Synthesis of small glycopeptides by decarboxylative condensation and insight into the reaction mechanism

Article abstract of DOI:10.1021/jo802278w

The chemical synthesis of homogeneous glycoproteins and glycopeptides facilitates progress toward understanding the functional role of carbohydrates attached to proteins and is important in the preparation of glycopeptide-based therapeutics. A series of protected and unprotected glycosyl dipeptides, glycopeptide I, which contained the α-ketoacid moiety at the C-terminus, were synthesized and ligated with a series of O-iert-butyl-proteeted N-hydroxylamino acids to afford O-tert-butyl-proteeted glycosyl tripeptides, glycopeptide II. The reactions were carried out under both anhydrous and aqueous conditions at neutral pH to produce glycopeptide products in yields ranging from 15% to 86% depending on the amino acids present at the ligation junction. The best yields were obtained when both the α-ketoacid and the N-hydroxylamino acid contained medium-sized side chains. In addition to the expected tripeptide product, 2,5-substituted oxazoles were isolated when O-tert-butyl protected N-hydroxylamines of glycine were employed in the reaction. The formation of the oxazole is believed to result from an intramolecular cyclization of the O-tert-butyl ester on a nitrilium ion intermediate followed by aromatization. A decarboxylative condensation between O¹⁸-labeled phenyl pyruvic acid and N-hydroxyphenethylamine oxalate salt resulted in amide products lacking the O¹⁸-label, providing further support for the nitrilium ion in the reaction pathway.

Full text of DOI:10.1021/jo802278w

Synthesis of Small Glycopeptides by Decarboxylative Condensation
and Insight into the Reaction Mechanism
Aditya K. Sanki, Rommel S. Talan, and Steven J. Sucheck*
Department of Chemistry, The UniVersity of Toledo, 2801 W. Bancroft Street, Toledo, Ohio 43606
steVe.sucheck@utoledo.edu
ReceiVed October 10, 2008
The chemical synthesis of homogeneous glycoproteins and glycopeptides facilitates progress toward
understanding the functional role of carbohydrates attached to proteins and is important in the preparation
of glycopeptide-based therapeutics. A series of protected and unprotected glycosyl dipeptides, glycopeptide
I, which contained the R-ketoacid moiety at the C-terminus, were synthesized and ligated with a series
of O-tert-butyl-protected N-hydroxylamino acids to afford O-tert-butyl-protected glycosyl tripeptides,
glycopeptide II. The reactions were carried out under both anhydrous and aqueous conditions at neutral
pH to produce glycopeptide products in yields ranging from 15% to 86% depending on the amino acids
present at the ligation junction. The best yields were obtained when both the R-ketoacid and the
N-hydroxylamino acid contained medium-sized side chains. In addition to the expected tripeptide product,
2,5-substituted oxazoles were isolated when O-tert-butyl protected N-hydroxylamines of glycine were
employed in the reaction. The formation of the oxazole is believed to result from an intramolecular
cyclization of the O-tert-butyl ester on a nitrilium ion intermediate followed by aromatization. A
decarboxylative condensation between O18-labeled phenyl pyruvic acid and N-hydroxyphenethylamine
oxalate salt resulted in amide products lacking the O18-label, providing further support for the nitrilium
ion in the reaction pathway.
Introduction
e40 amino acids.6 If larger peptides are needed or if chemoen-
zymatically derived glycopeptides are to be joined, chemical
methods for coupling glycopeptide fragments are frequently
required. The current standard reactions for joining two peptide
segments together to create a longer native peptide have
overwhelmingly been the native chemical ligation (NCL)7 and
more recently the application of Staudinger reaction8 in the form
Significant efforts have been made to access homogeneous
glycoproteins and glycopeptides over the past two decades.1
Chemical synthesis has the potential to fulfill this need, which
facilitates progress toward understanding the functional role of
carbohydrates attached to a particular protein2 and for the
preparation of glycopeptide- or glycoconjugate-based therapeu-
tics.3,4 Currently, solid-phase peptide synthesis methods5 are
considered practical for the preparation of peptides containing
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1997, 289, 221–245. (b) Dwek, R. A.; Butters, T. D. Chem. ReV. 2002, 102,
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1886 J. Org. Chem. 2009, 74, 1886–1896
10.1021/jo802278w CCC: $40.75 2009 American Chemical Society
Published on Web 01/30/2009

Glycopeptide Synthesis by DecarboxylatiVe Condensation
of the traceless Staudinger ligation.9 The reactivity of thioesters
used in the NCL provides practical ways to access full-length
proteins and glycoproteins. The potential of these powerful
techniques was recently demonstrated in the synthesis of a HIV
protease covalent dimer7t containing 203 amino acids in the
sequence. While each of these reactions has enjoyed great
success, each reaction has intrinsic limitations: the former has
an absolute reliance on a cysteine residue, present in <1.7% of
all residues in globular proteins,7h,10 at the ligation site, and
the latter encounters issues of solubility of the phosphinothiol
reagent in aqueous media9f and is most effective when one of
the two residues at the ligation junction is glycine.9e,f To bypass
the cysteine specific limitation of the NCL, a variety of
innovative auxiliaries have been developed.7c,f-i,k,l,o,r,s While
auxiliary-based efforts can deliver full-length peptides or
glycopeptides, they often require a less hindered amino acid
residues at the ligation junction for efficient ligation. In the
second phase of auxiliary development, a new approach
consisting of a glycopeptide segment with the sugar bearing
acetamidomethyl-thio-handle11 at its C-2 position has been
effective in delivering longer O-linked and N-linked glycopep-
tides and glycoproteins. The latter works by way of the thio-
auxiliary in the form of a sugar-assisted ligation. In the pursuit
of more general chemistry for glycopeptide synthesis, thiol-
auxiliary-free cysteine-free coupling protocols have been de-
veloped. In this regard, amide ligation by decarboxylative
FIGURE 1. General concept for decarboxylative condensation.
condensation between a hydroxylamine and a peptide bearing
an R-ketoacid moiety at C-terminus,12 metal using silver chloride
(AgCl) and metal-free (tris(2-carboxyethyl)phosphine, TCEP)
mediated coupling,13 and small peptide and thioacid-2,4-
dinitrobenzenesulfonamide couplings14 represent the latest
alternatives to cysteine-based and auxiliary-based ligation
protocols. Although the AgCl-assisted phenolic ester directed
amide coupling (PEDAC-AgCl) or PEDAC-TCEP13 has been
a reliable protocol for the synthesis of complex glycan-
containing polypeptides, it is not without limitation and an issue
of racemization has been noted. Moreover, compatibility of
thioacid-2,4-dinitrobenzenesulfonamide couplings14 has not been
extended to the context of glycopeptide synthesis, and chemose-
lectivity is an issue. Emerging chemistry that is capable of
linking two glycopeptide segments should in principle (a) avoid
amino-acid-specific limitations at the ligation junction, (b) be
water-tolerant, (c) be devoid of restricted access to peptide
thioesters,7u and (d) possess high chemoselectivity.
In this paper we describe the ligation of glycopeptide
fragments by way of the decarboxylative condensation reaction,
an amide ligation reported by Bode.12a-c We were intrigued
by this reaction because of its independence from thiol capture
methods, lack of requirement for external reagents, “traceless”
nature, water tolerance, and reported chemoselectivity. We
envisioned that this unique condensation chemistry would prove
ideal for ligating water-soluble, unprotected, and pH-sensitive
glycopeptides. To demonstrate the chemistry, we prepared
glycosyl dipeptides with C-terminal R-ketoacid moieties and a
series of amino acids containing N-hydroxylamino moieties. The
two components were used to study the amino acid requirements
at the ligation junction and evaluate the chemoselectivity in the
presence of unprotected glycans. The general concept for the
ligation is illustrated in Figure 1.
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Results and Discussion
To explore both the compatibility of the decarboxylative
condensation with glycans and systematically explore the amino
acid side chain requirement of the ligation junction a series of
keyintermediatesAc3GalNAc-R-O-Thr-Gly-C2O3H1,Ac3GalNAc-
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J. Org. Chem. Vol. 74, No. 5, 2009 1887

Sanki et al.
conditions (Scheme 1). Without further purification, cyano-
phosphoranes 13-15 having residual piperidine were coupled
with 7 in the presence of 2-(1H-benzotriazole-1-yl)-1,1,3,3-
tetramethyuronium hexafluorophosphate (HBTU), 1-hydroxy-
benzotriazole (HOBt), and 2,4,6-trimethylpyridine (TMP)20 in
DMF-CH2Cl2 (1:1) to generate cyano ketophosphorane deriva-
tives 16, 17, and 18 in 71%, 61%, and 60% yields, respectively.
During the HBTU condensation, a piperidine adduct A, Sup-
porting Information, was isolated in 25% yield in each case as
the byproduct. Cyano ketophosphorane derivatives 16, 17, and
18 were oxidized by dimethyldioxirane (DMDO) in acetone-
H2O21,22 to generate R-ketoacids 1, 2, and 3 in quantitative yield,
respectively (Scheme 1).
FIGURE 2. Key intermediates for amide ligation.
SCHEME 1. Synthesis of Glycosyl Dipeptide r-Ketoacids 1,
2, and 3 and Valine Hydroxyl Amine 6a
Having obtained the key R-ketoacid components 1, 2, and 3,
we next prepared N-hydroxylamines 4,12a,15 5,15 and 6. N-
Hydroxylamines 4 and 5 were prepared by the reported literature
protocols.12a,15 N-Hydroxyvaline derivative 6 was unknown and
was synthesized from valine tert-butyl ester 19. Protected valine
was alkylated with 2-bromoacetonitrile12a,15 to afford 20 in 64%
yield. Compound 20 was quantitatively converted to 6, via its
nitrone derivative 21 by oxidation with mCPBA followed by
aminolysis in the presence of hydroxylamine hydrochloride
(Scheme 1).
With R-ketoacids 1, 2, and 3 in hand, we condensed each of
them separately with appropriate N-hydroxyamino acid 4, 5, or
6 in anhydrous DMF or 5:1 DMF-water at 35-40 °C (Scheme
2). The results of decarboxylative condensations are summarized
in Table 1. Compound 1 with a glycine residue at the C-terminus
was reacted with a N-hydroxyglycine ester 4 in anhydrous DMF
at 40 °C to generate glycosyl tripeptide 22 and an oxazole
byproduct 23 in 41% and 22% yields, respectively (entry 1;
Table 1). Similarly, 45% and 22% yields, respectively, of 22
and 23 were isolated from the reaction between 1 and 4 at 40
°C in 5:1 DMF-water (entry 2; Table 1). Next, a slightly bulkier
N-hydroxyalanine ester 5 was reacted with 1 in both anhydrous
and aqueous DMF under the general reaction condition and
glycosyl tripeptide 24 was obtained in 40% and 23% yields,
respectively after 25 h (entries 3 and 4; Table 1). To our surprise
no oxazole byproduct formed under both anhydrous and aqueous
conditions. Next, we wished to employ this mild reaction
condition in a relatively hindered junction such as Ala-Gly,
Ala-Ala, or Gly-Val to ensure influence of the size of amino
acid side chain on the outcome of ligation. Therefore, R-ketoacid
2 bearing an alanine residue at the C-terminus was treated with
N-hydroxyglycine ester 4 in anhydrous DMF at 42 °C, and we
isolated glycopeptide 25 and an oxazole byproduct 26 in 43%
and 23% yields, respectively, in 6 h. In an effort to improve
yield, the latter reaction was carried out at room temperature,
and after 24 h, both 25 and 26 were isolated in 39% and 29%
yields, respectively. Similarly, in 5:1 DMF-water, reaction of
R-ketoacid 2 with 4 provided 25 and 26 in 46% and 23% yields,
respectively, after 48 h (entries 5, 6 and 7; Table 1). At this
point we were curious to know the outcome of ligation at the
“Ala-Ala” junction. Hence, R-ketoacid 2 was condensed with
N-hydroxylamine 5 in both anhydrous and aqueous DMF to
furnish 86% and 54% yields in 38 and 48 h, respectively (entries
8 and 9; Table 1). Interestingly, no oxazole byproduct formed
a Compounds 1-3 and 13-15 formed quantitatively and were used as
crude materials without further purification.
