The synthesis of (2S)-4,4-difluoroglutamyl γ-peptides based on Garner's aldehyde and fluoro-reformatsky chemistry

Article abstract of DOI:10.1055/s-2002-35616

The development of optically active fluorinated synthetic building blocks of general utility is a current goal of organo-fluorine chemists. The serine-derived Garner aldehyde was converted to a general 4,4-difluoroamino acid building block via fluoro-Reformatsky reaction with ethyl bromodifluoroacetate. The utility of this building block was demonstrated by the synthesis of derivatives of (2S)-4,4-difluoroglutamine, (2S)-4,4-difluoroglutamic acid, and its incorporation into a fluorophore-containing isopeptide 2 designed as a mechanistic probe of γ-glutamyl hydrolase. Compound 2 proved to be a substrate for γ-glutamyl hydrolase and was hydrolyzed at a rate significantly slower than the corresponding non-fluorinated analog.

Full text of DOI:10.1055/s-2002-35616

2616
SPECIAL TOPIC
The Synthesis of (2S)-4,4-Difluoroglutamyl -Peptides Based on Garner’s
Aldehyde and Fluoro-Reformatsky Chemistry
4
, 4-Difluoroglutam
a
yl
P
eptide
v
S
ynthesis an
i
d
B
ioc
d
hemical Studies W. Konas,a,1 Jessica J. Pankuch,a James K. Coward*a,b
a
Department of Chemistry, University of Michigan, Ann Arbor MI 48109, USA
b
Department of Medicinal Chemistry, University of Michigan, Ann Arbor MI 48109, USA
Fax +1(734)6474865; E-mail: jkcoward@umich.edu
Received 14 September 2002
fluorinated products, but they must also allow easy incor-
poration of these products into larger and more complex
molecules. In biochemical experiments involving fluoro-
glutamic acids, the most informative data are obtained
Abstract: The development of optically active fluorinated synthet-
ic building blocks of general utility is a current goal of organo-
fluorine chemists. The serine-derived Garner aldehyde was
converted to a general 4,4-difluoroamino acid building block via
fluoro-Reformatsky reaction with ethyl bromodifluoroacetate. The when using stereochemically pure synthetic substrates. In
utility of this building block was demonstrated by the synthesis of
derivatives of (2S)-4,4-difluoroglutamine, (2S)-4,4-difluoroglutam-
ic acid, and its incorporation into a fluorophore-containing isopep-
poration into much larger -linked polyglutamate mole-
tide 2 designed as a mechanistic probe of -glutamyl hydrolase.
Compound 2 proved to be a substrate for -glutamyl hydrolase and
addition, while some experiments involve single free
fluoroglutamates, many more studies require their incor-
cules.4 Thus, this situation represents an ideal application
for a fluorinated building block.
was hydrolyzed at a rate significantly slower than the corresponding
non-fluorinated analog.
Incorporation of fluoroglutamates into larger compounds
is now a priority given the recent development of a pep-
tide substrate 1, which is internally quenched by fluor-
escence resonance energy transfer (FRET). Enzyme-
catalyzed hydrolysis of this so-called FRET peptide re-
sults in release of the quenched fluorophore, N-(o-ami-
no)benzoyl-L-glutamic acid (Abz-Glu).15 This allows for
a convenient continuous assay of -glutamyl hydrolase
(E.C. 3.4.19.9, GH), an important enzyme involved in the
metabolism of folate and antifolate poly- -glutamate con-
jugates.16 In order to pursue a detailed kinetics examina-
tion of the effect of fluorination on the GH-catalyzed
hydrolysis reaction, synthesis of the corresponding
fluoropeptide, 2, containing (L)-4,4-difluoroglutamic acid
(4,4-F2Glu) as a part of the poly- -glutamate chain, was
desired (Figure 1).
