mp 98-101 °C for the unlabeled
and unlabeled commercial sample mp 132 °C); νmax/cm-1
3332 (NH), 1703 (CO), 1616 and 1548; δH (400 MHz, CDCl3)
7.72-7.19 (8H, m, Ar-H), 6.56-6.33 (3H, m), 4.55-4.12
(4H, m, CH2O, 2-H and 90-H), 3.25 (2H, m, 5-H2), 2.88
(2H, s, 300-H2), 2.56 (3H, s, CH3), 2.49 (3H, s, CH3), 2.06
(3H, s, CH3), 1.99-1.68 (4H, m, 3-H2 and 4-H2) and 1.42 (6H,
s, 2 ꢀ CH3); δC (100 MHz, CDCl3) 143.6, 141.2, 127.6, 127.0,
125.2, 119.9, 67.1 (CH2O), 54.3 and 53.8 (C-2, enhanced signal),
47.0 (C-90), 43.1 (C-300), 40.8 (C-5), 29.2 (C-3), 28.5 (CH3),
24.8 (C-4), 22.9, 19.3, 17.9, and 12.4 (all CH3); m/z (ESI) 672
(Mþ þ Na, 100%) and 650 (Mþ þ H, 90) [found (Mþ þ Na)
672.2563, C3313CH40N4NaO7S requires 672.2543; found: (Mþ þ
H) 650.2735, C3313CH41N4NaO7S requires 650.2724].
(3S,5S,6R)-4-(tert-Butoxycarbonyl)-3-[30-(tert-butyldimethyl-
silyloxy)prop-10-yl]-5,6-diphenylmorpholin-2-one, 17. First the
reaction was optimized with unlabeled material. To a solution
of unlabeled morpholine carbamate 16 (0.80 g, 2.25 mmol) and
iodide 15 (1.35 g, 4.50 mmol) in dry THF (20 mL) and HMPA
(2 mL) at -78 °C under nitrogen was added lithium bis-
(trimethylsilyl)amide (1.0 M solution in THF, 3.4 mL, 3.38
mmol). The resulting mixture was stirred at -78 °C for 10
min, before being allowed to warm to rt and left for a further
2.5 h. Ethyl acetate (50 mL) was added and washed with
saturated aqueous ammonium chloride (50 mL). The separated
aqueous layer was extracted with ethyl acetate (2 ꢀ 50 mL). The
combined organic extracts were washed with brine (2 ꢀ 50 mL),
then dried (MgSO4), filtered, and evaporated in vacuo. The
residue was purified by column chromatography (5% ethyl
acetate in petroleum ether) to give unlabeled carbamate 17
(0.50 g, 42%) as a colorless solid, which showed a mixture of
The above reaction was repeated with [13C]alcohol 6 (3.33 g,
7.46 mmol) and protected guanidine 12 (4.39 g, 7.57 mmol) to
give an inseparable mixture of protected [13C]arginine 7 and
hydrazodicarboxylate, which was used directly for the next step
without further purification; δC (100 MHz, CDCl3) 57.3 and
57.0 (C-3, enhanced signal); m/z (ESI) 1030 (Mþ þ Na, 19%),
879 (15), 603 (75), and 313 (100) [found (Mþ þ Na) 1030.3745,
C5613CH58N4NaO11S requires 1030.3749].
[2-13C]-(S)-N5-(20,20,40,60,70-Pentamethyldihydrobenzofuran-
50-sulfonyl)arginine, 8. To a solution of the mixture of diisopro-
pyl hydrazodicarboxylate and the [13C]-masked arginine 7 in
methanol (10 mL) and THF (10 mL) was added Pd/C (10%).
The resulting mixture was hydrogenated at 50 psi for 19 h, then
at atmospheric pressure for 2 days. The catalyst was filtered off.