R-O-Thr-Ala-C2O3H 2, and Ac3GalNAc-R-O-Thr-Val-C2O3H 3
were prepared. To explore the side chain requirement of
N-hydroxyamine components, N-hydroxyglycine tert-butyl ester
(4),12a,15 N-hydroxyalanine tert-butyl ester (5),15 and N-hy-
droxyvaline tert-butyl ester (6) were also prepared (Figure 2).
Glycosyl dipeptides 1-3 bearing C-terminal R-ketoacid
moieties were prepared by the reaction of a known glycosy-
lamino acid building block 716 (Figure 2) with glycine cyano-
phosphorane 13, alanine cyanophosphorane 14,17 and valine
cyanophosphorane 15, respectively (Scheme 1). Hitherto un-
known 13 and 15 were prepared by the same reaction conditions
employed for the synthesis of 14.17 Commercially available
Fmoc-protected glycine 8 was coupled with (cyanomethylen-
e)triphenylphosphorane 1018 in the presence of 1-ethyl-3-(3′-
dimethylaminopropyl)carbodiimide (EDCI) and 4-dimethylami-
nopyridine (DMAP) to afford Fmoc-protected glycine cyano-
phosphorane 11 in 79% yield. The Fmoc group of 11 was
subsequently removed by treatment with piperidine19 at ambient
temperature to furnish 13 as a crude material. Similarly, valine
cyanoketophosphorane 15 was synthesized quantitatively by the
condensation of 9 and 10, via 12, by the same reaction
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1888 J. Org. Chem. Vol. 74, No. 5, 2009

Glycopeptide Synthesis by DecarboxylatiVe Condensation
SCHEME 2. Decarboxylative Condensation Involving Protected Glycosyl Dipeptide r-Ketoacids and N-Hydroxyamino Acidsa
a Yields in parenthesis are from aqueous conditions.
TABLE 1. Products from Decarboxylative Condensation Involving Different Ligation Junctions
products
byproducts
yields (%)h
entry
R-ketoacids
N-hydroxyamines
reaction conditions
yields (%)h
1
2
3
4
5
6
7
8
R1 ) Ha
R1 ) Ha
R1 ) Ha
R1 ) Ha
R1 ) Meb
R1 ) Meb
R1 ) Meb
R1 ) Meb
R1 ) Meb
R1 ) Ha
R1 ) iPrc
R2 ) Ha
R2 ) Ha
R2 ) Meb
R2 ) Meb
R2 ) Ha
R2 ) Ha
R2 ) Ha
R2 ) Meb
R2 ) Meb
R2 ) iPrc
R2 ) iPrc
DMF, 24 hd
22 (41)
22 (45)
24 (40)
24 (23)
25 (43)
25 (39)
25 (46)
27 (86)
27 (54)
28 (15)
23 (22)
23 (22)
DMF/H2O (5:1), 15 hd
DMF, 25 hd
DMF/H2O (5:1), 25 hd
DMF,6 he
26 (23)
26 (29)
26 (23)
DMF, 24 hf
DMF/H2O (5:1), 48 hf
DMF, 38 hf
9
10
11
DMF/H2O (5:1), 48 hd
DMF, 19 hd
DMF, 36 hg
a Glycine. b Alanine. c Valine. d 35-40 °C. e 40-42 °C. f 20-25 °C. g 40-60 °C. h Isolated yield.
in either medium. Condensation of 1 with bulkier N-hydroxy-
valine ester 6 was low yielding, and 15% yield of the desired
glycosyl tripeptide 28 was isolated in anhydrous DMF after 19 h.
Again, we did not observe oxazole formation in this case. In
general we isolated the expected glycosyl tripeptides in moderate
to high yields from the decarboxylative condensation with
protected glycosyl dipeptide R-ketoacids with the exception of
the ligation involving glycosyl R-keto acid 3 and valine
hydroxylamine 6 for “Val-Val” junction (entry 11; Table 1,
Scheme 2) at the ligation site. In the latter case, reaction did
not proceed after 36 h under the general reaction condition
employed. Our efforts to initiate reaction by raising temperature
or by using p-toluenesulfonic acid (TsOH) as a catalyst did not
help.
With a notion to validate the chemoselectivity of this efficient
protocol for amide bond formation, next acetyl groups of 16
were deprotected in the presence of ammonia (1 N) in anhydrous
MeOH at 0 °C (Scheme 3) to provide the desired trihydroxy
derivative 29 in 56% yield accompanied by a small amount of
partially deprotected 6-O-acetyl derivative 30 (15% yield).
J. Org. Chem. Vol. 74, No. 5, 2009 1889

Sanki et al.
SCHEME 3. Synthesis of Deprotected Glycopeptide and Chemoselective Ligationa
a Conversion of 29 to 31 was quantitative, and the reaction mixture was concentrated and used without further purification. For decarboxylative condensation
of 31 with 4 under aqueous condition (5:1 DMF/H2O, 40 °C, 24 h), only 32 could be isolated (25% yield).
Compound 29 was converted to R-keto acid 31 with DMDO in
quantitative yield. R-Ketoacid 31 was subjected to decarboxy-
lative condensation in the presence of 4 in anyhydrous DMF to
deliver the glycosyl tripeptide 32 and an oxazole byproduct 33
in 26% and 18% yields, respectively. Condensation of 31 with
4 was also carried out in 5:1 DMF-water, and the desired
glycosyl tripeptide 32 was isolated in 25% yield. Oxazole
byproduct 33 in the latter case formed in low quantity, and
isolation was difficult due to paucity of the materials (Scheme
3).
Compounds 6, 11, 12, 16-18, 20-30, 32, and 33 were
1
1
characterized by H NMR, H-1H gCOSY, 13C NMR and
HRMS. DEPT experiments were carried out on a few of the
compounds to verify the number of methylene groups present
in the molecules.
1
Appearance of isolated singlets in H NMR of 23, 26, and
33 at δ ) 6.1, 6.13, and 6.24 ppm and carbons at δ ) 107.59,
107.41, and 108.93 ppm, respectively, in 13C NMR were
characteristics of the oxazole byproducts. The assignment of
the H-4 attached to the oxazole moiety was unambiguously
determined by carrying out the gHMQC NMR experiment on
33. The characteristic region of the gHMQC of 33 showing the
correlation between C-4 and H-4 of the oxazole moiety is
depicted in Figure 3.
On the basis of these results it is clear that hindered
C-terminal amino acids are much less reactive than the
moderately sized amino acids. Our outcome of ligations at
Gly-Ala and Ala-Ala junctions are consistent with those
known in the literature.7o,r,11
Next, we revisited the reported mechanism of the decarboxy-
lative condensation to rationalize the formation of the oxazoles
(23, 26, or 33). Two pathways, A and B, have been previously
proposed to arrive at amide products (Scheme 4).12a Common
to each pathway is the initial formation of a hemiaminal. In
pathway A, the hemiaminal loses a molecule of CO2 and water
to provide an imidic acid which undergoes tautomerisation to
afford the desired glycosyl tripeptides (dotted arrows; Scheme
4).
A mechanism arising from an intermediate along this pathway
leading to oxazoles is not readily apparent. A second plausible
mechanism for amide formation is illustrated along pathway
B. In this pathway the hemiaminal loses a molecule of water to
afford an (E/Z) mixture of nitrones. Loss of molecule of CO2
and water from the (E) nitrone would be expected to give rise
to a reactive nitrilium ion. The nitrilium species is in the right
setting to rapidly cyclize via attack of the tert-butyl ester
FIGURE 3. Characteristic region of gHMQC of oxazole byproduct
33.
carbonyl followed by aromatization to provide an oxazole
byproduct (dotted arrows; Scheme 4). The nitrilium ion could
also undergo addition of a water molecule to generate the imidic
acid of pathway A. Previous studies using various nucleophiles
failed to trap the speculated nitrilium intermediate suggesting
pathway A was the operative mechanism.12a Isolation of
oxazoles suggests that a nitrilium ion might be the key
intermediate in the decarboxylative ligation (vida infra). As it
appears, oxazole byproduct formed readily in the case of
Gly-Gly or Ala-Gly ligation junction. Exclusive isolation of
glycosyl tripeptide in the case of ligation between Ala-Ala and
Gly-Val suggests a role of steric bulk at the hydroxylamine
bearing amino acid. It is apparent that formation of an oxazole
byproduct is favored via a nitrilium ion intermediate when the
hydroxylamine residue at the ligation site is Gly. In addition to
this observation couplings were most effective when either
amino acid at the ligation junction lacked ꢀ-branching.
To further distinguish between path A and B, a model study
was performed involving decarboxylative condensation of
unlabeled and O18-labeled phenyl pyruvic acids 35 and 36,
respectively, with N-hydroxyphenethylamine oxalate salt 34.12a
In doing so, we synthesized N-hydroxyphenethylamine oxalate
salt 34 (Scheme 5) under the reaction conditions analogous to
the synthesis of 11. Commercially available phenyl pyruvic acid
1890 J. Org. Chem. Vol. 74, No. 5, 2009

Glycopeptide Synthesis by DecarboxylatiVe Condensation
To our surprise, amide 37 [m/z ) 240.2 (M + H)+] was
obtained with complete loss of label in 51% yield. Initially we
thought that loss of O18-oxygen in 3712a could be either the result
of an exchange with H2O16 or a problem of detection by the
mass spectrometer used. We verified the result by carrying out
an alternative synthesis where commercially available benzyl
cyanide 38 was hydrolyzed in a mixture of H2O18 and anhydrous
THF in the presence of HCl.24 The labeled carboxylic acid 39
[m/z ) 163 (M + Na)+] was condensed with commercially
available phenethylamine 40 in the presence of dicyclohexyl-
carbodiimide (DCC), and the crude product 41 (Scheme 5) was
analyzed by mass spectrometry.
SCHEME 4. Proposed Mechanisms Leading to Amides and
Oxazoles in the Decarboxylative Condensation
Compound 41 showed a peak at m/z ) 242.4 [M + H]+
indicating complete retention of O18-oxygen. The result suggests
that the O18-labeled amide is fairly stable once formed and does
not exchange with O16 in the mass spectrometry employed and
amide 37 loses its labeling during the amidation reaction.