Key words: fluoroamino acids, fluoropeptides, stereoselective syn-
thesis -glutamyl hydrolase, fluorescence enzyme assay
Introduction
Fluorinated amino acids are valuable tools for bioorganic
and medicinal chemists. Due to the properties of the
fluorine atom, substitution of fluorine for hydrogen within
a natural amino acid structure can impart dramatic effects
on the chemical reactivity of the resulting product. This
substitution is sterically conservative, and fluoroamino
acids often possess unique and interesting reactivities
within a biological system. Due to this fact, fluoroamino
acids and larger molecules containing them enjoy wide-
spread popularity and use in applications such as bio-
chemical probes, alternate enzyme substrates, and
enzyme inhibitors.2,3 The power and utility of fluoroami-
no acids is illustrated by this laboratory’s longstanding
history of employing fluorinated glutamic acids to study
the enzymatic synthesis and hydrolysis of folate and anti-
folate poly- -glutamate conjugates.4–8
NH2
O
NH
CO2H
OH
O
X
X
H
N
HO2C
N
NO2
H
O
CO2H
X=H (1), X=F (2)
A consequence of the popularity of fluoroamino acids is
the existence of a large body of literature describing meth-
ods for the synthesis of these compounds.9,10 As methods
for the synthesis of these fluorinated compounds continue
to evolve, more advanced secondary goals such as control
of product stereochemistry11–14 have gained importance.
Another such desirable goal is the development of general
fluorinated building blocks. Successful building blocks
must not only be useful for the synthesis of multiple
Figure 1 The structure of the peptide substrate 1 and its fluorinated
analogue 2.
Positioning fluorines adjacent to the amide bond undergo-
ing enzymatic hydrolysis in 1 is expected to alter its chem-
ical and electronic properties relative to the non-
fluorinated compound and thus have an effect on the hy-
drolysis reaction. In the past, synthesis of compounds
such as 2 first required the isolation of the free fluoro-
glutamates followed by re-protection and various peptide
coupling reactions.4 This approach involves numerous
synthetic steps and it is sometimes necessary to use differ-
Synthesis 2002, No. 17, Print: 02 12 2002.
Art Id.1437-210X,E;2002,0,17,2616,2626,ftx,en;C05102SS.pdf.
© Georg Thieme Verlag Stuttgart · New York
ISSN 0039-7881

SPECIAL TOPIC
4,4-Difluoroglutamyl Peptide Synthesis and Biochemical Studies
2617
ent protection schemes based on the position of the fluoro-
glutamate in the desired synthetic peptide sequence. Thus,
Preparation of the N-Cbz Garner Aldehyde
the synthesis of a universal fluorinated building block The original form of the Garner aldehyde contains a tert-
which can be used to synthesize 2 and other compounds butyl carbamate (Boc) as the protecting group on nitro-
incorporating fluoroamino acids into peptides and isopep- gen, and the majority of the work reported in the literature
tides at any position is desired.
using this type of serine aldehyde also utilizes Boc protec-
tion.17 Due to problems with selective acid-catalyzed ox-
azolidine hydrolysis encountered in preliminary
experiments as well as the protecting group strategy de-
signed for the synthesis of 2, the known but less widely
utilized Garner aldehyde derivative 3 containing a Cbz
group was used in this work. Preparation of 3 is shown in
Scheme 1. This route to 3 takes advantage of a number of
improvements, which have been reported in the literature
since Garner’s original synthesis as well as addressing
factors which are unique to the Cbz derivative. The start-
ing material, D-serine, was esterified and then protected
using benzyl chloroformate to yield the intermediate 8.
After Lewis acid-catalyzed condensation with 2,2-
dimethoxypropane (DMP) to obtain the cyclic product 9,
the methyl ester was carefully reduced using NaBH4 to
obtain the primary alcohol 10.20 Finally, this alcohol was
oxidized using catalytic 2,2,6,6-tetramethylpiperidine-N-
oxide (TEMPO) and NaOCl as the stoichiometric oxi-
dant.21,22 This oxidation method was found to be superior
to the more commonly used Swern oxidation for obtaining
3 due to the speed of the reaction, ease of carrying it out,
the fact that it does not require dry conditions, and be-
cause no unpleasant byproducts such as dimethyl sulfide
are produced.
The serine-derived oxazolidine known as Garner’s alde-
hyde 3 has become one of the most popular building
blocks in asymmetric synthesis due to its ease of prepara-
tion and flexibility for elaboration of the aldehyde carbo-
nyl.17 It has been used in a vast number of applications,
including the synthesis of difluorinated amino acid deriv-
atives.18,19 In the context of the current work, it was pro-
posed that reactions of the aldehyde carbonyl could be
used to create fluoroamino acid side-chain structures, and
then the oxazolidine ring could be employed as a differen-
tially protected N-terminal nitrogen and -carboxyl group
through strategic deprotection and oxidation (Figure 2).