The filtrate was concentrated in vacuo. The residue was parti-
tioned between ethyl acetate (50 mL) and water (100 mL). The
separated aqueous phase was freeze-dried to give [13C]arginine
two rotamers; [R]23D -35.6 (c 1.7, DCM); mp 122-124 °C; νmax
/
cm-1 1746 (CO) and 1702 (CO); δH (400 MHz, CDCl3)
7.26-6.56 (10H, m, Ar-H), 5.96 (1H, d, J = 2.9 Hz, 6-H),
[5.23 (0.5H, d, J=2.9 Hz) and 5.02 (0.5H, d, J=2.9 Hz), 5-H],
[5.05 (0.5H, dd, J=9.3 and 6.0 Hz) and 4.82 (0.5H, dd, J=10.2
and 4.4 Hz), 3-H], 3.78-3.64 (2H, m, 30-H2), 2.40-1.78
(4H, m, 10-H2 and 20-H2), 1.46 and 1.10 (9H, s, 3 ꢀ CH3),
0.92 and 0.91 (9H, s, 3 ꢀ CH3), 0.08 and 0.07 (6H, s, 2 ꢀ CH3);
δC (100 MHz, CDCl3) 169.4 and 169.3 (C-2), 153.6 and
152.9 (CO), 136.5 and 135.3 (C), 134.3 (C), 128.5, 128.1,
128.0, 127.8, 127.6, 127.5, 127.4, 127.3, 126.5, 126.4 (all CH),
81.5 and 81.0 (C), 79.4 and 78.8 (C-6), 62.3 and 62.2 (C-30), 61.5
and 60.3 (C-5), 57.7 and 56.4 (C-3), 32.4 and 31.6 (C-10), 29.4
and 29.2 (C-20), 28.2 and 27.7 (3 ꢀ CH3), 25.9 (3 ꢀ CH3), 18.3
and 18.2 (C), and -5.3 (2 ꢀ CH3); m/z (ESI) 548 (Mþ þ Na,
100%) [found (Mþ þ Na) 548.2811, C30H43NNaO5Si requires
548.2803].
(Pbf)-OH 8 (0.58 g, 18% over 2 steps) as a colorless solid; [R]24
D
-40.0 (c 0.3, H2O); mp 156-162 °C (lit.21 mp 154-157 °C for
the unlabeled); νmax/cm-1 3333, 1571, and 1546; δH (400 MHz,
DMSO-d6) 7.88-6.97 (5H, m, 5 ꢀ NH), 3.03 (2H, m, 5-H2), 2.96
(2H, s, 30-H2), 2.48 (3H, s, CH3), 2.42 (3H, s, CH3), 2.00 (3H, s,
CH3), 1.71-1.43 (4H, m, 3-H2 and 4-H2), and 1.40 (6H, s, 2 ꢀ
CH3) (2-H was obscured by the water peak); δC (100 MHz,
DMSO-d6) 53.8 (C-2, enhanced signal); m/z (ESI) 450 (Mþ
þ
Na, 34%), 442 (100) and 428 (Mþ þ H, 30) [found (Mþ þ H)
428.2040, C1813CH31N4O5S requires 428.2043].
The above reaction was repeated with [13C]morpholine car-
bamate 16 (1.80 g, 5.08 mmol) and [13C]iodide 15 to give
[13C2]carbamate 17 (1.30 g, 48%) as a colorless solid, which
showed a mixture of two rotamers; δH (400 MHz, CDCl3)
7.27-6.56 (10H, m, Ar-H), 5.96 (1H, d, J = 2.9 Hz, 6-H),
[5.23 (0.4H, d, J=2.9 Hz), 5.07-5.01 (1.2H, m) and 4.82 (0.4H,
m), 5-H and 3-H], 3.77-3.65 (2H, m, 30-H2), 2.57-1.80 (4H, m,
10-H2 and 20-H2), 1.46 and 1.09 (9H, s, 3 ꢀ CH3), 0.92 and
0.90 (9H, s, 3 ꢀ CH3), 0.08 and 0.07 (6H, s, 2 ꢀ CH3); δC
(100 MHz, CDCl3) 169.4 and 169.3 (C-2, enhanced signal), 32.4
and 31.6 (C-10, enhanced signal); m/z (ESI) 550 (Mþ þ Na,
100%), 528 (20), and 472 (10) [found (Mþ þ Na) 550.2877,
C2813C2H43NNaO5Si requires 550.2870].