Loss of labeling in 37 supports pathway B as the operative
mechanism for the decarboxylative condensation of N-hydroxy-
lamines and R-ketoacids.
In summary, the importance of this synthetic strategy for the
ligation of glycopeptide fragments stems from the fact that the
ligation proceeds under neutral aqueous conditions and occurs
at low molar concentrations. Decarboxylative ligation is proven
to be chemoselective in the presence of free hydroxyls. It is
now clear that conditions such as these would be useful for
joining unprotected glycopeptides that have been prepared by
chemoenzymatic methods. These studies are the first to take
advantage of the decarboxylative condensation to produce
glycopeptides. Isolation of oxazoles and isotope labeling experi-
ment on a model system provide reasonable evidence for a
nitrilium ion as the key intermediate in the decarboxylative
condensation. Work is in progress to explore its use in the
synthesis of glycopeptides of interest.
SCHEME 5. Isotope Labeling Experiment with O18-Labeled
Phenylpyruvic Acida
Experimental Section
General Methods. Amino acids and other fine chemicals were
purchased from commercial suppliers and were used without further
purification. All solvents used for reactions were dried following
the standard procedures.25 Thin-layer chromatography (TLC, silica
gel 60, f254) were performed in distilled solvents as specified and
visualized under UV light or by charring in the presence of 5%
H2SO4/MeOH. Flash column chromatography was performed on
1
silica gel (230-400 mesh) column using solvents as received. H
NMR were recorded on either a 400 or a 600 MHz spectrometer
in CDCl3, CD3OD, or DMSO-d6 using residual CHCl3, CH3OH,
or DMSO as internal references, respectively. 13C NMR were
recorded on either a 100.56 or a 150.83 MHz in CDCl3, CD3OD,
or DMSO-d6 using the triplet centered at δ77.273 for CDCl3, septet
centered at δ 49.0 for CD3OD, or septet centered at δ 39.5 for
DMSO-d6 as internal reference, respectively. 31P NMR was recorded
on a 161.9 MHz spectrometer using either CDCl3 or CD3OD as
a Compounds 39 and 41 were crude materials and no purification was
carried out.
1
1
solvent. H-1H gCOSY and H-13C gHMQC NMR were per-
formed on a 600 MHz spectrometer. Melting points of all crystalline
solids were determined using a capillary tube and are uncorrected.
High resolution mass spectrometry (HRMS) were performed on a
TOF mass spectrometer.
35 was used as an R-ketoacid. Compound 35 was first labeled
with O18-oxygen in the presence of H2O18 and 0.1 N HCl in
anhydrous THF to obtain 36 in 84% yield.23 Both 35 and 36
showed mass fragments of m/z ) 143.1 [M + Na - CO2]+ and
m/z ) 145.1 [M + Na - CO2]+, respectively, with a loss of
one molecule of CO2. Compound 35 was subjected to decar-
boxylative condensation in the presence of 34 in MeOH to afford
amide 37 [m/z ) 240.3 (M + H)+] in 41% yield (Scheme 5).
Next, O18-labeled 36 was reacted with hydroxylamine 34 under
the same reaction conditions.
Fmoc-Protected Glycine Cyanophosphorane 11. To a well-
stirred solution of Fmoc-protected glycine 8 (1.00 g, 3.36 mmol),
(24) Risley, J. M.; Van Etten, R. L. J. Am. Chem. Soc. 1980, 102, 4609–
4614.
(25) Armarego, W. L. F.; Chai, C. L. L. Purification of Laboratory Chemicals,
5th ed.; Butterworth-Heinemann: New York, 2003; pp 80-388.
(26) Murray, R. W.; Singh, M. Organic Synthesis; Wiley: New York, 1998;
Collect. Vol. IX; p 288; Org. Synth. 1997, 74, 91.
(23) Byrn, M.; Calvin, M. J. Am. Chem. Soc. 1966, 88, 1916–1922.
J. Org. Chem. Vol. 74, No. 5, 2009 1891

Sanki et al.
DMAP (0.04 g, 0.33 mmol), and EDCI (0.84 g, 4.37 mmol) in
CH2Cl2 (50 mL) was added (cyanomethylene)triphenylphophorane
10 (1.19 g, 3.53 mmol) at ambient temperature under N2 atmo-
sphere. The resulting solution was stirred at ambient temperature.
The reaction was monitored by TLC and appeared to stop after
20 h. The reaction mixture was diluted with CH2Cl2 (50 mL) and
washed successively with water (30 mL), saturated NaHCO3 (2 ×
30 mL) and water (30 mL). Combined organic phases were dried
(anhydrous Na2SO4) and filtered, and the filtrate was concentrated
to dryness under reduced pressure. Purification of the crude material
by silica gel flash column chromatography (10 × 5.5 cm) with 3:3:
14 and then 1:1:2 acetone/CHCl3/hexanes afforded the desired
product 11 as a fluffy mass, highly hygroscopic in nature: yield
1.51 g (79%); silica gel TLC Rf ) 0.45 (1.5:1 EtOAc/hexanes); 1H
NMR (600 MHz, CDCl3) δ 4.16 (t, 1H, J ) 7.2 Hz, Fmoc CH),
4.34 (d, 2H, J ) 7.2 Hz, Fmoc CH2), 4.42 (d, 2H, J ) 4.2 Hz,
R-CH2), 5.61 (br.s, 1H, NH), 7.25 (t, 2H, J ) 7.2 Hz, aromatic),
7.35 (t, 2H, J ) 7.8 Hz, aromatic), 7.53 (m, 6H, aromatic), 7.58
(m, 8H, aromatic), 7.64 (m, 3H, aromatic), 7.71 (d, 2H, J ) 7.8
Hz, aromatic); 13C NMR (150.83 MHz, CDCl3) δ 47.2, 47.7 (CH2),
66.8 (CH2), 119.9, 122.1, 122.7, 125.2, 127.0, 127.6, 129.3, 129.4,
133.5, 133.53, 133.56, 133.6, 141.2, 144.0, 156.1 (CdO), 189.9
(CdO); 31P NMR (161.9 MHz, CDCl3) δ 20.9 (s, PPh3); mass
spectrum (HRMS), m/z ) 603.1805 (M + Na)+ (C37H29N2NaO3P
requires 603.1814).
Fmoc-Protected Valine Cyanophosphorane 12. Fmoc-protected
valine 9 (1.1 g, 3.24 mmol) was reacted with (cyanomethylene)t-
riphenylphophorane 10 in the presence of EDCI (0.807 g, 4.21
mmol) and DMAP (0.04 g, 0.32 mmol) to furnish valine cyano-
phosphorane derivative 12 in 20 h, following the procedure
described for compound 11. Purification of the crude material by
silica gel flash column chromatography (10 × 5.5 cm) with 1:1:
7.1 acetone/CHCl3/hexanes produced 5 as a fluffy white solid: yield
2.0 g (99%); Rf ) 0.55 (1.5:1 EtOAc/hexanes); 1H NMR (400 MHz,
CDCl3) δ 0.83 (d, 3H, J ) 6.8 Hz, Val-CH3), 1.07 (d, 3H, J ) 6.8
Hz, Val-CH3), 2.43 (m, 1H, Val-CH), 4.22 (t, 1H, J ) 7.2 Hz,
Fmoc CH), 4.34 (m, 2H, Fmoc CH2), 4.91 (dd, 1H, J ) 4.0, 8.8
Hz, Val-R-CH), 5.60 (d, 1H, J ) 8.8 Hz, NH), 7.27 (t, 2H, J ) 7.2
Hz, aromatic), 7.37 (t, 2H, J ) 7.6 Hz, aromatic), 7.48-7.64 (m,
17H, aromatic), 7.75 (d, 2H, J ) 7.6 Hz, aromatic); 13C NMR
(100.56 MHz, CDCl3) δ 16.9, 20.2, 32.2, 47.4, 61.0, 61.1, 120.0,
122.4, 123.3, 125.4, 127.2, 127.7, 129.3, 129.4, 133.44, 133.47,
133.6, 133.7, 141.38, 141.40, 156.4, 194.0; 31P NMR (80.95 MHz,
CDCl3) δ 21.3 (s, PPh3); mass spectrum (ESI-MS), m/z ) 623.3
(M + H)+ (C40H36N2O3P requires 623.2); mass spectrum (HRMS),
m/z ) 645.2289 (M + Na)+ (C40H35N2O3PNa requires 645.2283).
Glycine Cyanophosphorane 13 and Valine Cyanophospho-
rane 15. Compound 11 (2.04 g, 3.52 mmol) was taken in anhydrous
piperidine (10 mL), and the resulting solution was stirred at ambient
temperature. The reaction was monitored by TLC and appeared to
stop within 15 min. Excess piperidine was removed under reduced
pressure, and the crude material 13 thus obtained was coevaporated
with anhydrous CH2Cl2 and triethylamine. The residue was dried
in high vacuum overnight before being used in the next step without
further purification. Crude dried material was off-white in color:
yield (quantitative); silica gel TLC Rf ) 0.41 (1:9 MeOH/CHCl3);
mass spectrum (ESI-MS), m/z ) 359.1 (M + H)+ (C22H20N2OP
requires 359.1). Similarly, Fomoc-protected valine cyanophospho-
rane 12 (0.15 g, 0.24 mmol) was reacted with neat piperidine to
generate valine cyanophosphorane 15 in quantitative yield and was
used in the next step without further purification. Crude dried
material was off-white in color: silica gel TLC Rf ) 0.44 (1:9
MeOH/CH2Cl2); mass spectrum (ESI-MS), m/z ) 401.5 (M +
H])+(C25H26N2OP requires 401.17).
dried for 0.5 h in high vacuum before addition of 1:1 anhydrous
DMF and CH2Cl2 (15 mL), and the resulting mixture was stirred
for 15 min at ambient temperature. Glycine cyanophosphorane
derivative 13 (0.85 g, 2.38 mmol) in CH2Cl2 (5 mL) was added
dropwise, followed by addition of TMP (174 µL, 1.31 mmol) under
N2 atmosphere, and stirring was continued at ambient temperature.
The reaction was monitored by TLC and appeared complete within
3 h. The reaction mixture was diluted with CH2Cl2 (ca. 60 mL)
and successively washed with cold water (30 mL), 1% citric acid
(30 mL), cold NaHCO3 (30 mL), and water (30 mL). Each aqueous
fraction was back extracted with CH2Cl2 (40 mL), the combined
organic phases were dried (anhydrous Na2SO4) and filtered, and
the filtrate was concentrated to dryness under reduced pressure.