This process could be used to synthesize both free amino
acids 5 or larger molecules such as 4,4-F2Glu-containing
peptides (e.g., 2) via intermediates having the general
structure of 6. For these reasons, Garner’s aldehyde ap-
peared to be an attractive starting material. This report
demonstrates the utility of an enantiomerically pure serine
aldehyde-derived 4,4-difluoroamino acid building block
for the synthesis of a variety of fluorinated compounds. Of
most significance is the 4,4-F2Glu-containing peptide
substrate 2, for use in studying the effect of fluorination
on the GH-catalyzed hydrolysis reaction.
NH2
HO2C
Fluorinated
Side Chain
5
Cbz
Cbz
N
N
O
O
O
O
Fluorinated
Side Chain
H
4
3
R
NH
F
F
H
N
HO2C
R'
O
6
Figure 2 Proposed use of the Garner aldehyde as a fluoroamino acid building block.
Cbz-Cl, NaHCO3
EtOAc, H2O
NH2
CO2H
NH2
CO2CH3
HCl
Acetyl Chloride
MeOH
HO
HO
7
(D)-Serine
2,2-Dimethoxypropane,
BF3.OEt2
Cbz
Cbz
HN
N
O
HO
Acetone
CO2CH3
CO2CH3
9
8
TEMPO, NaBr,
Cbz
NaBH4
THF, MeOH
Cbz
NaOCl
N
N
O
O
OH
H
PhCH3, EtOAc, H2O
87%
O
10
3
Scheme 1 Synthesis of the N-Cbz Garner aldehyde.
Synthesis 2002, No. 17, 2616–2626 ISSN 0039-7881 © Thieme Stuttgart · New York

2618
D. W. Konas et al.
SPECIAL TOPIC
Synthesis of N-Cbz-(2S)-4,4-Difluoroglutamine
4,4-Difluoroglutamine is a compound of interest to bio-
Preparation of a 4,4-Difluoroamino Acid Building
Block
Synthesis of the key 4,4-difluoroamino acid building chemists who study glutamine-dependent amidotrans-
block 13 is shown in Scheme 2. It uses the general strate- ferase enzymes.35–37 The interest in 4,4-difluoroglutamine
gy of nucleophilic attack by a reactive difluorinated com- lies in examining the effect of fluorination on the chemis-
pound at the aldehyde carbonyl followed by try of the glutamyl -carboxamido moiety in these enzy-
deoxygenation of the epimeric secondary alcohol formed matic reactions. Prior to beginning this work, the
in the addition product. In this case, the addition of ethyl preparation of optically active (2S)-4,4-difluoroglutamine
bromodifluoroacetate to the aldehyde carbonyl of 3 via a had not been reported in the literature, and the preparation
Reformatsky-type reaction23 followed by deoxygenation of the racemate had only appeared a single time in a pub-
leads to 13, which contains the proper side-chain structure lication from this laboratory.38 Since this previous synthe-
of 4,4-difluoroglutamic acid. Compound 13 is the desired sis took advantage of a regiospecific aminolysis of a 4,4-
4,4-difluoroamino acid building block (4, Figure 2) be- difluoroglutamate diester, it seemed that the difluorinated
cause it can be used to synthesize (2S)-4,4-difluoro- building block 13 would be well suited for conversion to
glutamate-containing products 6 or processed further to a single enantiomer of (2S)-4,4-difluoroglutamine. As this
obtain other structurally related non-glutamate fluoro- work was close to completion, a short report of the synthe-
amino acids and derivatives 5.
sis of (2S)-4,4-difluoroglutamine appeared in the litera-
ture.39 The reported synthesis utilizes the N-Boc protected
Garner aldehyde and a very similar strategy as that which
is described in this work. A second report40 also examined
this type of strategy for the synthesis of (2S)-4,4-difluoro-
glutamine, but in that case the difluoroglutamine was not
ultimately prepared.