[2-13C]-(S)-N2-(90-Fluorenylmethoxycarbonyl)-N5-(200,200,400,
600,700-pentamethyldihydrobenzofuran-500-sulfonyl)arginine, 1.
Fmoc-ONSu (54 mg, 0.16 mmol) was added to a mixture of L-
arginine (Pbf)-OH 8 (70 mg, 0.16 mmol) and sodium carbonate
(42 mg, 0.40 mmol) in water (4 mL) and dioxane (2 mL) at 0 °C.
After addition, the resulting mixture was allowed to warm to
ambient temperature and left for 2.5 h. Brine (100 mL) was
added and washed with diethyl ether (3 ꢀ 50 mL). The separated
aqueous phase was acidified with saturated citric acid (20 mL),
then extracted with ethyl acetate (3 ꢀ 50 mL). The combined
organic extracts were washed with water (3 ꢀ 50 mL), then brine
(100 mL), and then dried (MgSO4), filtered, and evaporated in
vacuo. The residue was precipitated by the addition of diethyl
ether to give Fmoc-[13C]arginine (Pbf)-OH 1 (78 mg, 75%) as a
(3S,5S,6R)-4-(tert-Butoxycarbonyl)-5,6-diphenyl-3-(30-hydro-
xyprop-10-yl)morpholin-2-one, 18. First the reaction was otpi-
mised with use of unlabeled material. Tetrabutylammonium
fluoride (1.0 M solution in THF, 1.7 mL, 1.70 mmol) was added
to a solution of unlabeled silyl ether 17 (92 mg, 0.17 mmol) in
(21) Du, X.; Zhang, P.; Zhu, Y.; Guo, C. Hua gong shi kan 2004, 18, 28–
29.
J. Org. Chem. Vol. 74, No. 23, 2009 8985

Song et al.
JOCArticle
DCM (10 mL). The resulting mixture was stirred at rt for 6 days.
The solvent was evaporated under reduced pressure. The residue
was purified by column chromatography (30%, then 50% ethyl
acetate in petroleum ether) to give unlabeled carbamate 18 (43
mg, 60%) as a colorless solid, which showed a mixture of two
unlabeled material. A mixture of unlabeled carbamate 19
(49 mg, 0.09 mmol) and trifluoroacetic acid (1 mL) in DCM
(10 mL) was stirred at rt for 1.5 h. Saturated aqueous sodium
hydrogen carbonate (50 mL) was added and the mixture was
stirred for a further 5 min. The layers were separated and the
aqueous layer was extracted with DCM (2 ꢀ 50 mL). The
combined organic extracts were dried (MgSO4), filtered, and
evaporated in vacuo. The residue was purified by column
chromatography (20% ethyl acetate in petroleum ether) to
give the unlabeled bicyclic compound 20 (23 mg, 91%) as a
colorless oil; [R]23D 135.6 (c 2.0, DCM) {lit.18 [R]25D -130.4 for
the opposite enantiomer (c 2.6, DCM)}; δH (400 MHz, CDCl3)
7.26-6.96 (10H, m, Ar-H), 5.60 (1H, d, J=3.8 Hz, 4-H), 4.30
(1H, d, J=3.8 Hz, 5-H), 4.17 (1H, dd, J=9.5 and 7.8 Hz, 1-H),
3.16 (1H, ddd, J=10.3, 7.1, and 3.4 Hz, 7-HH), 2.58 (1H, app.