The crude material thus obtained was purified by silica gel flash
column chromatography (10 × 5.5 cm). Elution with 0.6:0.6:1.2:
7.6 MeOH/acetone/CHCl3/hexanes generated desired product 16
as a colorless fluffy mass: yield 0.85 g (71%); silica gel TLC Rf )
0.3 (1:1:2:6 MeOH/acetone/CHCl3/hexanes); 1H NMR (600 MHz,
CDCl3) δ 1.2 (d, 3H, 6.6 Hz, Thr-CH3), 1.72 (s, 3H, CH3CO), 1.84
(s, 3H, CH3CO), 1.99 (s, 3H, CH3CO), 2.12 (s, 3H, CH3CO), 4.03
(m, 2H, H-6/H-6′), 4.21 (m, 4H, Thr-R-CH, ꢀ-CH, H-5 and Fmoc
CH), 4.38 (m, 3H, Fmoc CH2 and Gly-CH), 4.52 (m, 2H, H-2 and
Gly-CH), 4.94 (d, 1H, J ) 3.6 Hz, H-1), 5.02 (dd, 1H, J ) 3.0,
11.4 Hz, H-3), 5.33 (s, 1H, H-4), 5.92 (d, 1H, J ) 7.8 Hz, Fmoc
NH), 6.68 (d, 1H, J ) 9.6 Hz, NHAc), 7.03 (br. s, 1H, Gly-NH),
7.26 (t, 2H, J ) 7.8 Hz, aromatic), 7.36 (m, 2H, aromatic), 7.56
(m, 14H, aromatic), 7.65 (m, 3H, aromatic), 7.73 (d, 2H, J ) 7.2
Hz, aromatic); 13C NMR (100.56 MHz, CDCl3) δ 20.6, 20.71, 20.77,
22.8, 46.3 (CH2), 46.4, 47.0, 47.1, 47.5, 58.1, 62.1 (CH2), 67.1
(CH2), 67.2, 67.4, 68.5, 76.6, 77.4, 99.3 (C-1), 119.9, 120.2, 120.4,
121.5, 122.5, 125.2, 127.1, 127.7, 129.4, 129.5, 129.4, 129.5, 133.5,
133.6, 133.7, 141.2, 143.7, 143.8, 156.5 (CdO), 169.1 (CdO),
170.4 (3 × CdO), 170.5 (CdO), 171.0 (CdO), 189.5, 189.5; 31P
NMR (161.9 MHz, CDCl3) δ 20.6 (s, PPh3); mass spectrum
(HRMS), m/z ) 1033.3407 (M + Na)+ (C55H55N4NaO13 requires
1033.3401). When the reaction for compound 16 was repeated on
a 1.18 g scale, a byproduct A (0.328 g, 25%) was isolated as a
white fluffy mass after purification as described above: silica gel
TLC Rf ) 0.31 (1:1:2:6 MeOH/acetone/CHCl3/hexanes); 1H NMR
(600 MHz, CDCl3) δ 1.36 (d, 3H, J ) 6.0 Hz, Thr-CH3), 1.39-1.67
(m, 6H, piperidine Hs), 1.98 (s, 3H, CH3CO), 2.01 (s, 3H, CH3CO),
2.03 (s, 3H, CH3CO), 2.14 (s, 3H, CH3CO), 3.43 (m, 3H, piperidine
Hs), 3.62 (m, 1H, piperidine H), 3.86 (m, 1H, ꢀ-CH), 4.06 (m, 2H,
Fmoc CH2), 4.23 (dd, 2H, J ) 7.2, 12.6 Hz, H-5 and Fmoc CH),
4.35 (dd, 1H, J ) 7.2, 10.2 Hz, H-6), 4.41 (dd, 1H, J ) 7.2, 10.2
Hz, H-6′), 4.58 (ddd, 1H, J ) 3.0, 9.0, 9.0 Hz, H-2), 4.68 (dd, 1H,
J ) 1.8, 9.6 Hz, Thr-R-CH), 4.74 (d, 1H, J ) 3.0 Hz, H-1), 5.06
(dd, 1H, J ) 3.0, 11.4 Hz, H-3), 5.4 (d, 1H, J ) 2.4 Hz, H-4), 5.92
(d, 1H, J ) 9.0 Hz, Fmoc NH), 6.39 (d, 1H, J ) 9.6 Hz, NHAc),
7.31 (dd, 2H, J ) 7.8, 15.6 Hz, aromatic), 7.38 (d, 2H, J ) 7.2,
13.8 Hz, aromatic), 7.63 (d, 2H, J ) 7.8 Hz, aromatic), 7.75 (d,
2H, J ) 7.2 Hz, aromatic); 13C NMR (150.83 MHz, CDCl3) δ 18.8
(Thr-CH3), 20.7, 20.8, 20.84, 23.1, 24.2 (CH2), 25.6 (CH2), 26.7
(CH2), 43.5 (CH2), 46.8 (CH2), 47.1, 47.2, 55.1, 62.3 (CH2), 67.4
(CH2), 67.5, 67.6, 69.2, 78.2, 101.3 (C-1), 120.0, 125.2, 125.3,
127.2, 127.7, 129.3, 129.4, 133.6, 133.65, 141.3, 143.8, 143.8, 156.8
(CdO), 168.2 (CdO), 170.4 (2 × CdO), 170.7 (CdO), 170.8
(CdO); mass spectrum (HRMS), m/z ) 760.3043 (M + Na)+ (C3
8H47N3NaO12 requires 760.3057).
Glycodipeptide Cyanophosphorane Analog 17. Glycosylamino
acid 7 (0.15 g, 0.22 mmol) was reacted with alanine cyanophos-
phorane derivative 14 (0.13 g, 0.34 mmol) in the presence of HBTU
(0.178 g, 0.469 mmol), HOBt (0.071 g, 0.464 mmol), and TMP
(33 µL, 0.246 mmol) in 3:7 anhydrous DMF/CH2Cl2 (9 mL) to
produce compound 17 as colorless amorphous solid, following the
procedure described for compound 16. The reaction appeared
complete within 3.5 h. The crude material was purified by silica
gel flash column chromatography (7 × 4.5 cm) using 0.5:0.5:1:8
and then 1:1:2:6 MeOH/acetone/CHCl3/hexanes to afford 17 as a
Glycodipeptide Cyanophosphorane Analog 16 and Glycosy-
lamino Acid-Piperidine Byproduct A. NR-(Fluoren-9-ylmethoxy-
carbonyl)-O-(3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-R-D-galacto-
pyranosyl)-L-threonine 7 (0.8 g, 1.19 mmol), HBTU (0.9 g, 2.38
mmol), and HOBt (0.32 g, 2.38 mmol) were taken together and
1892 J. Org. Chem. Vol. 74, No. 5, 2009

Glycopeptide Synthesis by DecarboxylatiVe Condensation
white amorphous solid: yield 0.14 g (61%); silica gel TLC Rf )
0.31 (1:1:2:6 MeOH/acetone/CHCl3/hexanes); 1H NMR (600 MHz,
CDCl3) δ 1.16 (d, 3H, J ) 6.6 Hz, Thr-CH3), 1.50 (d, 3H, J ) 7.2
Hz, Ala-CH3), 1.81 (s, 3H, CH3CO), 1.85 (s, 3H, CH3CO), 1.99
(s, 3H, CH3CO), 2.13 (s, 3H, CH3CO), 4.03 (d, 2H, J ) 5.4 Hz,
H-6 and H-6′), 4.13-4.22 (m, 4H, Thr-R-CH, ꢀ-CH, H-5 and Fmoc
CH), 4.36 (m, 2H, Fmoc CH2), 4.52 (m, 1H, H-2), 4.84 (dd, 1H, J
) 3.0, 11.4 Hz, H-3), 4.86 (d, 1H, J ) 3.6 Hz, H-1), 5.13 (m, 1H,
Ala-R-CH), 5.28 (d, 1H, J ) 2.4 Hz, H-4), 5.83 (d, 1H, J ) 7.2
Hz, Fmoc NH), 6.81 (d, 1H, J ) 9.6 Hz, NHAc), 7.10 (d, 1H, J )
6.6 Hz, Ala-NH), 7.27 (m, 2H, aromatic), 7.36 (t, 2H, J ) 7.2 Hz,
aromatic), 7.55 (m, 14H, aromatic), 7.64 (t, 3H, J ) 6.6 Hz,
aromatic), 7.73 (d, 2H, J ) 7.8 Hz, aromatic); 13C NMR (150.83
Purification of the crude residue with silica gel flash chromatography
(8 × 3.5 cm) with 1:9 EtOAc/hexanes generated 20 as a white
amorphous solid: yield 0.325 g (64%); silica gel TLC Rf ) 0.37
(1:4 EtOAc/hexanes); mp 25.5-26 °C; 1H NMR (600 MHz, CDCl3)
δ 0.85 (d, 3H, J ) 7.2 Hz, Val-CH3), 0.93 (d, 3H, J ) 6.6 Hz,
Val-CH3), 1.45 (s, 9H, tBu), 1.85 (br.s, 1H, NH), 1.93 (m, 1H,
Val-CH), 2.97(d, 1H, J ) 4.2 Hz, Val-R-CH), 3.50 (dd, 2H, J )
17.4, 33.0 Hz, CH2CN); 13C NMR (150.83 MHz, CDCl3) δ 17.8,
19.3, 28.2 (3 × CH3 of tBu), 31.8, 36.9, 66.6, 82.1, 82.1, 117.9
(CN), 173.0 (CdO); mass spectrum (HRMS), m/z ) 235.1423 (M
+ Na)+ (C11H20NaN2O2 requires 235.1422).
N-Cyanomethyl N-oxide Valine tert-Butyl Ester 21. To a well-
stirred solution of compound 20 (0.284 g, 1.34 mmol) in CH2Cl2
(15 mL) at 0 °C was added mCPBA (70-75%, 0.627 g, 2.72 mmol)
in six portions at 5 min intervals. The resulting solution was allowed
to stir at ambient temperature. Completion of the reaction was
detected by TLC and appeared complete within 30 min. Sodium
thiosulfate Na2S2O3 ·5H2O (0.664 g, 2.68 mmol) dissolved in water
(2.9 mL) was added followed by addition of saturated NaHCO3
(7.2 mL) at 0 °C, and the resulting solution was stirred for 1 h.
The reaction mixture was diluted with CH2Cl2 (100 mL) and washed
with saturated NaHCO3 (60 mL). The aqueous layer was back-
extracted with CH2Cl2 (3 × 100 mL), and the organic layers were
washed with brine (100 mL), dried (anhydrous Na2SO4), and
filtered. The filtrate was concentrated to dryness under reduced
pressure. The crude material thus obtained was purified by silica
gel flash column chromatography (9 × 4.6 cm) with 3:7 EtOAc/
hexanes to yield 21 as white amorphous solid: yield 0.285 g (94%);
silica gel TLC Rf ) 0.28 (1:4 EtOAc/hexanes); mp 60-61 °C; 1H
NMR (600 MHz, CDCl3) δ 1.01 (d, 3H, J ) 6.6 Hz, Val-CH3),
1.03 (d, 3H, J ) 7.2 Hz, Val-CH3), 1.48 (s, 9H, tBu), 2.44 (m, 1H,
Val-CH), 4.17 (d, 1H, J ) 10.2 Hz, Val-R-CH), 6.97 (s, 1H,
CHCN); 13C NMR (100.56 MHz, CDCl3) δ 18.7, 18.9, 27.9 (3 ×
CH3 of tBu), 31.1, 84.6, 86.0, 107.5 (CHCN), 112.2 (CN), 164.9
(CdO); mass spectrum (HRMS), m/z ) 249.1216 (M + Na)+
(C11H18NaN2O3 requires 249.1215).