The addition of ethyl bromodifluoroacetate to aldehyde 3
in the presence of freshly activated zinc powder proceed-
ed in good yield to give the product 11 as a mixture of dia-
stereomeric alcohols. One of the two diastereomers was
clearly formed in excess to the other, but the stereochem-
ical identity and individual yields of the two isomers of 11
were not determined as the newly formed chiral alcohol The synthetic pathway developed here to obtain N-Cbz-
was immediately removed in the subsequent step. The (2S)-4,4-difluoroglutamine from 13 is shown in
most popular method for the mild and selective deoxygen- Scheme 3. The first step is hydrolysis of the oxazolidine
ation of alcohols is the radical-based Barton–McCombie ring to give the primary alcohol. As mentioned earlier, the
reaction.24–26 Since its introduction, a variety of related Cbz derivative of the Garner aldehyde was used to help
procedures have been published describing alternatives to improve the selectivity of the hydrolysis reaction. In the
the sometimes-problematic tin hydrides utilized in the case of 13, undesired removal of the Cbz group was un-
original Barton–McCombie method.27–34 After evaluating likely, but competing hydrolysis and/or transesterification
these various deoxygenation reactions, it was determined at the activated , -difluoro ethyl ester required consider-
that treatment of 11 with 1,1 -thiocarbonyldiimidazole in ation. After examining the hydrolysis reaction using prot-
THF to obtain 12, followed by deoxygenation with the tri- ic acids (TsOH, AcOH, HCl) in various solvents as well
ethylsilane/benzoyl peroxide system29 (in dioxane for in- as Lewis acid-catalyzed hydrolysis (BF3 2AcOH), it was
creased solubility of the starting material and product) determined that the best way to achieve selective hydrol-
was preferred. This provided the most reliable deoxygen- ysis and ring-opening of 13 was by using p-toluenesulfon-
ation reaction and highest yields of the desired optically ic acid (TsOH) in refluxing 95% EtOH so any
active 4,4-difluoroamino acid building block 13.
transesterification which took place still led to 14. After
hydrolysis, the alcohol product was immediately isolated
and subjected to oxidation with periodic acid and catalytic
Cbz
Cbz
BrCF2CO2Et, Zn
N
(Im)2C=S
F
F
N
O
O
H
CO2Et
THF
79%
THF
85% crude
O
OH
3
11
Cbz
F
Cbz
F
Et3SiH, benzoyl
peroxide
N
F
O
N
F
O
CO2Et
dioxane
CO2Et
O
62%, 2 steps
S
13
12
Im
Scheme 2 Synthesis of a 4,4-difluoroamino acid building block.
Synthesis 2002, No. 17, 2616–2626 ISSN 0039-7881 © Thieme Stuttgart · New York

SPECIAL TOPIC
4,4-Difluoroglutamyl Peptide Synthesis and Biochemical Studies
2619
Cbz
Cbz
F
HN
1. TsOH, EtOH
2. CrO3, H5IO6, CH3CN-H2O
F
F
N
F
HO
O
CO2Et
CO2Et
O
13
14
Cbz
HN
F
F
NH4OH
MeOH
HO
CONH2
54%, 3 steps
O
15
Scheme 3 Synthesis of N-Cbz-(2S)-4,4-difluoroglutamine.
chromium trioxide41 to give 14. This minimizes any pos- into the final product in a convergent manner. Segment A,
sibility of intramolecular attack of the intermediate prima- N-Boc-ortho-aminobenzoic acid is commercially avail-
ry alcohol on the activated , -difluoro ethyl ester to give able. Segment B is compound 13, already prepared from
an undesired lactone.
