(R)-tert-butoxycarbonylamino-fluorenylmethoxycarbonyl-glycine from (S)-benzyloxycarbonyl-serine or from papain resolution of the corresponding amide or methyl ester

Article abstract of DOI:10.1021/jo000736e

The enantiospecific synthesis of (R)-Boc-(Fmoc)-aminoglycine 7 was achieved. (S)-Cbz-serine 1 was reacted with diphenylphosphoryl azide in the presence of triethylamine to yield cyclic(S) carbamate 2. The ring nitrogen of 2 was protected with a Boc group.

Full text of DOI:10.1021/jo000736e

J . Org. Chem. 2000, 65, 6595-6600
6595
(R)-ter t-Bu toxyca r bon yla m in o-flu or en ylm eth oxyca r bon yl-glycin e
fr om (S)-Ben zyloxyca r bon yl-ser in e or fr om P a p a in Resolu tion of
th e Cor r esp on d in g Am id e or Meth yl Ester †
Michal Sypniewski,‡ Botond Penke,§ Lajos Simon,| and J ean Rivier*
Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 North Torrey Pines Road,
La J olla, California 92037
jrivier@salk.edu
Received May 15, 2000
The enantiospecific synthesis of (R)-Boc-(Fmoc)-aminoglycine 7 was achieved. (S)-Cbz-serine 1 was
reacted with diphenylphosphoryl azide in the presence of triethylamine to yield cyclic (S) carbamate
2. The ring nitrogen of 2 was protected with a Boc group (3). The cyclic carbamate of 3 was
hydrolyzed with benzyltrimethylammonium hydroxide to yield the (R)-enantiomer of alcohol 4.
The oxidation of 4 with pyridinium dichromate yielded the enantiomerically pure (97% ee) (R)-
Boc-(Cbz)-aminoglycine 5, which was converted to 7 with retention of optical purity. Similarly,
starting from (S)-Boc-serine 9, cyclic (S) carbamate 10 was obtained. The ring nitrogen of 10 was
protected with a Cbz group (11) with retention of configuration. The cyclic carbamate of 11 was
base hydrolyzed to yield 12, the (S)-enantiomer alcohol. Independently, Boc-(Fmoc)-aminoglycine
amide 13 and Boc-(Fmoc)-aminoglycine methyl ester 14 were resolved using papain. The
stereochemistry of the isolated acid was determined to be (R) by coelution on HPLC of its derivative
with Marfey’s reagent and that of an authentic sample (7) obtained by enantiospecific synthesis.
In tr od u ction
Interest in monomeric building blocks that mimic the
peptide backbone has dramatically increased with the
coming of age of combinatorial chemistry. Peptoids,1
azoles,2 2-isooxazoles,3 oligosulfones and oligosulfoxides,4
pyrrolidones,5 vinylogous backbones,6 â-methyl-amino
F igu r e 1. Representative structure of betidamino acids.
*Indicates a chiral center.
acids,7 and â-amino acids8,9 are a few examples of such
compounds. We have found aminoglycine (Agl) to offer
great versatility and compatibility with biological systems
when selectively alkylated and/or acylated to yield
betidamino acids.10,11 (Figure 1).
* Corresponding author.Phone: (858) 453-4100. Fax: (858) 552-
1546.
Orthogonally protected R-(N-tert-butoxycarbonylamino)-
NR-(fluorenylmethoxycarbonyl)glycine, [Boc-(Fmoc)-Agl-
OH] was first synthesized by Qasmi et al. as the smallest
of the dibasic amino acids.12 An advantageous property
of the aminoglycine scaffold over many other building
blocks is that it is a versatile precursor of a wide variety
of betidamino acids that can be prepared directly during
a solid-phase peptide synthesis. After incorporation of the
orthogonally protected aminoglycine in the peptide chain
and the deprotection of Boc, the amino function can be
acylated (R1 ) R2 ) H), or alkylated13 (R1 and/or R2 )
alkyl group)14 and acylated with the desired acyl deriva-
tive (R-CO-). Whereas betidamino acids (R1 ) R2 ) H)
may assume conformational states reminiscent of amino
† Abbreviations: Agl ) aminoglycine, (Boc)2O ) di-tert-butyl dicar-
bonate, t-BuOH ) tert-butyl alcohol, DMAP ) dimethylaminopyridine,
DABCO ) 1,4-diazabicyclo[2.2.2]octane, DPPA ) diphenylphosphoryl
azide, Et3N ) triethylamine, EtOAc ) ethyl acetate, Fmoc-OSu )
9-fluorenylmethylsuccinimidyl carbonate, HOBt ) N-hydroxybenzo-
triazole, Marfey’s reagent ) 1-fluoro-2,4-dinitrophenyl-5-alanine amide,
MeCN ) acetonitrile, PDC ) pyridinium dichromate, Ph ) phenyl.
‡ Current address: Pharmaceutical Research Institute, ul. Rydygi-
era 8, 01-793 Warsaw, Poland.
§Current address: Albert Szent-Gyo¨rgyi Medical University, De-
partment of Medicinal Chemistry, Szeged, Hungary.
| Current address: Institute of Pharmaceutical Chem., Albert Szent-
Gyo¨rgyi Medical University, Szeged, Hungary.
(1) Simon, R. J .; Kania, R. S.; Zuckermann, R. N.; Huebner, V. D.;
J ewell, D. A.; Banville, S.; Ng, S.; Wang, L.; Rosenberg, S.; Marlowe,
C. K.; Spellmeyer, D. C.; Tan, R.; Frankel, A. D.; Santi, D. V.; Cohen,
F. E.; Bartlett, P. A. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 9367-
9371.