MHz, CDCl3) δ 16.5, 19.8, 20.6, 20.6, 20.7, 22.8, 47.0 (d, JCP
)
2.9 Hz), 51.0 (d, JCP ) 9.3 Hz), 57.2, 62.1, 66.9, 67.2, 67.2, 68.6,
75.5, 98.5 (C-1), 119.9, 119.91, 122.0 (d, JCP ) 93.2 Hz), 125.1,
127.0, 127.0, 127.6, 129.2 (d, JCP ) 12.9 Hz), 133.5 (d, JCP ) 2.6
Hz), 133.5 (d, JCP ) 10.4 Hz), 141.2, 141.2, 143.6, 143.8, 156.2
(CdO), 167.8 (CdO), 170.1 (CdO), 170.3 (CdO), 170.4 (CdO),
170.7 (CdO), 193.9 (d, JCP ) 3.6 Hz, CdO); 31P NMR (161.9
MHz, CDCl3) δ 21.1 (s, PPh3); mass spectrum (HRMS), m/z )
1047.3565 (M + Na)+ (C56H57NaN4O13P requires 1047.3557).
Glycodipeptide Cyanophosphorane Analogue 18. Glycosylamino
acid 7 (0.1 g, 0.149 mmol) was reacted with crude valine
cyanophosphorane derivative 15 (0.96 g, 0.24 mmol) in the presence
of HBTU (0.0.85 g, 0.223 mmol) and HOBt (0.030 g, 0.223 mmol)
in 1:1 anhydrous DMF/CH2Cl2 (5 mL) to produce a crude mixture,
following the procedure described for compound 16. The reaction
was monitored by TLC and appeared complete within 2 h. The
crude material was purified by silica gel flash column chromatog-
raphy (7 × 3 cm) with 0.5:0.5:1:8 and then 0.7:0.7:1.4:7.2 MeOH/
acetone/CHCl3/hexanes to generate 18 as colorless fluffy mass: yield
0.95 g (60%); silica gel TLC Rf ) 0.35 (1:1:2:6 MeOH/acetone/
1
CHCl3/hexanes); H NMR (600 MHz, CDCl3) δ 0.78 (d, 3H, J )
6.6 Hz, Val-CH3), 1.06 (d, 3H, J ) 6.6 Hz, Val-CH3), 1.23 (d, 3H,
J ) 6.6 Hz, Thr-CH3), 1.92 (s, 3H, CH3CO), 1.93 (s, 3H, CH3CO),
2.04 (s, 3H, CH3CO), 2.18 (s, 3H, CH3CO), 2.51 (m, 1H, Val-
CH), 4.05-4.11 (m, 3H, Thr-ꢀ-CH, H-6 and H-6′), 4.19 (t, 1H, J
) 6.6 Hz, H-5), 4.24 (t, 1H, J ) 7.2 Hz, Fmoc CH), 4.30 (dd, 1H,
J ) 1.8, 7.8 Hz, Thr-R-CH), 4.40 (m, 2H, Fmoc CH2), 4.51 (ddd,
1H, J ) 3.6, 10.8, 10.8 Hz, H-2), 4.74 (d, 1H, J ) 3.0 Hz, H-1),
4.82 (dd, 1H, J ) 3.0, 11.4 Hz, H-3), 5.11 (dd, 1H, J ) 3.6, 7.0
Hz, Val-R-CH), 5.31 (d, 1H, J ) 2.4 Hz, H-4), 5.84 (d, 1H, J )
7.8 Hz, Fmoc NH), 6.59 (d, 1H, J ) 10.2 Hz, NHAc), 6.66 (d, 1H,
J ) 8.4 Hz, Val-NH), 7.33 (m, 2H, aromatic), 7.40 (m, 2H,
aromatic), 7.55 (m, 6H, aromatic), 7.59-7.68 (m, 11H, aromatic),
7.76 (d, 2H, J ) 7.8 Hz, aromatic); 13C NMR (150.83 MHz, CDCl3)
δ 16.7, 17.3, 20.6, 20.8, 20.9, 20.99, 23.2, 32.4, 47.1, 47.3, 57.9,
59.6, 59.6, 62.4, 67.1, 67.5, 67.6, 69.2, 76.7, 99.5, 120.1, 120.2,
122.1, 122.7, 125.4, 125.4, 129.4, 129.4, 133.6, 133.7, 133.8, 133.9,
141.4, 143.9, 144.0, 156.5 (CdO), 169.1 (CdO), 170.4 (CdO),
170.4 (CdO), 170.6 (CdO), 171.0 (2 × CdO), 193.5, 193.5; 31P
NMR (161.9 MHz, CDCl3) δ 21.0 (s, PPh3); mass spectrum
(HRMS), m/z ) 1075.3815 (M + Na)+ (C58H61N4NaO13P requires
1075.3870).
N-Hydroxyvaline tert-Butyl Ester 6. To a well-stirred solution
of compound 21 (0.266 g, 1.18 mmol) in MeOH (30 mL) was added
hydroxylamine hydrochloride (0.409 g, 5.88 mmol), and the
resulting solution was stirred at 35-40 °C. The reaction was
monitored by TLC and appeared complete after 36 h. The reaction
mixture was allowed to attain room temperature, diluted with
CH2Cl2 (30 mL), and stirred for 5 min. Saturated NaHCO3 (70 mL)
was added, and the organic layer was separated. The aqueous layer
was extracted with CH2Cl2 (3 × 100 mL). The combined organic
phases were washed with brine (100 mL), dried (anhydrous
Na2SO4), and filtered. The filtrate was concentrated to dryness under
reduced pressure to provide the crude material. To the above crude
material was added oxalic acid (0.212 g, 2.36 mmol) in MeOH
(3.5 mL), and the resulting solution was triturated with hexanes.
The liquid containing solids was centrifuged to render 6 as a white
powder: yield 0.276 g (100%); silica gel TLC Rf ) 0.28 (3:7 EtOAc/
1
hexanes); mp 66-67 °C; H NMR (600 MHz, DMSO-d6) δ 0.87
(d, 3H, J ) 6.6 Hz, Val-CH3), 0.94 (d, 3 H, J ) 6.6 Hz, Val-CH3),
1.43 (s, 9H, tBu), 1.87 (m, 1H, Val-CH), 3.28 (d, 1H, J ) 6.6 Hz,
Val-R-CH); 13C NMR (100.56 MHz, DMSO-d6) δ 18.5, 19.6, 27.8,
27.9, 70.9, 81.2 (R-CH), 161.8 (CdOoxalate), 170.6 (CdO); mass
spectrum (HRMS), m/z ) 212.1261 (M + Na)+ (C9H19NaNO3
requires 212.1263).
N-Cyanomethylvaline tert-Butyl Ester (20). To a well-stirred
suspension of valine tert-butyl ester hydrochloride 19 (0.5 g, 2.38
mmol) in anhydrous acetonitrile (10 mL) was added DIPEA (0.87
mL, 5.25 mmol) dropwise over a period of 15 min, and the resulting
solution was stirred under N2 atmosphere for 5 min. Bromoaceto-
nitrile (0.17 mL, 2.38 mmol) was added dropwise over a period of
15 min, and stirring was continued. The reaction was monitored
by TLC and appeared to stop after 3 days. Excess solvent was
evaporated to dryness under reduced pressure to get a crude material
which was dissolved in CH2Cl2 (50 mL) and washed with saturated
NaHCO3. Aqueous layer was back-extracted with CH2Cl2 (2 × 50
mL). Combined organic layers were washed with brine (1 × 50
mL), dried (anhydrous Na2SO4), and filtered. The filtrate was
concentrated under reduced pressure to get the crude residue.
Glycotripeptide tert-Butyl Ester 22 and Glycopeptide-Derived
Oxazole Byproduct 23. Cyanophosphorane 16 (0.08 g, 0.0791
mmol) was first converted to R-ketoacid 1 in quantitative yield with
DMDO (4 mL in acetone, approximately 2 equiv) in acetone (0.5
mL) and water (200 µL). Crude material 1 was dried in high vacuum
for 0.5 h and reacted with N-hydroxy glycine tert-butyl ester ·oxalate
salt 4 (0.037 g, 0.158 mmol) in anhydrous DMF (2 mL) at 40 °C
to furnish glycotripeptide 22 after 24 h, following the general
procedure described in Supporting Information Part-I. Crude
material having two new UV active spots was separated by silica
J. Org. Chem. Vol. 74, No. 5, 2009 1893

Sanki et al.
gel flash column chromatography (10 × 3 cm). Elution with 1:1:2
acetone/CHCl3/hexanes and then 0.75:0.75:1.5:7 MeOH/acetone/
CHCl3/hexanes yielded desired product 22 as a white fluffy mass:
yield 0.027 g (41%); silica gel TLC Rf ) 0.26 (1:1:2:6 MeOH/
58.01, 62.31, 67.37, 67.51, 68.32, 75.78, 82.80, 99.11 (C-1), 120.27,
125.29, 125.34, 127.36, 128.02, 141.54, 143.87, 143.96, 156.46
(CdO), 167.94 (CdO), 169.58 (CdO), 170.69 (2 × CdO), 170.87
(CdO), 171.13 (CdO), 171.97 (CdO); mass spectrum (HRMS),
m/z ) 877.3519 (M + Na)+ (C42H54N4NaO15 requires 877.3483).