the N-Cbz Garner aldehyde 3 (Scheme 2). Segment C is L-
glutamic acid- -tert-butyl-ester- -(2-trimethylsilyl)ethyl
ester which can be prepared via known glutamate protect-
ing group chemistry15 or a commercially available pro-
tected glutamic acid. Finally, segment D, L-3-
nitrotyrosine-tert-butyl ester, has been prepared previous-
ly in this laboratory.15
At this point, compound 14 can be converted to (2S)-4,4-
difluoroglutamic acid by hydrolysis of the ethyl ester and
removal of the Cbz group. This provides another synthetic
route to this compound in addition to those already pub-
lished.13,40,42 Similarly, aminolysis of ester 14 gave the de-
sired primary amide, N-Cbz-(2S)-4,4-difluoroglutamine
15. Compound 15 can be used in peptide synthesis or The synthesis of the important protected (2S)-4,4-difluo-
deprotected to obtain the free amino acid, (2S)-4,4-difluo- roglutamate-containing Glu- -Glu analog 22 from seg-
roglutamine, by hydrogenolysis according to the proce- ments B and C is shown in Scheme 4. Commercially
dure developed previously in this laboratory for the available 16 was protected at the -carboxyl as a (2-trim-
deprotection of racemic 15.38 Unfortunately, the yield of ethylsilyl)ethyl ester so that particular protecting group
this deprotection is significantly lower than that reported would have a different reactivity profile from the tert-bu-
recently for the final deprotection of N-Boc-(2S)-4,4-di- tyl esters used throughout the rest of the molecule.15 Re-
fluoroglutamine.39 In contrast, the oxazolidine hydrolysis, moval of the Cbz group from 17 by hydrogenolysis gave
primary alcohol oxidation, and aminolysis of the more the free amine 18 (Segment C, Figure 3). The preparation
acid-stabile Cbz-protected 13 in this work appears to be of 18 was carried out just prior to using this compound in
more efficient and less problematic than the same se- order to avoid any possible cyclization and undesired py-
quence with the N-Boc derivative.39a These data, taken to- roglutamate formation. To convert compound 13 to the
gether, should ultimately lead to a more efficient synthesis corresponding carboxylate for coupling with 18, the ethyl
of (2S)-4,4-difluoroglutamine.39b
ester was hydrolyzed with NaOH and the sodium salt 19
was isolated.
Compounds 18 and 19 were then coupled using DCC to
form the peptide bond of the (2S)-4,4-F2Glu- -Glu pre-
cursor, 20. The best method for the selective hydrolysis of
the oxazolidine ring in 20 was determined by once again
testing the methods outlined earlier for the hydrolysis of
13. In this case, however, harsh conditions such as reflux-
ing alcohols were avoided and a focus was placed on
milder methods. Ultimately, the use of TsOH in wet ace-
tonitrile at room temperature was successful for obtaining
the desired alcohol, 21, which was once again oxidized
using periodic acid and catalytic chromium trioxide41 to
give the intermediate carboxylic acid. The acid was con-
verted to its tert-butyl ester by alkylation with N,N -di-
cyclohexyl-O-tert-butylisourea43 to give the important
differentially protected (2S)-4,4-F2Glu- -Glu building
block 22.
Synthesis of a (2S)-4,4-Difluoroglutamate-Contain-
ing Peptide Substrate 2 for the Study of -Glutamyl
Hydrolase
As described earlier, the (2S)-4,4-difluoroglutamate-con-
taining peptide 2 is an important synthetic target for use in
the study of GH. The building block 13 is equivalent to a
differentially protected (2S)-4,4-difluoroglutamic acid
molecule that can be utilized in a general method for syn-
thesizing compounds such as 2. Therefore, a goal of this
research was to further demonstrate the general utility of
13 by using it in the synthesis of a (2S)-4,4-difluoro-
glutamate-containing Glu- -Glu analog and incorporating
that precursor into the desired FRET peptide, 2, a poten-
tial GH substrate.
The strategy for the synthesis of 2 is shown in Figure 3.
This target can be divided into four basic segments (A–D),
which were obtained independently and then assembled
Final assembly of the FRET peptide 2 is shown in
Scheme 5. The Cbz group of 22 was removed by hydro-
genolysis and the amine was immediately used for DCC-
Synthesis 2002, No. 17, 2616–2626 ISSN 0039-7881 © Thieme Stuttgart · New York

2620
D. W. Konas et al.
SPECIAL TOPIC
Figure 3 Strategy for the synthesis of the GH FRET substrate 2.
mediated coupling with N-Boc-ortho-aminobenzoic acid HPLC, and LC-MS analysis indicates that both peaks con-
to give 23. To prepare for the final DCC coupling, the tain only the expected product mass. Based on the behav-
TMSE ester of 23 was removed using TBAF. The inter- ior of this compound as a GH substrate (See Biochemistry
mediate carboxylic acid was purified by silica gel column Section), we conclude that the two peaks may be due to
chromatography and then coupled to (L)-3-nitrotyrosine- the presence of conformational isomers of 2 in solution.
tert-butyl ester again using DCC to give the fully protect-
ed FRET substrate 24. Finally, the protecting groups of 24
were removed in one step using anhydrous TFA in CH2Cl2
Biochemistry
to yield the final desired product 2. After removal of the
excess TFA, the final product 2 was purified by DEAE ion Compound 2 was incubated extensively with GH until
exchange chromatography. Compound 2 exhibits two complete hydrolysis was demonstrated by LC-MS. The
closely eluting peaks of almost equal area (1.5:1) by analysis of the hydrolysis mixture showed complete con-
Scheme 4 Synthesis of the protected Glu- -Glu analog 22.