(2) Gordon, T. D.; Singh, J .; Hansen, P. E.; Morgan, B. A. Tetrahe-
dron Lett. 1993, 34, 1901-1904.
(9) Matthews, J . L.; Braun, C.; Guibourdenche, C.; Overhand, M.;
Seebach, D. In Enantioselective Synthesis of â-Amino Acids; J uaristi,
E., Ed.; Wiley-VCH: New York, 1997; pp 105-126.
(10) Rivier, J . E.; J iang, G.-C.; Koerber, S. C.; Porter, J .; Craig, A.
G.; Hoeger, C. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 2031-2036.
(11) J iang, G.; Miller, C.; Koerber, S. C.; Porter, J .; Craig, A. G.;
Bhattacharjee, S.; Kraft, P.; Burris, T. P.; Campen, C. A.; Rivier, C.
L.; Rivier, J . E. J . Med. Chem. 1997, 40, 3739-3748.
(12) Qasmi, D.; Rene´, L.; Badet, B. Tetrahedron Lett. 1993, 34,
3861-3862.
(3) Kim, B. H.; Chung, Y. J .; Keum, G.; Kim, J .; Kim, K. Tetrahedron
Lett. 1992, 33, 6811-6814.
(4) Moran, E. J .; Wilson, T. E.; Cho, C. Y.; Cherry, S. R.; Schultz, P.
G. Biopolymers 1995, 37, 213-219.
(5) Smith, A. B., III; Hirschmann, R.; Pasternak, A.; Akaishi, R.;
Guzman, M. C.; J ones, D. R.; Keenan, T. P.; Sprengeler, P. A.; Darke,
P. L.; Emini, E. A.; Holloway, M. K.; Schleif, W. A. J . Med. Chem. 1994,
37, 215-218.
(6) Hagihara, M.; Anthony, N. J .; Stout, T. J .; Clardy, J .; Schreiber,
S. L. J . Am. Chem. Soc. 1992, 114, 6568-6870.
(13) Kaljuste, K.; Unde´n, A. Int. J . Pept. Protein Res. 1993, 42, 118-
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(7) Kazmierski, W. M.; Urbanczyk-Lipkowska, Z.; Hruby, V. J . J .
Org. Chem. 1994, 59, 1789-1795.
(14) J iang, G.-C.; Simon, L.; Rivier, J . E. Protein Pept. Lett. 1996,
3, 219-224.
(8) Gmeiner, P. Tetrahedron Lett. 1990, 31, 5717-5720.
10.1021/jo000736e CCC: $19.00 © 2000 American Chemical Society
Published on Web 09/14/2000

6596 J . Org. Chem., Vol. 65, No. 20, 2000
Sch em e 1. Syn th etic Sch em e for (R)-Boc-(Cbz)-Agl-OH (5)
Sypniewski et al.
Sch em e 2. P r op osed Mech a n ism for Ba se
acids, methylation of either of the two nitrogens will
induce unique conformational preferences difficult to
mimic otherwise.10 For example, betidamino acids meth-
ylated at R2 will readily mimic â-methylated amino acids
that are synthetically challenging to obtain in an optically
pure form. Aminoglycine itself is achiral and unstable
in its unprotected form due to the geminal diamino
function. Mono-N-protected derivatives are more stable,
chiral and can be handled or stored around 0 °C.15 Di-
N-protected derivatives of aminoglycine are stable at
room temperature and are chiral when the N-substitu-
tions are different. We describe the enantiospecific
synthesis of (R)-Boc-(Fmoc)-Agl-OH from (S)-serine and
the resolution of Boc-(Fmoc)-Agl-OMe and of Boc-(Fmoc)-
Agl-NH2 mediated by papain. The stereochemistry of the
hydrolyzed acid (R) was determined by chromatographic
comparison of Marfey’s condensation products of authentic/
synthetic and Boc-(Fmoc)-Agl-OH isolated after papain
treatment.
Elim in a tion of Cbz Gr ou p
zyl ester 4. These low-temperature conditions for base-
induced ring opening of the oxazolidone turned out to be
essential to avoid the undesired N-deprotection of the
benzyloxycarbonyl group observed earlier by Ishizuka
and Kunieda21 in similar systems. We found in agreement
with related examples of Ishizuka and Kunieda that 3,
treated with LiOH or K2CO3 in MeOH/H2O, gave a
mixture of the desired protected amino alcohol and a
compound characterized as (R)-4-tert-butoxycarbonylami-
no-2-oxazolidone (the R enantiomer of 10, (R)-10, see
Schemes 2 and 5) as determined by identical physico-
chemical properties (1H NMR, and melting point) except
for their optical rotation of opposite signs.
These observations led us to propose a mechanism for
the elimination of the Cbz group whereby an alkoxylate
is formed first (resulting in the opening of the oxazoli-
done) followed by an intramolecular nucleophilic dis-
placement of the Cbz group resulting in a change in
configuration Scheme 2. The same reaction was observed
in preliminary experiments with (S)-3-benzyloxycarbonyl-
4-tert-butoxycarbonylamino-2-oxazolidone 11 (see below).
The oxidation of 4 with pyridinium dichromate (PDC)
in DMF at room temperature22 led to (R)-benzyloxycar-
bonylamino-tert-butoxycarbonylaminoacetic acid [(R)-
Boc-(Cbz)-Agl-OH, 5].
Resu lts a n d Discu ssion
We focused on (S)-serine (L-serine) as a synthetic
template for the enantiospecific synthesis of Boc-(Fmoc)-
Agl-OH. Each of the three carbons of serine bears a
functionality that can be selectively protected or modified
with retention of configuration at the R-carbon as evi-
denced by an abundant literature on this topic.16-19
Our synthetic strategy (Scheme 1) took advantage of
the carboxylic moiety to introduce the second amino
function through its conversion to a carbamino group via
the acyl azide-isocyanate pathway. The hydroxymethyl
group was then oxidized to a carboxyl functionality.