td, J=9.7 and 6.4 Hz, 7-HH), 2.42 (1H, dddd, J=12.7, 10.1,
7.8, and 2.7 Hz, 9-HH), 2.21 (1H, dddd, J=12.7, 10.6, 9.5, and
7.7 Hz, 9-HH), and 1.92-1.73 (2H, m 8-H2); δC (100 MHZ,
CDCl3) 172.9 (C-2), 136.9, 135.2 (both C), 128.7, 128.4, 128.3,
128.0, 127.9, 127.8 (all CH), 84.0 (C-4), 66.6 (C-5), 60.3 (C-1),
55.0 (C-7), 29.9 (C-9), and 23.7 (C-8); m/z (EI) 293 (Mþ, 4%),
180 (2), 159 (50), and 58 (100) [found (Mþ) 293.1414,
rotamers; [R]23 -49.1 (c 2.2, DCM); νmax/cm-1 3277 (OH),
D
1745 (CO) and 1698 (CO); δH (400 MHz, CDCl3) 7.27-6.56
(10H, m, Ar-H), [5.96 (0.25H, d, J=3.0 Hz) and 5.94 (0.75H, d,
J=3.0 Hz), 6-H], [5.23 (0.25H, d, J=3.0 Hz) and 5.01 (0.75H, d,
J=3.0 Hz), 5-H], [5.05 (0.75H, dd, J=10.4 and 4.4 Hz) and 4.89
(0.25H, dd, J=10.4 and 4.4 Hz), 3-H], 3.94-3.77 (2H, m, 30-H2),
2.39-1.85 (4H, m, 10-H2 and 20-H2), 1.46 and 1.11 (9H, s, 3 ꢀ
CH3); δC (100 MHz, CDCl3) 169.6 and 169.0 (C-2), 154.8 and
153.8 (CO), 136.2, 134.2 (both C), 128.6, 128.1, 127.8, 127.7,
127.6, 127.4, 127.3, 126.5, 126.4 (all CH), 81.5 (C), 79.1 (C-6),
61.4 (C-5), 61.2 (C-30), 55.2 (C-3) 31.5 (C-10), 28.3 (C-20), and
27.8 (3 ꢀ CH3); m/z (ESI) 434 (Mþ þ Na, 100%) [found (Mþ þ
Na) 434.1944, C24H29NNaO5 requires 434.1938].
The above reaction was repeated with [13C2]silyl ether 17 (1.45
g, 2.75 mmol) to give [13C2]carbamate 18 (0.65 g, 57%) as a
colorless solid, which showed a mixture of two rotamers; δH
(400 MHz, CDCl3) 7.28-6.58 (10H, m, Ar-H), [5.99 (0.2H, d,
J=2.9 Hz) and 5.96 (0.8H, d, J=2.9 Hz), 6-H], [5.23 (0.2H, d, J=
2.9 Hz) and 5.01 (0.8H, d, J=2.9 Hz), 5-H], 5.10-4.87 (1H, m,
3-H), 3.95-3.78 (2H, m, 30-H2), 2.54-1.85 (4H, m, 10-H2 and
20-H2), 1.46 and 1.11 (9H, s, 3 ꢀ CH3); δC (100 MHz, CDCl3)
169.4 and 169.2 (C-2, enhanced signal), 32.2 and 31.4 (C-10,
enhanced signal); m/z (ESI) 436 (Mþ þ Na, 100%) [found (Mþ
þ Na) 436.1995, C2213C2H29NNaO5 requires 436.2005].
C
19H19NO2 requires 293.1416].