Synthesis 2002, No. 17, 2616–2626 ISSN 0039-7881 © Thieme Stuttgart · New York

SPECIAL TOPIC
4,4-Difluoroglutamyl Peptide Synthesis and Biochemical Studies
2621
Scheme 5 Final assembly of the GH FRET substrate 2.
sumption of the starting material (TR 34 min) with the GH when compared to the corresponding Glu- -Glu pep-
concomitant appearance of a peak (TR 17 min) with a tides.45,46 If the rate difference observed here is due to an
mass of 356.1, corresponding to [M + H]+ of a Glu- - alteration of the rate of breakdown of a reaction interme-
Tyr(NO2) product. A mass of 303.1, corresponding to [M diate rather than a difference in binding affinity, 2 will
+ H]+ for the presumed second product, Abz-F2Glu, was provide a particularly interesting mechanistic probe of the
observed as a minor mass in a peak at TR = 4 minutes. GH reaction. Further investigation of the basis for the
Since this compound was not available as an intermediate slow hydrolysis of 2 is currently underway.
in the synthesis of 2 for use as an authentic HPLC stan-
dard, we were unable to confirm the formation of Abz-
F2Glu by LC-MS. No LC peaks or [M + H]+ ions corre-
sponding to the hydrolysis products were observed in con-
trol reactions without GH. These data demonstrate that 2
is a substrate for GH and appears to be hydrolyzed at the
internal -glutamyl bond, as expected from results with
similar substrates.15,44 Having demonstrated that com-
pound 2 is a substrate for GH, it remained to determine if
the placement of fluorines adjacent to the amide bond un-
dergoing enzymatic hydrolysis had an effect on the rate of
the reaction relative to the non-fluorinated peptide sub-
strate 1.
The progress of the hydrolysis reaction was monitored by
observing the increase in fluorescence of the sample as 2
was hydrolyzed and the intramolecular fluorescence
quenching was relieved (See Experimental Section). A di-
rect comparison of the initial rates (fluorescence counts
per s) between 0.5, 2.5, and 10 M 1 and 2 under identical
conditions is shown in Figure 4. Although 2 was hydro-
lyzed, the rate was significantly slower than for the corre-
Figure 4 A comparison of initial rates between 0.5 (l), 2.5 (p) and
10 M (n) fluorogenic peptide 1 and 0.5 (†), 2.5 (r), and 10
) fluoropeptide 2. Each point shown for the 6 runs is an average of
duplicate exeriments. Inset: Expanded view of data for fluoropeptide
2.
sponding non-fluorinated substrate 1. This is consistent
with preliminary results obtained by an HPLC assay
M
(
method demonstrating that methotrexate-based
-
glutamyl peptides containing (2RS,4RS)-4,4-F2-Glu- -
Glu and (2S,4S)-4-F-Glu- -Glu are slow substrates for
Synthesis 2002, No. 17, 2616–2626 ISSN 0039-7881 © Thieme Stuttgart · New York

2622
D. W. Konas et al.
SPECIAL TOPIC
product as a yellow oil which was used without further purification.
THF was freshly distilled from sodium/benzophenone. CH2Cl2 was
freshly distilled from CaH2. 1,4-Dioxane was distilled from LiAlH4.
Zinc powder was washed with 1 N HCl for 1 min followed by wash-
ings with distilled water, EtOH, Et2O, and drying completely under
high vacuum. N-Boc-o-Aminobenzoic acid (Boc-Abz-OH) was ob-
tained from Advanced ChemTech, Louisville, KY, USA. All other
reagents and starting materials were obtained from Sigma–Aldrich
or Fisher–Acros and used without further purification. Air- and
moisture-sensitive reactions were run in flame- or oven-dried (T
>100 °C overnight) glassware under dry nitrogen or argon.