More specifically, (S)-benzyloxycarbonyl-serine [(S)-
Cbz-serine] 1 was converted into (S)-4-benzyloxycarbon-
ylamino-2-oxazolidone 2 by refluxing with diphenylphos-
phoryl azide (DPPA) and triethylamine (Et3N) in t-
BuOH.20 The ring nitrogen of 2 was protected with Boc
using di-tert-butyl dicarbonate (Boc)2O and Et3N in THF,
at low temperature. The critical ring opening of the (R)-
4-benzyloxycarbonylamino-3-tert-butoxycarbonyl-2-ox-
azolidone 3 was performed with benzyltrimethylammo-
nium hydroxide in THF at -40 °C, yielding (R)-2-hy-
droxy-1-tert-butoxycarbonylaminoethylcarbamic acid ben-
The optical purity of 5 was measured after catalytic
hydrogenation of the Cbz group and coupling of Marfey’s
reagent23 in acetone in the presence of Et3N. The dia-
(15) Bock, M. P.; DiPardo, R. M.; Freidinger, R. M. J . Org. Chem.
1986, 51, 3718-3720.
(16) Tanner, D. Angew. Chem., Int. Ed., Engl. 1994, 33, 599-619.
(17) Miller, M. J . Acc. Chem. Res. 1986, 19, 49-56.
(18) J urczak, J .; Golebiowski, A. Chem. Rev. 1989, 89, 149-164.
(19) Kulkarni, Y. S. Aldrichimica Acta 1999, 32, 18-27.
(20) Shioiri, T.; Ninomiya, K.; Yamada, S. J . Am. Chem. Soc. 1972,
94, 6203-6205.
(21) Ishizuka, T.; Kunieda, T. Tetrahedron Lett. 1987, 28, 4185-
4188.
(22) Corey, E. J .; Schmidt, G. Tetrahedron Lett. 1979, 5, 399-402.
(23) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591-596.

Enantiospecific Synthesis of (R)-Boc-(Fmoc)-aminoglycine
J . Org. Chem., Vol. 65, No. 20, 2000 6597
peptide synthesis protocol,20 could not be used in our case.
The excess would compete with Boc-Agl-OH for Marfey’s
reagent, and the separation of piperidine from labile Boc-
Agl-OH would be very difficult. Other reagents known
to cleave the Fmoc-protecting group, like tetrabutylam-
monium fluoride,21 did not give satisfactory results in our
case. Instead, we successfully used DABCO. Two equiva-
lents of DABCO per 1 equiv of Fmoc-protected amine in
DMF solution at room-temperature liberated the amino
function within 30 min. As a tertiary base, DABCO does
not react with Marfey’s reagent, but provides the neces-
sary basic conditions for coupling. To our knowledge this
is the first report of the use of DABCO to cleave the
Fmoc-protecting group. As was shown above, the reagent
did not cause noticeable racemization of the chiral center
of the protected aminoglycine.
Sch em e 3. Der iva tiza tion of P a r tia lly P r otected
Agl-Sca ffold s for Deter m in a tion of Op tica l P u r ity
We used (S)-Boc-serine 9 for the synthesis of 12, the
(S) enantiomer alcohol (Scheme 5). (S)-Boc-serine reacted
with DPPA and Et3N in t-BuOH to yield the (S)-4-tert-
butoxycarbonylamino-2-oxazolidone 10. The ring nitrogen
of 10 was then reacted with an excess of benzyloxycar-
bonyl chloride in a THF/Et3N solution at -40 °C to yield
the (S)-3-benzyloxycarbonyl-4-tert-butoxycarbonylamino-
2-oxazolidone 11. Introduction of Fmoc on the ring
nitrogen of 10 would have been convenient, but it would
not have been stable under the basic conditions used for
the ring opening. Compound 11 was then converted to
the (S)-2-hydroxy-1-tert-butoxycarbonylaminoethylcar-
bamic acid benzyl ester 12 after hydrolysis with benzyl-
trimethylammonium hydroxide in toluene at -40 °C. It
is noteworthy that the tendency for the undesired course
of the reaction shown in Scheme 2 was greater with 11
than with 3, hence the changes in experimental protocols.
The alcohol 12 was the (S)-enantiomer of 4 with physical
Sch em e 4: Con ver sion of (R)-Boc-(Cbz)-Agl-OH to
(R)-Boc-(F m oc)-Agl-OH
stereoisomeric mixture of Boc-aminoglycine derivatives
6 was then analyzed on reverse phase HPLC. The optical
purity of (R)-Boc-(Cbz)-Agl-OH 5 (97% ee) was based on
the integration of the absorbances corresponding to (R,S)
and (S,S) diastereoisomers of 6. Racemic Boc-(Cbz)-Agl-
OH was prepared using the same protocol as that used
to make racemic Boc-(Fmoc)-Agl-OH10 and subjected to
the same reactions producing a 1:1 mixture of (R,S) and
(S,S) diastereoisomers of 6 (Scheme 3).
(R)-Boc-(Cbz)-Agl-OH 5 was converted to (R)-Boc-
(Fmoc)-Agl-OH, 7 in two steps (Scheme 4): first the Cbz
group was cleaved by catalytic hydrogenation in metha-
nol at 0 °C and then, without isolation of the formed Boc-
Agl-OH, excess Fmoc-OSu was added. This was followed
by the slow addition of 1 equiv of Et3N.