The reaction was repeated with [13C2]carbamate 19 (545 mg,
0.96 mmol) to give the [13C2]bicyclic compound 20 (178 mg,
63%) as a colorless oil; δH (400 MHz, CDCl3) 7.25-6.95 (10H,
m, Ar-H), 5.59 (1H, dd, J=4.9 and 3.9 Hz, 4-H), 4.27 (1H, d, J=
3.9 Hz, 5-H), 4.15 (1H, ddt, J=9.5, 7.8, and 4.6 Hz, 1-H), 3.12
(1H, m, 7-HH), 2.55 (1H, ddt, J=9.3, 6.6, and 4.4 Hz, 7-HH),
and 2.40-1.70 (4H, m, 8-H2 and 9-H2); δC (100 MHZ, CDCl3)
172.6 (C-2, enhanced signal) and 29.7 (C-9, enhanced signal); m/
z (EI) 295 (Mþ, 3%), 180 (35), and 160 (100) [found (Mþ)
295.1481, C1713C2H19NO2 requires 295.1483].
(3S,5S,6R)-4-(tert-Butoxycarbonyl)-5,6-diphenyl-3-[30-(400-
methylphenylsulfonyloxy)prop-10-yl]morpholin-2-one, 19. First
the reaction was optimized with use of unlabeled material. A
mixture of unlabeled alcohol 18 (43 mg, 0.10 mmol), DMAP (28
mg, 0.23 mmol), and tosyl chloride (40 mg, 0.21 mmol) in dry
DCM (5 mL) was stirred at rt for 27 h. Water (20 mL) was
added, and the separated aqueous phase was extracted with
DCM (2 ꢀ 50 mL). The combined organic extracts were dried
(MgSO4), filtered, and evaporated in vacuo. The residue was
purified by column chromatography (20%, then 30% ethyl
acetate in petroleum ether) to give unlabeled carbamate 19 (49
mg, 83%) as a colorless solid, which showed a mixture of two
[1,3-13C2]-L-Proline, 21. A mixture of the [13C2]bicyclic com-
pound 20 (179 mg, 0.60 mmol), palladium chloride (53 mg, 0.30
mmol), THF (2.5 mL), and ethanol (4.5 mL) was hydrogenated
at 50 psi for 21.5 h. The catalyst was filtered through a pad of
Celite. The filtrate was evaporated in vacuo. The residue was
partitioned between water (20 mL) and diethyl ether (20 mL).
The separated aqueous phase was washed with diethyl ether (20
mL) and then evaporated in vacuo. The residue was purified by
column on Dowex ion-exchange resin (50WX8-100), flushing
with 1 M aqueous ammonia to give [1,3-13C2]-L-proline 21 (55
mg, 78%) as a colorless solid; mp 206-208 °C; δH (400 MHz,
D2O) 4.32 (1H, m, 2-H), 3.40-3.26 (2H, m, 5-H2), and
2.57-1.86 (4H, m, 3-H2 and 4-H2); δC (100 MHz, D2O) 172.2
(C-1, enhanced signal) and 28.4 (C-3, enhanced signal); m/z (EI)
117 (Mþ, 40%) and 91 (100) [found (Mþ) 117.0699,
C313C2H9NO2 requires 117.0700].
rotamers; [R]23 -28.0 (c 0.5, DCM); mp 185-186 °C; νmax
/
D
cm-1 1747 (CO) and 1701 (CO); δH (400 MHz, CDCl3)
7.81-6.53 (14H, m, Ar-H), 5.89 (1H, d, J = 2.9 Hz, 6-H),
[5.21 (0.3H, d, J=2.9 Hz) and 4.99 (0.7H, d, J=2.9 Hz), 5-H],
[4.94 (0.7H, m) and 4.75 (0.3H, m), 3-H], 4.18-4.10 (2H, m,
30-H2), 2.44 and 2.42 (3H, s, CH3), 2.26-1.95 (4H, m, 10-H2 and
20-H2), 1.44 and 1.09 (9H, s, 3 ꢀ CH3); δC (100 MHZ, CDCl3)
169.0 (C-2), 157.3 (CO), 144.9 and 142.0 (C), 136.3 and 135.1
(C), 134.1 and 133.0 (C), 129.9, 128.6, 128.1, 128.1, 127.9, 127.8,
127.7, 127.6, 127.4, 127.3, 126.5, 126.4 (all CH), 81.3 (C), 78.9
(C-6), 69.8 (C-30), 61.4 (C-5), 56.0 (C-3), 31.2 (C-10), 28.2 anþd
27.8 (3 ꢀ CH3), 25.6 (C-20), and 21.6 (CH3); m/z (ESI) 588 (M
þ Na, 100%) [found (Mþ þ Na) 588.2038, C31H35NNaO7S
requires 588.2026].