Conclusions
The development of optically active fluorinated synthetic
building blocks of general utility is a current goal of orga-
nofluorine chemists. The serine-derived Garner aldehyde
was successfully converted to the general 4,4-difluoro-
amino acid building block 13, through a fluoro-Refor-
matsky reaction with ethyl bromodifluoroacetate. The
utility of this building block for synthesizing difluoroam-
ino acids was demonstrated by the synthesis of N-Cbz-
(2S)-4,4-difluoroglutamine (15), via an intermediate 14,
which could also be converted to (2S)-4,4-difluoro-
glutamic acid. The ability of 13 to be conveniently incor-
(4R)-4-Formyl-2,2-dimethyloxazolidine-3-carboxylic Acid Ben-
zyl Ester (3)
(S)-4-Hydroxymethyl-2,2-dimethyloxazolidine-3-carboxylic acid
porated into larger structures was demonstrated with the benzyl ester 1020 (1.85 g, 7.0 mmol) and NaBr (720 mg, 7.0 mmol,
1 equiv) were dissolved in a vigorously stirred solvent mixture of
toluene–EtOAc–H2O (6:6:1) and then cooled to 0 °C in an ice-water
bath before the addition of solid TEMPO (22 mg, 0.14 mmol, 0.02
equiv). An aqueous solution of NaOCl (573 mg, 7.7 mmol, 1.1
synthesis of a (2S)-4,4-difluoroglutamate-containing
isopeptide 2, designed as a potential mechanistic probe of
-glutamyl hydrolase. The initial biochemical evaluation
of 2 demonstrates that it is a substrate for -glutamyl hy-
equiv, 0.35 M) and NaHCO3 (1.7 g, 20.3 mmol, 2.9 equiv) was con-
drolase and is hydrolyzed at a rate significantly slower
than the corresponding non-fluorinated FRET peptide 1.
Given the utility of the Garner aldehyde and building
block 13, this chemistry and the various intermediates in-
volved should continue to be of interest to organofluorine
chemists and biochemists.
tinuously added dropwise over 1 h. The reaction mixture was stirred
an additional 15 min after complete addition of the oxidant solution
and then H2O was added to the flask. The layers were separated and
the aqueous layer was extracted with Et2O. The combined organic
layers were washed with solutions of KI (4 mg/mL) in 10% aq
KHSO4, 10% aq Na2S2O3, and then brine, and dried (Na2SO4). The
solvents were removed in vacuo and the resulting crude product was
purified by silica gel column chromatography (hexanes–EtOAc,
1:1) to give the desired aldehyde 3, as a colorless oil (1.60 g, 87%);
Rf 0.56 (hexanes–EtOAc, 2:1).
1H NMR (DMSO-d6, 300 MHz, 80 °C): = 1.50 (s, 3 H), 1.56 (s, 3
H), 4.13 (d, 2 H, J = 5.1 Hz), 4.52 (dt, 1 H, J = 5.1, 1.4 Hz), 5.11 (s,
2 H), 7.35 (m, 5 H), 9.57 (s, 1 H).
13C NMR (DMSO-d6, 75.5 MHz, 80 °C): = 23.46, 25.24, 62.80,
64.08, 66.00, 94.00, 127.06, 127.42, 127.92, 136.03, 151.50,
198.60.
TLC was performed with standard Sigma-Aldrich brand silica gel
60 F-254 plates. Column chromatography was performed with sili-
ca gel 60 (230–400 mesh). Melting points were obtained on a Tho-
1
mas–Hoover Mel-Temp apparatus and are uncorrected. H NMR,
13C and 19F NMR spectra were recorded on Bruker Avance
DRX300 and DRX500 spectrometers using the X-WinNMR soft-
ware. Chemical shifts are reported in parts per million (ppm) upfield
1
or downfield from tetramethylsilane (internal standard for H and
13C) or trifluoroacetic acid (external standard for 19F ). The NMR
spectra of some intermediates containing the oxazolidine structure
derived from 3 exhibited line broadening and doubling of some sig-
nals due to slow or restricted rotation within the molecule. NMR
spectra of these compounds were obtained at elevated temperatures
when it aided in simplifying their appearance. IR spectra were re-
corded on a Nicolet 5-DX spectrometer. Mass spectra were ob-
tained with a VG 70-250-S mass spectrometer made by Micromass
(UK) and an Opus data system. HPLC chromatograms were ob-
tained with Waters 510 and 6000A model pumps with a 996 diode
array detector using Millenium version 3.20 software and a Vydac
C18 column. A gradient elution of 15% to 20% solvent A (0.1%
AcOH, 0.02% TFA in MeCN) in solvent B (0.1% AcOH, 0.02%
TFA in H2O) over 25 min followed by an increase to 95% solvent
A in 15 min was used unless otherwise specified. LC-MS data were
collected from ThermoFinnigan Surveyor HPLC components with
a Vydac C18 column and a Finnigan LCQ.