Determination of the optical purity of the (R)-Boc-
(Fmoc)-Agl-OH 7 was carried out using the procedure
described earlier for 5. After cleavage of the Fmoc-group
by DABCO, Boc-Agl-OH was coupled with Marfey’s
reagent, and the resulting mixture was analyzed by
reverse-phase HPLC. Integration of the absorbances
corresponding to (R,S) and (S,S) diastereoisomers of 6
showed 97% ee of the (R)-Boc-(Fmoc)-Agl-OH (Scheme
3).
1
properties identical to that of 4 (mp, H NMR spectrum,
elemental composition) with the exception of the specific
rotation which was of identical absolute value but op-
posite in sign. Oxidation of 12 to (S)-tert-butoxycarbonyl-
amino-benzyloxycarbonylaminoacetic acid, [(S)-Boc-(Cbz)-
Agl-OH] and conversion to (S)-tert-butoxycarbonylamino-
fluorenylmethoxycarbonylaminoacetic acid [(S)-Boc-(Fmoc)-
Agl-OH] can be achieved by the same steps that were
described above for compound 4.
It would have been evident to synthesize (S)-Boc-(Cbz)-
Agl-OH from (R)-Cbz-serine using the synthetic protocol
shown in Scheme 1. Alternatively, we identified modified
synthetic protocols (changes of solvent and temperature
as compared to those shown in Scheme 1) using (S)-Boc-
serine for the synthesis of (S)-2-hydroxy-1-tert-butoxy-
carbonylaminoethylcarbamic acid benzyl ester 12 (Scheme
Piperidine, which is the most widely used reagent for
cleavage of the Fmoc-protecting group in solid-phase
Sch em e 5: Syn th etic Sch em e for (S)-2-Hyd r oxy-1-ter t-bu toxyca r bon yla m in oeth ylca r ba m ic Acid
Ben zyl Ester 12

6598 J . Org. Chem., Vol. 65, No. 20, 2000
Sypniewski et al.
12 h, centrifuged, and finally filtered before use. Other
reagents were used without further purification. Analytical
thin-layer chromatography was performed using Merck Kie-
selgel 60 F254 aluminum sheets (EM Science, Gibbstown, NJ ).
For the reverse-phase analytical HPLC, a Vydac (The Separa-
tions Group, Hesperia, CA) C18 column (0.46 × 25 cm, 5 µm
particle size, 300 Å pore size) was used. Mobile phases A and
B were 0.1% TFA in H2O and 0.1% TFA in (60% MeCN/40%
H2O), respectively. Gradient shapes and flow rates are de-
scribed in the text. UV detection was at 214 nm.
5). In that case the introduction of the Cbz protecting
group to the nitrogen of the oxazolidone maintained the
order of priority of the groups around the chiral carbon
atom resulting in retention of the (S) configuration.
We hypothesized that enzymatic resolution of amino
acid derivatives could be more easily scaled up than the
enantiospecific synthesis because of difficulties experi-
enced during the oxidation step. Therefore, we sought
conditions that would resolve (R,S)-Boc-(Fmoc)-Agl-OH.
We found that both (R,S)-Boc-(Fmoc)-Agl-NH2 13 and
(R,S)-Boc-(Fmoc)-Agl-OMe 14 were susceptible to papain
hydrolysis, and detailed conditions are given in the
Experimental Section. The extraction steps are delicate
due to the fact that Boc-(Fmoc)-Agl-NH2, Boc-(Fmoc)-Agl-
OMe, and the resolved Boc-(Fmoc)-Agl-OH are relatively
insoluble in most solvents. Despite these difficulties,
significant quantities of (R)-Boc-(Fmoc)-Agl-OH (7) with
high enantiomeric purity (>92%, >50 g) were obtained
from racemic Boc-(Fmoc)-Agl-NH2(13) or Boc-(Fmoc)-Agl-
OMe (14). The stereochemistry of the isolated acid was
determined to be (R) by coelution on HPLC of its adduct
with Marfey’s reagent and that of an authentic sample
obtained by enantiospecific synthesis. Although we have
focused our efforts toward the isolation of significant
quantities of (R)-Boc-(Fmoc)-Agl-OH (7), the correspond-
ing (S)-Boc-(Fmoc)-Agl-OMe and (S)-Boc-(Fmoc)-Agl-NH2
were also isolated. In principle, both the methyl ester and
amide could be hydrolyzed using a nonspecific hydrolase
such as Pronase. This approach was not pursued because
both betidamino-enantiomers are readily available from
the same (R)-Boc-(Fmoc)-Agl-OH scaffold depending on
whether the acylating agent used to mimic an amino acid
side chain is built on the Boc- or Fmoc-bearing nitrogen
of aminoglycine. It was hoped that the differences in
solubility of either (S)-Boc-(Fmoc)-Agl-NH2 or (S)-Boc-
(Fmoc)-Agl-OMe and the desired (R)-Boc-(Fmoc)-Agl-OH
during extractions at basic and acidic pHs would be such
that one derivative could be favored over the other. This
was not the case.
(S)-4-Ben zyloxyca r bon yla m in o-2-oxa zolid on e (2). (S)-
Cbz-serine 1 (12.5 g, 52.3 mmol) and DPPA (15.8 g, 57.5 mmol)
were dissolved in t-BuOH (200 mL). The reaction flask was
flushed with argon, and Et3N (5.82 g, 57.5 mmol) was added.