1
acetone/CHCl3/hexanes); H NMR (600 MHz, CDCl3) δ 1.88 (d,
3H, J ) 6.0 Hz, Thr-CH3), 1.44 (s, 9H, tBu), 1.9 (s, 3H, CH3CO),
1.98 (s, 3H, CH3CO), 1.99 (s, 3H, CH3CO), 2.13 (s, 3H, CH3CO),
3.89 (t, 2H, J ) 3.6 Hz, CH2COOtBu), 3.93 (dd, 1H, J ) 4.2, 16.8
Hz, Gly-CH), 4.06 (m, 3H, H-6, H-6′ and Gly-CH), 4.23 (dd, 2H,
J ) 7.2, 12.5 Hz, Fmoc CH and H-5), 4.28 (m, 2H, Thr-R-CH and
ꢀ-CH), 4.44 (m, 2H, Fmoc CH2), 4.54 (m, 1H, H-2), 5.09 (m, 2H,
H-1 and H-3), 5.36 (br.s, 1H, H-4), 5.96 (d, 1H, J ) 7.8 Hz, Fmoc
NH), 6.48 (br.s, 1H, NHCH2CO2tBu), 6.9 (d, 1H, J ) 9 Hz, NHAc),
7.1 (br.s, 1H, NHCH2-), 7.3 (t, 2H, J ) 7.2 Hz, aromatic), 7.38 (t,
2H, J ) 7.2 Hz, aromatic), 7.59 (d, 2H, J ) 7.8 Hz, aromatic),
7.74 (d, 2H, J ) 7.2 Hz, aromatic); 13C NMR (150.83 MHz, CDCl3)
δ 16.9 (Thr-CH3), 20.9, 20.9, 21.0, 23.0, 28.2 (3 × CH3 of tBu),
29.9, 42.2 (CH2), 42.7 (CH2), 47.4, 47.9, 58.2, 62.3 (CH2), 67.3
(CH2), 67.4, 67.5, 68.3, 75.8, 83.1, 99.1 (C-1), 120.2, 125.3, 127.3,
127.4, 128.0, 128.4, 129.2, 141.5, 141.5, 143.86, 143.88, 156.5
(CdO), 168.6 (CdO), 168.7 (CdO), 169.8 (CdO), 170.6 (2 ×
CdO), 171.0 (CdO), 171.1 (CdO); mass spectrum (HRMS), m/z
) 863.3309 (M + Na)+ (C41H52N4NaO15 requires 863.3327). Also
produced was a byproduct 23 as a colorless amorphous solid: yield
0.014 g (22%); silica gel TLC Rf ) 0.34 (1:1:2:6 MeOH/acetone/
Glycotripeptide tert-Butyl Ester 25 and Glycopeptide-Derived
Oxazole Byproduct 26. Cyanophosphorane 17 (0.053 g, 0.0517
mmol) was first converted to R-ketoacid 2 in quantitative yield with
DMDO (4 mL, approximately 2 equiv) in acetone (2 mL) and water
(250 µL). Crude material 2 was dried in high vacuum for 0.5 h
and reacted with N-hydroxy glycine tert-butyl ester ·oxalate salt 4
(0.015 g, 0.0620 mmol) in anhydrous DMF (1.5 mL) at 40-42 °C
to afford glycotripeptide 25 and 26 after 6 h, following the general
procedure described in Supporting Information Part-I. Crude
material was purified by silica gel flash column chromatography
(10 × 3 cm). Elution with 1.5:98.5 MeOH/CH2Cl2 yielded desired
product 25 as white amorphous solid: yield 0.019 g (43%); silica
gel TLC Rf ) 0.22 (2.5:97.5 MeOH/CH2Cl2, run twice); 1H NMR
(600 MHz, CDCl3) δ 1.15 (d, 3H, J ) 6.6 Hz, Thr-CH3), 1.41 (d,
3H, J ) 7.2 Hz, Ala-CH3), 1.44 (s, 9H, tBu), 1.98 (s, 3H, CH3CO),
1.99 (s, 3H, CH3CO), 1.997 (s, 3H, CH3CO), 2.13 (s, 3H, CH3CO),
3.91 (t, 2H, J ) 4.8 Hz, Gly-CH2), 4.05 (d, 2H, J ) 6.6 Hz, H-6
and H-6′), 4.17 (dd, 1H, J ) 3.6, 6.6 Hz, Thr-ꢀ-CH), 4.20 (t, 2H,
J ) 6.6 Hz, H-5 and Fmoc CH), 4.28 (dd, 1H, J ) 3.6, 7.2 Hz,
Thr-R-CH), 4.38 (d, 2H, J ) 7.2 Hz, Fmoc CH2), 4.56 (m, 2H,
H-2 and Ala-CH), 5.07 (dd, 1H, J ) 3.0, 12.0 Hz, H-3), 5.16 (d,
1H, J ) 3.0 Hz, H-1), 5.36 (d, 1H, J ) 2.4 Hz, H-4), 5.93 (d, 1H,
J ) 7.2 Hz, Fmoc NH), 6.54 (t, 1H, J ) 4.2 Hz, Gly-NH), 7.13 (d,
1H, J ) 6.6 Hz, Ala-NH), 7.17 (d, 1H, J ) 8.4 Hz, NHAc), 7.29
(t, 2H, J ) 7.2 Hz, aromatic), 7.38 (t, 2H, J ) 7.2 Hz, aromatic),
7.58 (d, 2H, J ) 7.2 Hz, aromatic), 7.74 (d, 2H, J ) 7.2 Hz,
aromatic); 13C NMR (150.83 MHz, CDCl3) δ 16.5, 20.0, 20.88,
20.97, 20.99, 23.2, 28.2 (3 × CH3 of tBu), 29.9, 42.4, 47.3, 47.8,
49.2, 57.4, 62.2, 67.2, 67.4, 67.45, 68.2, 75.1, 83.0, 98.8 (C-1),
120.2, 120.2, 125.3, 125.3, 127.3, 127.9, 141.5, 143.8, 143.9, 156.2
(CdO), 168.5 (CdO), 168.7 (CdO), 170.6 (2 × CdO), 170.8
(CdO), 171.0 (CdO), 172.3 (CdO); mass spectrum (HRMS), m/z
) 877.3501 (M + Na)+ (C42H54NaN4O15 requires 877.3483) and
26 as white amorphous solid: yield 0.010 g (23%); Rf ) 0.24 (2.5:
1
CHCl3/hexanes); H NMR (600 MHz, CDCl3) δ 1.19 (d, 3H, J )
6.6 Hz, Thr-CH3), 1.36 (s, 9H, tBu), 1.8 (s, 3H, CH3CO), 1.99 (s,
6H, 2 × CH3CO), 2.14 (s, 3H, CH3CO), 4.06 (d, 2H, J ) 6.6 Hz,
H-6 and H-6′), 4.24 (m, 2H, Fmoc CH and H-5), 4.29 (m, 1H,
Thr-R-CH), 4.35 (m, 2H, Gly-CH and Thr-ꢀ-CH), 4.46 (m, 2H,
Fmoc CH2), 4.56 (m, 2H, H-2 and Gly-CH), 5.13 (d, 1H, J ) 3.6
Hz, H-1), 5.16 (dd, 1H, J ) 3.0, 11.4 Hz, H-3), 5.35 (br.s, 1H,
H-4), 6.02 (d, 1H, J ) 7.2 Hz, Fmoc NH), 6.1 (s, 1H, oxazole
H-4), 7.07 (br.s, 1H, NHCH2-), 7.30 (m, 3H, NH and aromatic),
7.38 (m, 2H, aromatic), 7.61 (t, 2H, J ) 7.2 Hz, aromatic), 7.74 (t,
2H, J ) 7.8 Hz, aromatic); 13C NMR (150.83 MHz, CDCl3) δ 14.3,
16.9, 20.9, 20.9, 21.0, 22.8, 28.2 (3 × CH3 of tBu), 29.9, 37.4,
47.4, 47.9, 58.0, 62.2, 67.23, 67.26, 67.4, 68.2, 75.6, 99.0 (C-1),
107.6 (oxazole C-4), 120.2, 125.3, 127.3, 127.3, 127.9, 128.0, 141.5,
141.5, 143.8, 143.9, 153.2, 156.5 (CdO), 157.2, 169.5 (CdO),
170.6 (CdO), 170.7 (CdO), 170.9 (2 × CdO); mass spectrum
(HRMS), m/z ) 845.3204 (M + Na)+ (C41H50N4NaO14 requires
845.3221).
1
97.5 MeOH-CH2Cl2, run twice); H NMR (600 MHz, CDCl3) δ
1.15 (d, 3H, J ) 6.0 Hz, Thr-CH3), 1.37 (s, 9H, tBu), 1.49 (d, 3H,
J ) 6.6 Hz, Ala-CH3), 1.87 (s, 3H, CH3CO), 1.99 (s, 3H, CH3CO),
2.00 (s, 3H, CH3CO), 2.14 (s, 3H, CH3CO), 4.06 (d, 2H, J ) 6.6
Hz, H-6 and H-6′), 4.22 (m, 3H, H-5, Thr-ꢀ-CH and Fmoc CH),
4.28 (dd, 1H, J ) 3.0, 6.6 Hz, Thr-R-CH), 4.39 (dd, 2H, J ) 3.0,
6.6 Hz, Fmoc CH2), 4.63 (m, 1H, H-2), 5.04 (m, 1H, Ala-CH),
5.18 (dd, 1H, J ) 3.0, 12.0 Hz, H-3), 5.21 (d, 1H, J ) 3.0 Hz, 1H;
H-1), 5.35 (d, 1H, J ) 2.4 Hz, H-4), 5.84 (d, 1H, J ) 6.6 Hz,
Fmoc NH), 6.13 (s, 1H, oxazole H-4), 7.26 (br.s, 1H, Ala-NH),
7.30 (m, 2H, aromatic), 7.38 (t, 2H, J ) 7.2 Hz, aromatic), 7.59 (t,
3H, J ) 7.8 Hz, NHAc and aromatic), 7.74 (d, 2H, J ) 7.2 Hz,
aromatic); 13C NMR (100.56 MHz, CDCl3) δ 16.2, 20.7, 20.9,
20.98, 21.0, 22.9, 28.2 (3 × CH3 of tBu), 29.9, 44.8, 47.3, 47.8,
57.0, 62.2, 67.1, 67.3, 67.5, 68.3, 74.4, 84.9, 98.2 (C-1), 107.4
(oxazole C-4), 120.2, 120.2, 125.3, 125.3, 127.3, 127.9, 141.5,
143.8, 144.0, 156.1, 156.7 (CdO), 157.0, 168.6 (CdO), 170.6
(CdO), 170.7 (CdO), 170.7 (CdO), 170.9 (CdO); mass spectrum
(HRMS), m/z ) 859.3375 (M + Na)+ (C42H52NaN4O14 requires
859.3378).
Glycotripeptide tert-Butyl Ester 27. Cyanophosphorane 17 (0.056
g, 0.0546 mmol) was first converted to R-ketoacid 2 in quantitative
yield with DMDO (4 mL in acetone, approximately 2 equiv) in
acetone (2 mL) and water (250 µL). Crude material 2 was dried in
high vacuum for 0.5 h and reacted with N-hydroxyalanine tert-
butyl ester ·oxalate salt 5 (0.016 g, 0.0656 mmol) in anhydrous DMF
(1.5 mL) at 35-40 °C to produce glycotripeptide 27 after 38 h as
the sole product, following the general procedure described in
Supporting Information Part-I. Crude material was purified by silica
gel flash column chromatography (6 × 3.5 cm). Elution with 0.5:
Glycotripeptide tert-Butyl Ester 24. Cyanophosphorane 16 (0.08
g, 0.0791 mmol) was first converted to R-ketoacid 1 in quantitative
yield with DMDO (4 mL in acetone, approximately 2 equiv) in
acetone (0.5 mL) and water (200 µL). Crude material 1 was dried
in high vacuum for 0.5 h and reacted with N-hydroxyalanine tert-
butyl ester ·oxalate salt 5 (0.030 g, 0.118 mmol) in anhydrous DMF
(2 mL) at 40 °C to furnish glycotripeptide 24 after 25 h, following
the general procedure described in Supporting Information Part-I.