[1,3-13C2] N-(90-Fluorenylmethoxycarbonyl)-L-proline, 22. 9-
Fluorenylmethyl chloroformate (115 mg, 0.45 mmol) was added
portionwise to a solution of [1,3-13C2]-L-proline 21 (55 mg, 0.47
mmol) and potassium carbonate (162 mg, 1.18 mmol) in water
(10 mL) and dioxane (3 mL) at 0 °C. After addition, the resulting
mixture was stirred for 17 h without further cooling. The
mixture was diluted with water (20 mL) and extracted with
diethyl ether (2 ꢀ 20 mL). The separated aqueous phase was
acidified with 1 M hydrochloric acid, and then extracted with
DCM (3 ꢀ 20 mL). The combined organic extracts were dried
(MgSO4), filtered, and evaporated in vacuo to give Fmoc-
[1,3-13C2]-L-proline 22 (129 mg, 81%) as a colorless solid, which
showed a mixture of two rotamers; mp 115-116 °C (lit19 mp
114-115), [R]25D -35.8 (c 1.0, DMF) {lit.19 [R]25D -33.4 for the
unlabeled (c 1.0, DMF)}; δH (400 MHz, CDCl3) 7.77-7.27 (8H,
m, Ar-H), 4.50-4.13 (4H, m, 2-H, 90-H, and 100-H2), 3.63-3.45
(2H, m, 5-H2), and 2.46-1.90 (4H, m, 3-H2 and 4-H2); δC (100
MHz, CDCl3) 177.6 and 175.6 (C-1, enhanced signal), 31.0 and
29.1 (C-3, enhanced signal); m/z (ESI) 362 (Mþ þ Na, 100%)
The reaction was repeated with [13C2]alcohol 18 (619 mg, 1.5
mmol) to give [13C2]carbamate 19 (566 mg, 67%) as a colorless
solid, which showed a mixture of two rotamers; δH (400 MHz,
CDCl3) 7.84-6.55 (14H, m, Ar-H), 5.91 (1H, d, J=2.8 Hz,
6-H), [5.23 (0.3H, d, J=2.8 Hz) and 5.02 (0.7H, d, J=2.8 Hz),
5-H], 4.87 (1H, m, 3-H), 4.22-4.12 (2H, m, 30-H2), 2.46 and 2.45
(3H, s, CH3), 2.43-1.96 (4H, m, 10-H2 and 20-H2), 1.46 and 1.11
(9H, s, 3 ꢀ CH3); δC (100 MHZ, CDCl3) 169.0 and 168.9 (C-2,
enhanced signal), 31.7 and 31.3 (C-10, enhanced signal); m/z
(ESI) 590 (Mþ þ Na, 100%) [found (Mþ þ Na) 590.2090,
C2913C2H35NNaO7S requires 590.2094].
(1S,4R,5S)-6-Aza-4,5-diphenyl-2-oxo-3-oxabicyclo[4.3.0]-
nonane, 20. First the reaction was optimized with use of
8986 J. Org. Chem. Vol. 74, No. 23, 2009

Song et al.
JOCArticle
[found (Mþ þ Na) 362.1264, C1813C2H19NNaO4 requires
(C.S. and A.M.) and to the Royal Thai Government for a
Scholarship to S.T.
362.1273].
Supporting Information Available: 1H- and 13C NMR
spectra for all new compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
Acknowledgment. We are grateful to the EPSRC
(Platform Grant No. EP/E000/77/1) and BBSRC for funding
J. Org. Chem. Vol. 74, No. 23, 2009 8987