The reaction mixture was refluxed under argon for 3 h and
then cooled to room temperature. EtOAc (50 mL) was added,
and solvents were evaporated under reduced pressure. A
saturated solution of NaHCO3 (500 mL) was then added to
the residue, and the product was extracted with EtOAc (3 ×
100 mL). The combined extracts were washed with saturated
NaHCO3 solution, 2% NaHSO4 solution, water, and finally
dried over anhydrous MgSO4. After evaporation of the solvent
under reduced pressure, the solid residue was subjected to
crystallization from EtOAc to yield 2 (10.3 g, 84%): mp 169-
171 °C (EtOAc); [R]25 ) -87.3° (c ) 1, MeOH); 1H NMR
D
(DMSO-d6) 4.01-4.03 (m, 1H); 4.46-4.50 (m, 1H); 5.05 (s, 2H);
5.35-5.39 (m, 1H); 7.3-7.4 (m, 5H); 8.24 (d, 1H, J ) 8.3 Hz);
8.30 (s, 1H). Elemental composition calculated for C11H12N2O4:
C, 55.93%; H, 5.12%; N, 11.86%; found: C, 55.90%; H, 5.04%;
N, 11.8.
(R)-4-Ben zyloxyca r bon yla m in o-3-ter t-bu toxyca r bon yl-
2-oxa zolid on e (3). Compound 2 (33.0 g, 0.14 mol), DMAP (360
mg), and Et3N (17.0 g, 0.168 mol) were dissolved in dry THF
(380 mL) and cooled in an ice bath. A solution of (Boc)2O (32.0
g, 0.147 mol) in THF (120 mL) was added to the stirred
reaction mixture over 30 min. The temperature of the reaction
mixture was kept between 5 and 10 °C, and the progress of
the reaction was monitored by TLC. As soon as the starting
material had been consumed, a solution of NaHSO4 (20.13 g,
0.168 mmol) in water (150 mL) was added. The THF was
evaporated under reduced pressure. The white suspension of
the crude product was then dissolved in EtOAc, and the
solution was washed with dilute NaHSO4 solution, water, and
finally dried with anhydrous MgSO4. After evaporation of the
solvent under reduced pressure, an oily residue was im-
mediately dissolved in diethyl ether (200 mL). Within a few
minutes, white crystals of the product started to precipitate.
After filtration and concentration of the filtrate under reduced
pressure, an additional amount of 3 was obtained (44.8 g,
95%): mp 128-130 °C; [R]20D ) -8.7° (c ) 1, MeOH); 1H NMR
(acetone) 1.44 (s, 9H); 4.19-4.21 (m, 1H); 4.59-4.63 (m, 1H);
5.09-5.13 (m, 2H); 5.93-5.96 (m, 1H); 7.30-7.40 (m, 5H); 7.51
(bs, 1H). Elemental composition calculated for C16H20N2O6: C,
57.14%; H, 5.99%; N, 8.33%; found: C, 57.46%; H, 5.90%; N,
8.36%.
Con clu sion
We describe the enantiospecific synthesis of optically
enriched (97% ee) (R)-tert-butoxycarbonylamino-benzyl-
oxycarbonylaminoacetic acid [(R)-Boc-(Cbz)-Agl-OH] 5
and (R)-tert-butoxycarbonylamino-fluorenylmethoxycar-
bonylaminoacetic acid [(R)-Boc-(Fmoc)-Agl-OH] 7. We
have also described synthetic protocols yielding the (S)-
enantiomer of 4 precursor of 5 and 7. In each case the
chiral center of the protected aminoglycine is originated
from commercially available (S)-serine derivatives. We
have also described a practical, large-scale method for
the enzymatic resolution of orthogonally protected Boc-
(Fmoc)-Agl-methyl ester and amide.
(R)-2-H yd r oxy-1-ter t-b u t oxyca r b on yla m in oet h ylca r -
ba m ic Acid Ben zyl Ester (4). Compound 3 (40.0 g, 0.119
mol) was dissolved in dry THF (1200 mL) and cooled to -40
°C, under argon atmosphere. Benzyltrimethylammonium hy-
droxide (59.3 mL of 40% solution in methanol, 0.13 mol) was
then added over 20 min. After an additional 20 min of stirring
at -40 °C, acetic acid (25 mL) and water (60 mL) were added.
The reaction flask was transferred to a water bath (+35 °C)
for 20 min followed by evaporation of the organic solvents. The
white residue was dissolved in EtOAc (1.2 L), washed with
2% NaHSO4 solution and water, dried with anhydrous MgSO4,
and evaporated. The solid 4 was dissolved in a small volume
of hot EtOAc and precipitated with petroleum ether (yield was
Exp er im en ta l Section
Melting points were determined on a capillary melting point
apparatus and were not corrected. 1H nuclear magnetic
resonance spectra were recorded on a 500 MHz instrument.
THF was distilled from potassium before use. Et3N and
t-BuOH were distilled from CaH2. Other solvents were used
without special purification. Papain was purchased from Acros,
(Fisher Scientific, Pittsburgh, PA). Commercially available (S)-
Cbz-serine and (S)-Boc-serine (Bachem, CA) were dried over
P2O5 in a vacuum for 12 h before use. Benzyltrimethylammo-
nium hydroxide (40% solution in methanol) was purchased
from Aldrich (Milwaukee, WI) and was dried over CaH2 for
30.4 g, 82%): mp 140-141 °C; [R]25 ) +8.0° (c ) 1, MeOH);
D
1H NMR (DMSO) 1.37 (s, 9H); 3.30-3.37 (m, 2H); 4.79 (t, 1H,
J ) 6.0 Hz); 4.96-4.99 (m, 1H); 5.01 (s, 2H); 6.82 (bs, 1H);
7.26 (d, 1H, J ) 6.1 Hz); 7.29-7.36 (m, 5H). Elemental
composition calculated for C15H22N2O5: C, 58.05%; H, 7.15%;
N, 9.03%; found: C, 58.14%; H, 6.98%; N, 8.96%.