The crude material was purified by silica gel flash column
chromatography (10 × 3 cm). Elution with 0.5:0.5:1:8 and then
0.7:0.7:1.4:7.2 MeOH/acetone/CHCl3/hexanes yielded desired prod-
uct 24 as a colorless fluffy mass: yield 0.027 g (40%); silica gel
1
TLC Rf ) 0.3 (1:1:2:6 MeOH/acetone/CHCl3/hexanes); H NMR
(600 MHz, CDCl3) δ 1.21 (d, 3H, J ) 6.0 Hz, Thr-CH3), 1.38 (d,
3H, J ) 6.6 Hz, Ala-CH3), 1.46 (s, 9H, tBu), 1.92 (s, 3H, CH3CO),
2.01 (s, 3H, CH3CO), 2.02 (s, 3H, CH3CO), 2.15 (s, 3H, CH3CO),
3.95 (m, 1H, Gly-CH), 4.08 (m, 3H, Gly-CH, H-6 and H-6′),
4.22-4.33 (m, 4H, H-5, Fmoc CH, Thr-ꢀ-CH and Thr-R-CH), 4.35
(4.47 (m, 3H, Fmoc CH2 and Ala-CH), 4.58 (m, 1H, H-2), 5.14 (d,
1H, J ) 3.0 Hz, H-1), 5.15 (dd, 1H, J ) 3.0, 11.4 Hz, H-3), 5.40
(br.s, 1H, H-4), 5.96 (d, 1H, J ) 7.2 Hz, Fmoc NH), 6.63 (br.s,
1H, Ala-NH), 7.07 (d, 1H, J ) 9.0 Hz, NHAc), 7.21 (br.s, 1H,
Gly-NH), 7.32 (t, 2H, J ) 6.0 Hz, aromatic), 7.40 (t, 2H, J ) 6.6
Hz, aromatic), 7.61 (d, 2H, J ) 7.2 Hz, aromatic), 7.76 (d, 2H, J
) 7.2 Hz, aromatic); 13C NMR (150.83 MHz, CDCl3) δ 16.78,
18.44, 20.91, 23.08, 28.08, 28.15, 29.93, 42.79, 47.37, 47.93, 49.31,
1894 J. Org. Chem. Vol. 74, No. 5, 2009

Glycopeptide Synthesis by DecarboxylatiVe Condensation
0.5:1:8 MeOH/acetone/CHCl3/hexanes yielded desired product 27
as white amorphous solid: yield 0.041 g (86%); silica gel TLC Rf
) 0.21 (1:1:1:7 MeOH/acetone/CHCl3/hexanes); H NMR (600
CH3CONH), 2.80 (br. hump, 1H, OH), 3.69 (m, 1H, H-3), 3.75
(m, 1H, H-6), 3.80 (t, 1H, J ) 4.8 Hz, H-5), 3.87 (dd, 1H, J ) 4.8,
11.4 Hz, H-6′), 3.90 (br.s, 1H, H-4), 4.11 (m, 1H, H-2), 4.17 (m,
2H, Fmoc CH and Thr-ꢀ-CH), 4.20 (d, 1H, J ) 8.4 Hz, Thr-R-
CH), 4.36 (d, 2H, J ) 7.2 Hz, Fmoc CH2), 4.41 (dd, 2H, J ) 3.6,
12.6 Hz, Gly-CH2), 4.88 (d, 2H, J ) 3.0 Hz, H-1 and OH), 5.75
(d, 1H, J ) 6.0 Hz, Fmoc NH), 7.10 (br.s, 1H, Gly-NH), 7.25 (t,
2H, J ) 7.2 Hz, aromatic), 7.34 (ddd, 2H, J ) 3.0, 6.0, 6.0 Hz,
aromatic), 7.47 (d, 1H, J ) 6.6 Hz, NHAc), 7.54 (m, 14H,
aromatic), 7.71 (d, 2H, J ) 7.2 Hz, aromatic); 13C NMR (150.83
MHz, CDCl3) δ 17.4, 22.4, 29.3, 29.7, 46.2, 46.3, 47.1, 47.2, 48.1,
50.8, 67.2, 69.4, 69.7, 70.6, 75.7, 99.1 (C-1), 119.9, 120.5, 120.6,
121.7, 122.3, 125.2, 127.2, 127.7, 129.4, 129.5, 133.6, 133.7,
133.73, 141.2, 141.3, 143.7, 143.9,156.6 (CdO), 169.8 (CdO),
173.4 (2 × CdO), 189.5, 189.5; 31P NMR (161.9 MHz, CDCl3) δ
20.7 (s, PPh3); mass spectrum (HRMS), m/z ) 907.3056 (M +
Na)+ (C49H49N4NaO10P requires 907.3084). For compound 30: yield
34 mg (15%); Rf ) 0.44 (1.5:1.5:3:4 MeOH/acetone/CHCl3/
hexanes); 1H NMR (600 MHz, CDCl3) δ 1.20 (d, 3H, J ) 6.6 Hz,
Thr-CH3), 2.00 (s, 3H, CH3CO), 2.02 (s, 3H, CH3CO), 3.10 (br.
hump, 1H, OH), 3.72 (dd, 1H, J ) 2.4, 10.2 Hz, H-3), 3.81 (br.s,
1H, H-4), 3.97 (t, 1H, J ) 5.4 Hz, H-5), 4.08 (m, 2H, H-2 and
Thr-ꢀ-CH), 4.19 (t, 1H, J ) 6.6 Hz, Fmoc CH), 4.22-4.29 (m,
3H, Thr-R-CH, H-6 and H-6′), 4.37 (d, 2H, J ) 6.6 Hz, Fmoc
CH2), 4.40 (d, 2H, J ) 4.2 Hz, Gly-CH2), 4.78 (d, 1H, J ) 3.6 Hz,
H-1), 5.09 (br. hump, 1H, OH), 5.71 (d, 1H, J ) 9.0 Hz, Fmoc
NH), 7.05 (br.s, 1H, Gly-NH), 7.25 (m, 2H, aromatic), 7.35 (m,
2H, aromatic), 7.46 (d, 1H, J ) 7.2 Hz, NHAc), 7.55 (m, 14H,
aromatic), 7.64 (m, 3H, aromatic), 7.72 (d, 2H, J ) 7.2 Hz,
aromatic); 13C NMR (150.83 MHz, CDCl3) δ 18.0, 21.0, 22.8, 29.9,
46.3, 46.4, 47.3, 51.6, 58.4, 64.2, 67.5, 68.4, 68.5, 71.2, 99.9 (C-
1), 120.1, 120.2, 120.5, 120.6, 121.8, 122.5, 125.2, 127.3, 127.31,
127.9, 129.6, 129.7, 133.7, 133.74, 133.8, 133.81, 133.9, 133.92,
141.4, 141.5, 143.8, 144.0, 156.7 (CdO), 170.5 (CdO), 170.9
(CdO), 174.7 (CdO), 189.1, 189.1; 31P NMR (161.9 MHz, CDCl3)
δ 20.7 (s, PPh3); mass spectrum (HRMS), m/z ) 949.3192 (M +
Na)+ (C51H51N4NaO11 requires 949.3190).
1
MHz, CDCl3) δ 1.17 (d, 3H, J ) 6.0 Hz, Thr-CH3), 1.40 (d, 3H,
J ) 7.2 Hz, Ala-CH3), 1.45 (s, 12H, tBu and Ala-CH3), 2.00 (s,
3H, CH3CO), 2.02 (s, 6H, CH3CO), 2.15 (s, 3H, CH3CO), 4.08 (d,
2H, J ) 6.6 Hz, H-6 and H-6’), 4.23 (m, 3H, H-5, Thr-ꢀ-CH and
Fmoc CH), 4.32 (dd, 1H, J ) 3.0, 6.6 Hz, Thr-R-CH), 4.38 (m,
1H, Ala-CH), 4.40 (d, 2H, J ) 7.2 Hz, Fmoc CH2), 4.55 (m, 1H,
Ala-CH), 4.60 (m, 1H, H-2), 5.11 (dd, 1H, J ) 3.0, 12.0 Hz, H-3),
5.17 (d, 1H, J ) 3.0 Hz, H-1), 5.40 (br.s, 1H, H-4), 5.99 (d, 1H,
J ) 6.6 Hz, Fmoc NH), 6.63 (d, 1H, J ) 6.6 Hz, Ala-NH), 7.25
(d, 1H, J ) 6.6 Hz, Ala-NH), 7.30 (m, 3H, NHAc and aromatic),
7.40 (t, 2H, J ) 7.2 Hz, aromatic), 7.60 (d, 2H, J ) 7.2 Hz,
aromatic), 7.76 (d, 2H, J ) 7.2 Hz, aromatic); 13C NMR (150.83
MHz, CDCl3) δ 16.3, 18.2 (Ala-CH3), 20.1, 20.8, 20.9, 23.1, 28.1
(3 × CH3 of tBu), 29.9, 47.3, 47.8, 49.1, 49.3, 57.2, 62.2, 67.2,
67.4, 67.4, 68.2, 74.73, 82.5, 98.6 (C-1), 120.2, 120.22, 125.3,
125.33, 127.3, 127.9, 141.47, 141.48, 143.8, 143.9, 156.1 (CdO),
168.3 (CdO), 170.6 (CdO), 170.6 (CdO), 170.8 (CdO), 171.0
(CdO), 171.8 (CdO), 171.8 (CdO); mass spectrum (HRMS), m/z
) 891.3650 (M + Na)+ (C43H56NaN4O15 requires 891.3640).
Glycotripeptide tert-Butyl Ester 28. Cyanophosphorane 16 (0.08
g, 0.0791 mmol) was first converted to R-ketoacid 1 in quantitative
yield with DMDO (4 mL in acetone, approximately 2 equiv) in
acetone (0.5 mL) and water (200 µL). Crude material 1 was dried
in high vacuum for 0.5 h and reacted with N-hydroxyvaline tert-
butyl ester ·oxalate salt 6 (0.033 g, 0.118 mmol) in anhydrous DMF
(2 mL) at 40 °C to generate glycotripeptide 28 after 19 h, following
the general procedure described in Supporting Information Part-I.
Crude material was purified by silica gel flash column chromatog-
raphy (6.5 × 3 cm). Elution with 0.4:0.4:0.8:8.4 and then 0.6:0.6:
1.2:7.6 MeOH/acetone/CHCl3/hexanes yielded desired product 28
as a colorless fluffy mass: yield 0.011 g (15%); silica gel TLC Rf
1
) 0.35 (1:1:2:6 MeOH/acetone/CHCl3/hexanes); H NMR (600
MHz, CDCl3) δ 0.93 (t, 6H, J ) 7.2 Hz, 2 × Val-CH3), 1.21 (d,
3H, J ) 6.0 Hz, Thr-CH3), 1.47 (s, 9H, tBu), 1.93 (s, 3H, CH3CO),
2.01 (s, 3H, CH3CO), 2.02 (s, 3H, CH3CO), 2.16 (s, 4H, CH3CO
and Val-CH), 3.97 (dd, 1H, J ) 4.2, 16.8 Hz, Gly-CH), 4.08 (d,
2H, J ) 6.0 Hz, H-6 and H-6′), 4.13 (dd, 1H, J ) 4.2, 16.8 Hz,
Gly-CH), 4.25 (m, 2H, Fmoc CH and H-5), 4.31 (d, 2H, J ) 6.0
Hz, Thr-R-CH and Thr-ꢀ-CH), 4.37 (dd, 1H, J ) 4.2, 7.8 Hz, Val-
R-CH), 4.45 (m, 2H, Fmoc CH2), 4.59 (m, 1H, H-2), 5.12 (d, 1H,
J ) 3.0 Hz, H-1), 5.14 (dd, 1H, J ) 2.4, 12.0 Hz, H-3), 5.41 (br.s,
1H, H-4), 5.85 (d, 1H, J ) 6.6 Hz, Fmoc NH), 6.40 (d, 1H, J )
8.4 Hz, Val-NH), 7.01 (d, 1H, J ) 8.4 Hz, NHAc), 7.14 (br.s, 1H,
Gly-NH), 7.33 (t, 2H, J ) 7.2 Hz, aromatic), 7.41 (t, 2H, J ) 7.2
Hz, aromatic), 7.62 (d, 2H, J ) 7.2 Hz, aromatic), 7.77 (d, 2H, J
) 7.8 Hz, aromatic); 13C NMR (150.83 MHz, CDCl3) δ 16.7, 18.0,
18.9, 20.9, 21.0, 22.9, 23.1, 28.3, 29.9, 31.5, 42.9, 47.41, 47.9, 58.0,
58.1, 62.3, 67.4, 67.5, 68.3, 75.6, 82.9, 99.0 (C-1), 120.2, 120.29,
125.3, 125.3, 127.4, 128.0, 141.6, 143.8, 143.9, 156.4 (CdO), 168.3
(CdO), 169.5 (CdO), 170.7 (2 × CdO), 170.74 (CdO), 170.8
(CdO), 171.1 (CdO); mass spectrum (HRMS), m/z ) 905.3792
(M + Na)+ (C45H62NaN4O15 requires 905.3796).