Enantiospecific Synthesis of (R)-Boc-(Fmoc)-aminoglycine
J . Org. Chem., Vol. 65, No. 20, 2000 6599
(R)-Ben zyloxycar bon ylam in o-ter t-bu toxycar bon ylam i-
n oa cetic a cid , [(R)-Boc-(Cbz)-Agl-OH] (5). Compound 4 (1.0
g, 3.22 mmol) was dissolved in DMF (3.2 mL). Solid PDC (3.64
g, 9.67 mmol) was then added, and the reaction mixture was
stirred for 20 h at room temperature. The reaction mixture
was then poured into water (100 mL), acidified to pH 3, and
extracted with EtOAc (4 × 30 mL). The organic phase was
washed with water. Upon the addition of a solution of
saturated NaHCO3 solution (3 × 30 mL), the product trans-
ferred to the aqueous phase which was washed once with
EtOAc and then acidified with solid NaHSO4 to pH 3. The
precipitated product was then extracted with EtOAc (3 × 30
mL), and the combined extracts were washed with water, dried
over anhydrous MgSO4, and evaporated to yield 5 (596 mg,
scribed for compound 2. Compound 10 was dissolved in a small
amount of hot EtOAc and precipitated with ether resulting in
2.45 g (61%) pure product: mp 187-189 °C (EtOAc/Et2O);
[R]25D ) -107.1° (c ) 1, MeOH); 1H NMR (DMSO) 1.39 (s, 9H);
2.48-2.50 (m, 1H); 3.96-4.00 (m, 1H); 4.42-4.46 (m, 1H);
5.27-5.31 (m, 1H); 7.82 (d, 1H, J ) 8.4 Hz); 8.21 (s, 1H).
Elemental composition calculated for C8H14N2O4: C, 47.52%;
H, 6.98%; N, 13.85%; found: C, 47.59%; H, 6.78%; N, 13.81%.
(S)-3-Ben zyloxyca r bon yla m in o-4-ter t-bu toxyca r bon yl-
a m in o-2-oxa zolid on e (11). Compound 10 (2.02 g, 10 mmol)
was dissolved in THF (30 mL) and Et3N (10 mL). The reaction
mixture was cooled under argon to -40 °C. A solution of benzyl
chloroformate, 95% pure (3.92 g, 20 mmol), in THF (3.3 mL)
was slowly added at -40 °C. The stirred reaction mixture was
allowed to warm to -15 °C within 40 min and then kept at
-15 °C overnight. The reaction mixture was then quickly
neutralized with a stoichiometric amount of cold 5% aqueous
solution of NaHSO4 and extracted with EtOAc (3 × 50 mL).
The combined organic extracts were washed, dried, and
evaporated similarly to 4. The solid residue was dissolved in
a small amount of hot EtOAc and diluted with a large volume
of ether. White crystals of the product started to precipitate
within 10 min. The precipitate was cooled at 4 °C for 1 h before
filtration of the crystals. The filtrate was concentrated under
reduced pressure, and a second crop of product was isolated.
59%): mp 149-150 °C; [R]25 ) +5.5° (c ) 2, DMF); 1H NMR
D
(DMSO) 1.38 (s, 9H); 5.04 (s, 2H); 5.26-5.28 (m, 1H); 7.30-
7.36 (m, 6H); 7.83-7.85 (m, 1H), 12.95 (bs, 1H). Elemental
composition calculated for C15H20N2O6: C, 55.55%; H, 6.22%;
N, 8.64%; found: C, 55.62%; H, 6.04%; N, 8.54%.
Deter m in a tion of th e Op tica l P u r ity of 5. Palladium on
activated carbon (10%) (1 mg) was added to a solution of 5 (12
mg) in methanol (0.5 mL). Hydrogenation was performed at
50 psi for 30 min. The above solution (5 µL) was then added
to the solution of the Marfey’s reagent (1 mg) in acetone (0.1
mL) and 10 µL of Et3N. The reaction mixture was heated for
1 h at 40 °C to give a quantitative yield of 6. Then the above
solution (5 µL) was injected on the HPLC column. At a flow
rate of 1 mL/min, and a gradient of MeCN from 0 to 60% in
25 min, retention times for the (S,S) diastereomer was 12.7
and 14.0 min for the (R,S) diastereomer. Integration of the
two absorbances (λ ) 340 nm) corresponding to the diastereo-
isomeric derivatives 6 of tert-butoxycarbonylaminoacetic acid
showed 97% ee for 5.
(R)-ter t-Bu toxyca r bon yla m in o-flu or en ylm eth oxyca r -
bon yla m in oa cetic Acid , [(R)-Boc-(F m oc)-Agl-OH] (7).
Compound 5 (324 mg, 1.0 mmol) was dissolved in methanol
(7 mL), and palladium on activated carbon (10% Pd) (200 mg)
was added. The reaction flask was flushed with hydrogen, and
then hydrogenation was performed at 1 psi for 30 min at 0 °C
with the reaction flask immersed in ice/water. A solution of
Fmoc-OSu (505 mg, 1.5 mmol) was then dissolved in DMF (2
mL) and was added to the reaction mixture. The slow addition
(20 min) of Et3N (101 mg, 1 mmol) diluted with THF (0.5 mL)
followed. The reaction mixture was stirred at 0 °C for 1 h and
at room temperature for an additional 1 h. The solvents were
evaporated, and the residue was suspended in water and
extracted with EtOAc (3 × 30 mL). The combined extracts were
washed, dried, and evaporated similar to 4. The solid residue
containing 7 and fulvene was triturated with a small amount
of diethyl ether, filtered, washed with diethyl ether over a
The total yield of 11 was 3.17 g, 94%: mp 141-143 °C (EtOAc/
1
Et2O); [R]25 ) +23.4° (c ) 1, MeOH); H NMR (DMSO) 1.35
D
(s, 9H); 4.03-4.06 (m, 1H); 4.49-4.53 (m, 1H); 5.21-5.26 (m,
2H), 5.70-5.80 (m, 1H); 7.32-7.41 (m, 5H); 8.06 (d, 1H, J )
6.6 Hz). Elemental composition calculated for C16H20N2O6: C,
57.14%; H, 5.99%; N, 8.33%; found: C, 57.40%; H, 5.96%; N,
8.36%.
(S)-2-H yd r oxy-1-ter t-b u t oxyca r b on yla m in oet h ylca r -
ba m ic Acid Ben zyl Ester (12). Compound 11 (1.68 g, 5.0
mmol) was dissolved quickly in warm toluene (100 mL) and
cooled to -45 °C. Benzyltrimethylammonium hydroxide (2.5
mL of 40% solution in methanol, 5.5 mmol) was added over
10 min to the suspension of 11 in toluene and stirred for an
additional 45 min at -45 °C. Acetic acid (0.5 mL) was then
added, the reaction mixture was warmed to room temperature,
and the solvent was evaporated under reduced pressure. The
residue was dissolved in EtOAc (50 mL), which was washed,
dried, and evaporated similarly to 4. The solid residue was
subjected to crystallization from EtOAc to yield 12 (68%): mp
1
139-141 °C (EtOAc); [R]25 ) -8.0° (c ) 1, MeOH); H NMR
D
(DMSO) 1.37 (s, 9H); 3.30-3.37 (m, 2H); 4.79 (t, 1H, J ) 6.0
Hz); 4.96-4.99 (m, 1H); 5.01 (s, 2H); 6.82 (bs, 1H); 7.26 (d,
1H, J ) 6.1 Hz); 7.29-7.36 (m, 5H). Elemental composition
calculated for C15H22N2O5: C, 58.05%; H, 7.15%; N, 9.03%;
found: C, 58.26%; H, 6.86%; N, 8.90%.
filter, and dried under vacuum to yield 7 (363 mg, 88%): mp
Syn th esis of (R,S)-ter t-Bu toxyca r bon yla m in o-flu or en -
ylm eth oxyca r bon yl-a m in oa ceta m id e [(R,S)-Boc-(F m oc)-
Agl-NH2] (13). To a stirred solution of (R,S)-Boc-(Fmoc)-Agl-
OH10,12 (10.3 g, 25 mmol) in anhydrous DMF (40 mL) and
EtOAc (100 mL) at 0 °C was added DCC (5.3 g, 25 mmol),
followed by HOBt (3.5 g, 27 mmol) 5 min later. The mixture
was slowly warmed to room temperature and stirred for an
additional 30 min. After activation, 10 M cc. NH3 solution in
water (2.8 mL, ∼28 mmol) was added to the reaction mixture,
with stirring, at room temperature for 30 min. The reaction
mixture was then diluted to 400 mL with EtOAc. The
precipitate (DCU) was filtered off, and the filtrate was washed
with 5% NaHSO4 solution, H2O, 5% KHCO3 solution, and H2O.
As DMF was removed from the EtOAc phase with aqueous
washes, some precipitation of Boc-(Fmoc)-Agl-NH2 occurred
because of poor solubility in EtOAc. Boc-(Fmoc)-Agl-NH2 was
kept in solution by the addition of EtOAc and dried for a short
time over anhydrous Na2SO4 to avoid precipitation of the
product onto the Na2SO4 crystals. After filtration the solution
was concentrated to 1/3 of its original volume; the suspension
was allowed to stand at 4 °C overnight to yield 9.5 g (92%) of
(R,S)-Boc-(Fmoc)-Agl-NH2 as white powder: TLC Rf 0.7 (CHCl3:
MeOH:AcOH ) 90:10:0.5); MS FAB: m/e 412.03 (M + H),
calcd: 412.19 (M + H); tR ) 3.3 min for the amide and 4.0
min for the starting material (acid) determined by HPLC under
1
190 °C dec (EtOAc); [R]25 ) +10.5° (c ) 2, DMF); H NMR
D
(DMSO) 1.39 (s, 9H); 4.22-4.28 (m, 3H); 5.26-5.29 (m, 1H);
7.30-7.35 (m, 3H); 7.40-7.43 (m, 2H); 7.72-7.74 (m, 2H)7.88-
7.90 (m, 2H), 8.02 (d, 1H, J ) 7.3 Hz). Elemental composition
calculated for C22H24N2O6: C, 64.07%; H, 5.87%; N, 6.79%;
found: C, 63.74%; H, 5.75%; N, 6.60%.
Deter m in a tion of th e Op tica l P u r ity of 7. Compound 7
(20 mg) was dissolved in DMF (0.1 mL), DABCO (18 mg) was
added, and the reaction mixture was stirred at room temper-
ature for 30 min. The above solution (3.5 µL) was then added
to the solution of Marfey’s reagent (1 mg) in acetone (0.1 mL)
and treated as described earlier. Integration of the two
absorbances corresponding to diastereoisomeric derivatives 6
of tert-butoxycarbonylaminoacetic acid showed a 97% ee for
7.
(S)-4-ter t-Bu toxyca r bon yla m in o-2-oxa zolid on e (10).24
(S)-Boc-serine 9 (4.10 g, 20 mmol) and DPPA (6.05 g, 22 mmol)
were dissolved in t-BuOH (80 mL). The reaction mixture was
flushed with argon, and Et3N (2.77 g, 22 mmol) was added.
The reaction conditions and workup were the same as de-
(24) Gordon, E. M.; Ondetti, M. A.; Pluscec; J elka; Cimarusti, C.