Glycotripeptide tert-Butyl Ester 32 and Glycopeptide-Derived
Oxazole Byproduct 33. Cyanophosphorane 29 (0.07 g, 0.0791
mmol) was first converted to R-ketoacid 31 in quantitative yield
with DMDO (4 mL in acetone, approximately 2 equiv) in acetone
(0.5 mL) and water (200 µL). Crude material 31 was dried in high
vacuum for 0.5 h and reacted with N-hydroxy glycine tert-butyl
ester·oxalate salt 4 (0.037 g, 0.158 mmol) in anhydrous DMF (2
mL) at 40 °C to produce glycotripeptide 32 and an oxazole
byproduct 33 after 16 h, following the general procedure described
in Supporting Information Part-I. Purification of the crude material
by silica gel flash column chromatography (6.0 × 3.0 cm) with
1:1:2:6 and then 1.5:1.5:3:4 MeOH/Acetone/CHCl3/hexanes gener-
1
ated 32 as colorless glassy solid: yield 15 mg (26%); H NMR
(600 MHz, CD3OD) δ 1.19 (d, 3H, J ) 6.0 Hz, Thr-CH3), 1.40 (s,
t
9H, Bu), 1.92 (s, 3H, CH3CO), 3.67 (m, 3H, H-3 and two other
protons), 3.79 (dd, 2H, J ) 17.4, 46.8 Hz, Gly-CH2), 3.84 (t, 2H,
J ) 3.6 Hz), 3.87 (d, 1H, J ) 3.6 Hz, H-4), 4.15 (dd, 1H, J ) 4.2,
11.4 Hz, H-2), 4.19 (d, 2H, J ) 2.4 Hz), 4.21 (t, 1H, J ) 6.0 Hz,
H-5), 4.24 (dd, 1H, J ) 1.8, 6.0 Hz), 4.41 (dd, 1H, J ) 6.6, 10.8
Hz, H-6), 4.51 (dd, 1H, J ) 6.6, 10.8 Hz, H-6′), 4.87 (s, 1H, H-1),
7.28 (m, 2H, aromatic), 7.35 (m, 2H, aromatic), 7.65 (t, 2H, J )
7.8 Hz, aromatic), 7.76 (d, 2H, J ) 7.2 Hz, aromatic); 13C NMR
(150.83 MHz, CD3OD) δ 19.1, 23.2, 28.4 (3 × CH3 of tBu), 42.8,
43.3, 51.6, 60.7, 62.8, 68.0, 70.3, 70.5, 73.0, 77.1, 83.1, 100.1 (C-
1), 121.1, 121.1, 126.3, 126.33, 128.3, 128.38, 128.97, 128.99,
142.8, 145.2, 145.4, 159.2 (CdO), 170.6 (CdO), 171.8 (CdO),
173.1 (CdO), 174.3 (CdO); mass spectrum (HRMS), m/z )
737.2963 (M + Na)+ (C35H46N4NaO12 requires 737.3010). Also
produced was 33 as off-white amorphous solid: yield 10 mg (18%);
1H NMR (600 MHz, CD3OD) δ 1.20 (d, 3H, J ) 6.6 Hz, Thr-
Glycodipeptide Cyanophosphorane Analog 29 and 6-O-Acetyl-
glycodipeptide Cyanophosphorane Analog 30. Starting material 16
(0.25 g, 0.247 mmol) was dissolved in anhydrous MeOH (8.0 mL),
and the temperature was lowered to 0 °C. Ammonia in MeOH (2.1
mL, 7 N) was added dropwise under N2 atmosphere. The resulting
solution was stirred at 0 °C. Reaction was monitored by TLC and
appeared to stop after 5.5 h. Solvent was removed under reduced
pressure, and the crude material thus obtained was purified by silica
gel flash column chromatography (6.0 × 3.0 cm) with 1:1:2:6 (300
mL) and then 1.5:1.5:3:4 (200 mL) MeOH/acetone/CHCl3/hexanes
to yield 29 and 30, respectively, as white amorphous solids. For
compound 29: yield 123 mg (56%); silica gel TLC Rf ) 0.26 (1.5:
1.5:3:4 MeOH/acetone/CHCl3/hexanes); 1H NMR (600 MHz,
CDCl3) δ 1.15 (d, 3H, J ) 6.6 Hz, Thr-CH3), 1.94 (s, 3H,
t
CH3), 1.32 (s, 9H, Bu), 1.89 (s, 3H, NHCOCH3), 3.68 (m, 3H),
3.85 (m, 2H), 4.16 (dd, 1H, J ) 3.6, 10.8 Hz, H-2), 4.22 (m, 3H,
J. Org. Chem. Vol. 74, No. 5, 2009 1895

Sanki et al.
H-5 and two other protons), 4.33 (dd, 2H, J ) 16.8, 30.0 Hz, Gly-
CH2), 4.40 (dd, 1H, J ) 6.0, 10.8 Hz, H-6), 4.51 (dd, 1H, J ) 6.6,
10.8 Hz, H-6′), 4.87 (s, 1H, H-1), 6.24 (s, 1H, oxazole H-4), 7.28
(m, 2H, aromatic), 7.36 (t, 2H, J ) 7.2 Hz, aromatic), 7.66 (t, 2H,
J ) 7.8 Hz, aromatic), 7.77 (d, 2H, J ) 7.2 Hz, aromatic); 13C
NMR (150.83 MHz, CD3OD) δ 19.2, 23.1, 28.5, 37.8, 51.6, 60.6,
62.8, 68.0, 70.4, 70.5, 73.0, 77.6, 101.0 (C-1), 108.9 (oxazole C-4),
121.1, 126.3, 126.33, 128.3, 128.4, 128.9, 129.0, 142.8, 145.2,
145.5, 159.1 (CdO), 173.0 (CdO), 174.3 (CdO); mass spectrum
(HRMS), m/z ) 719.2860 (M + Na)+ (C35H44N4NaO11 requires
719.2904).
reaction conditions in 41% yield. The 1H and 13C NMR of 37 from
either route were found identical to those reported in the literature
(ref Bode et al.).
[O18] N-(2-Phenylethyl)phenylacetamide (41). To a stirred solu-
tion of benzyl cyanide 38 (55 µL, 0.477 mmol) in anhydrous THF
(0.5 mL) was added H2O18 (0.478 g, 23.9 mmol) dropwise under
N2 atmosphere. HCl (g) was bubbled into the reaction mixture for
10 min. The resulting mixture was refluxed at 50 °C for 12 h under
N2 atmosphere. The desired O18-labeled phenylacetic acid was
detected by ESI-MS [m/z ) 163.15 (M + Na)+]. The mixture was
concentrated under reduced pressure in a N2-flushed rotary evapora-
tor. The residual solvent was removed in high vacuum to afford
the crude product 39 (0.070 g) which was used in the next step
without further purification. To a well-stirred solution of 39 (0.070
g) in anhydrous CH2Cl2 (2 mL) was added freshly distilled
dicyclohexylcarbodiimide (0.095 g, 0.460 mmol), phenethylamine
40 (0.060 mL, 0.476 mmol) and DIPEA (0.16 mL, 0.968 mmol).
The resulting mixture was stirred at room temperature for 36 h
under N2 atmosphere. ESI-MS of the reaction mixture revealed
labeled amide 41 with m/z ) 242.4 (M + H)+ (C16H18N18O requires
242.14).
[O18] 3-Phenyl-2-oxopropanoic Acid (36). To a vacuum-dried
phenylpyruvic acid 35 (0.1 g, 0.609 mmol) under N2 atmosphere
was added 0.5 mL of 0.1 N HCl in anhydrous THF and H2O18 (1.0
g, 49.95 mmol). The mixture was stirred at room temperature, and
the O18-exchange was complete in 15 min (monitored by ESI-MS).
The mixture was concentrated under reduced pressure in a N2-
flushed rotary evaporator and the residual solvent was removed in
high vacuum to afford 36 as a light yellow amorphous solid (0.087
1
g, 84%). H NMR (400 MHz, DMSO-d6) δ 6.40 (s, 1H, HCdC-
OH), 7.24 (t, 1H, J ) 7.2 Hz, aromatic), 7.34 (t, 3H, J ) 7.6 Hz,
aromatic), 7.75 (d, 2H, J ) 7.6 Hz, aromatic), 9.26 (br.s, 1H,
HC)COH), 13.21 (br.s, 1H, COOH); 13C NMR (100.57 MHz,
DMSO-d6) δ 109.5 (C)C-OH)) 127.2 (C ) C-OH), 128.3, 129.3,
135.0, 141.9, 166.4 (CdO); mass spectrum (ESI-MS), phenylpyru-
vic acid was decarboxylated at 320 °C to give 2-phenylethanal,
m/z ) 145.1 (M + Na)+ (C8H8ONa requires 145.05).
Acknowledgment. This work was supported in part by grants
from The University of Toledo deArce Memorial Endowment
Fund, Elsa U. Pardee Foundation, and The Ohio Cancer
Research Associates. We are grateful to Professor J. W. Bode
for helpful suggestions.
N-(2-Phenylethyl)phenylacetamide (37). A vacuum-dried mixture
of O18-labeled phenylpyruvic acid 36 (0.063 g, 0.370 mmol) and
phenethyl hydroxyl amine oxalate salt 34 (0.081 g, 0.444 mmol)
was dissolved in anhydrous MeOH (8 mL). The resulting mixture
was stirred at 35-40 °C under N2 atmosphere. After 18 h, an aliquot
of the reaction mixture was analyzed by mass spectroscopy (ESI-
MS). Excess solvent was removed under reduced pressure. The
crude material was purified by silica gel flash column chromatog-
raphy (9 × 3.5 cm) using 1:99 MeOH/CH2Cl2 to furnish 37 as a
light yellow solid; yield 0.045 g (51%). Similarly, compound 35
(0.076 g, 0.457 mmol) was converted to amide 37 under the same
Supporting Information Available: General procedure for
DMDO oxidation, Copies of 1H, 13C and 31P NMR for
compounds 11, 12, 16-18, 29, and 30; 1H and 13C NMR
for compounds 6, 20, 21, 22-28, 32, 33, 36, and byproduct A;
and mass spectra (ESI-MS) for 36, 37, 39, and 41; and 1H-1H
gCOSY NMR. This material is available free of charge via the
Internet at http://pubs.acs.org.
JO802278W
1896 J. Org. Chem. Vol. 74, No. 5, 2009