M.; Bonner, D. P.; Sykes, R. B. J . Am. Chem. Soc. 1982, 104, 6053-
6060.

6600 J . Org. Chem., Vol. 65, No. 20, 2000
Sypniewski et al.
isocratic condition (48% MeCN in 0.1% TFA) at a flow rate of
2.0 mL/min.
g, 100 mmol) in DMF (150 mL) was stirred in a tightly closed
flask at room temperature for 10 min. Periodically the CO2
that was produced was vented. The mixture was then cooled
to 4 °C (ice bath), and CH3I (9.35 mL, 150 mmol) was added
to it and closed tightly. The stirring was continued in an ice
bath for 2 h followed by 2 h at room temperature. The reaction
was monitored by HPLC with a gradient running from 30%
to 54% MeCN over 20 min at a flow rate of 1.0 mL/min. When
the reaction was complete, the solid was filtered and washed
with DMF. The filtrate was evaporated to dryness in a vacuum
and crystallized from ether/petroleum ether to yield 14
(>90%): mp 104-106 °C, MS FAB: m/e 427.00 (M + H),
calcd: 427.19 (M + H).
P a p a in Hyd r olysis of 13.25 Activation of papain:
A
mixture of EDTA (0.01 mol) in water (1000 mL; pH adjusted
to 6.2 with 1 M NaOH), 0.05 mol cystein‚HCl in water (1000
mL, pH adjusted to 6.2 with 1 M NaOH), H2O (240 mL, MiliQ
pure), and 0.06 mol of mercaptoethanol (10 mL) were used to
dissolve and activate 1 g of papain at 25 °C for 2 h.
Enzymatic hydrolysis: To a filtered solution of 13 (50 g, 122
mmol) in DMF (850 mL) and MeCN (1400 mL, HPLC grade)
was added the activated papain solution (2250 mL) which was
gently shaken under argon atmosphere at 37 °C for 17-20 h.
The hydrolysis was monitored by RP-HPLC. After 19 h, the
mixture contained 50% amide and 50% acid according to
HPLC. This ratio was not modified with either additional
enzyme or reaction time. After evaporation of the MeCN, the
pH of the reaction mixture was adjusted to 7.5-8.0 with 1 M
NaOH, and the resulting solution was further diluted with
water then left overnight at 0 °C, resulting in the precipitation
of the original amide. Unhydrolyzed, chiral (S)-Boc-(Fmoc)-
Agl-NH2 (20.8 g, 42%) was collected by filtration: mp 162-6
P a p a in Hyd r olysis of 14.25 Using conditions similar to
those used for the papain hydrolysis of 13, compound 14 (21.5
g, 50 mmol) was suspended and stirred in a phosphate buffer
(pH ) 6.2) consisting of 0.1 M Na2HPO4‚7H2O and 0.1 M citric
acid with 1.5 mM EDTA.Na2 in water (300 mL) and 50% DMF/
MeCN (80 mL). Activated aliquots of papain (100 mg) were
added at regular intervals (12-24 h) until completion of
hydrolysis monitored by HPLC. Solid (S)-Boc-(Fmoc)-Agl-OMe
(9 g) was filtered after dilution of the above with water (900
mL). MS FAB: m/e 427.00 (M + H), calcd: 427.19 (M + H).
Filtrate was treated as above (evaporation of MeCN, extraction
with EtOAc and acidification) to yield (R)-Boc-(Fmoc)-Agl-OH
7 (10 g): mp 191-193 °C (dec); MS FAB: m/e 412.99 (M + H),
calcd: 413.17 (M + H); [R]25D ) +9.8° (c ) 2, DMF). The optical
purity of 7, tested as earlier, showed (R)-Boc-(Fmoc)-Agl-OH
to be in 95% enantiomeric excess (ee).
°C (dec); [R]25 ) -6.5° (c ) 2, DMF), MS FAB: m/e 412.03
D
(M + H), calcd: 412.19 (M + H). The aqueous solution was
extracted with EtOAc (to separate any remaining amide) and
acidified to pH 2 with solid NaHSO4. The solution was left
standing at 0 °C overnight to yield the hydrolyzed chiral (R)-
Boc-(Fmoc)-Agl-OH 7 (24 g, 48%) after filtration: mp 185-7
°C (dec), MS FAB: m/e 413.01 (M + H), calcd: 413.17 (M +
H); [R]25 ) +8.5° (c ) 2, DMF). The optical purity of chiral
D
(R)-Boc-(Fmoc)-Agl-OH 7 was tested as described earlier. Ratio
of the integration of the two absorbances showed one of the
two diastereomers of 7 to be in 92% enantiomeric excess (ee).
Syn th esis of (R,S)-ter t-Bu toxyca r bon yla m in o-flu or en -
ylm eth oxyca r bon yla cetic Acid Meth yl Ester [(R,S)-Boc-
(F m oc)-Agl-OMe]15 (14). A mixture of (R,S)-Boc-(Fmoc)-Agl-
OH10,12 (21.0 g, 50 mmol) and powdered anhydrous K2CO3 (10.0
Ack n ow led gm en t. Research supported by NIH
grants HD-1-3527, DK-26741, DK-50124, and the Hearst
Foundation. The authors thank J udit Erchegyi and
Ryan DeBoard for careful editing of the manuscript and
duplication of some synthetic steps, and Debbie Doan
for manuscript preparation. We are indebted to A.G.
Craig, T. Goedken and Ryan DeBoard for mass spectral
analyses.
(25) Clapes, P.; Valverde, I.; J aime, C.; Torres, J . L. Tetrahedron
Lett. 1996, 37, 417-418.
J O000736E