(4R)-4-[(1RS)-2-Ethoxycarbonyl-2,2-difluoro-1-hydroxyethyl]-
2,2-dimethyloxazolidine-3-carboxylic Acid Benzyl Ester (11)
A stirred suspension of zinc powder (2.8 g, 43.2 mmol, 3.0 equiv)
in THF was heated to reflux temperature in a flask equipped with a
condenser and drying tube and a solution of the aldehyde 3 (3.8 g,
14.4 mmol) and ethyl bromodifluoroacetate (3.79 g, 18.7 mmol, 1.3
equiv) in THF was added dropwise over 15 min. After complete ad-
dition, the reaction mixture was stirred an additional 15 min before
being cooled to r.t. and quenched with 1 N aq KHSO4. The mixture
was diluted with CH2Cl2 and then filtered through Celite. The fil-
trate was condensed in vacuo and the resulting residue was parti-
tioned between EtOAc and H2O. The EtOAc layer was washed with
brine, dried (Na2SO4), and condensed in vacuo. The resulting crude
product was purified by silica gel column chromatography
(CH2Cl2–EtOAc, 16:1) and the desired product 11, was obtained as
a colorless oil consisting of a 3:1 mixture of diastereomers (4.4 g,
79%); Rf 0.58, 0.50 (CH2Cl2– EtOAc, 16:1). A sample of only the
major (lower Rf) diastereomer was isolated and utilized for spectral
characterization.
1H NMR (DMSO-d6, 500 MHz, 80 °C): = 1.28 (t, 3 H, J = 7.1 Hz),
1.47 (s, 3 H), 1.54 (s, 3 H), 3.97 (dd, 1 H, J = 8.7, 7.1 Hz), 4.15 (dd,
1 H, J = 8.8, 3.5 Hz), 4.20 (m, 1 H), 4.29 (q, 2 H, J = 7.1 Hz), 4.53
(m, 1 H), 5.14 (s, 2 H), 6.0 (br, 1 H), 7.33–7.89 (m, 5 H).
13C NMR (DMSO-d6, 125.75 MHz, 80 °C): = 13.64, 23.76 (br),
25.49, 55.99, 62.19, 62.34, 65.91, 68.30 (m), 93.08, 114.90 (t,
1JC-F = 256 Hz), 127.27, 127.43, 127.91, 136.08, 151.63, 162.40 (t,
2JC-F = 31.3 Hz).
Compound 1 was prepared as previously described.15 (S)-4-Hy-
droxymethyl-2,2-dimethyloxazolidine-3-carboxylic acid benzyl es-
ter 10 was synthesized from D-serine according to the procedure
described by Delle Monache et al.20 for the preparation of its enan-
tiomer. N-Cbz-(L)-glutamic acid- -tert-butyl ester dicyclohexylam-
monium salt 16 was purchased from Chem Impex International,
Inc., Wood Dale, IL, USA. N,N -Dicyclohexyl-O-tert-
butylisourea43 was prepared by stirring DCC in tert-butyl alcohol
(1.5 equiv) at 35 °C with CuCl (catalytic). The reaction was com-
plete (approximately 24 h) when the IR spectrum indicated the loss
of the carbodiimide (2100 cm–1) and the appearance of the isourea
(1660 cm–1). The mixture was diluted with CHCl3 and all volatile
components were evaporated in vacuo to give the desired isourea
Synthesis 2002, No. 17, 2616–2626 ISSN 0039-7881 © Thieme Stuttgart · New York

SPECIAL TOPIC
4,4-Difluoroglutamyl Peptide Synthesis and Biochemical Studies
2623
19F NMR (DMSO-d6, 282.38 MHz, 60 °C): = –44.5 (dm,
2JF-F = 258 Hz), –35.8 (d, 2JF-F = 258 Hz).
CI-MS (NH3): m/z (%) = 388.1 (MH+, 90.8), 330.1 (100.0), 108.1
N-Cbz-(L)-4,4-Difluoroglutamine (15)
Compound 13 (86 mg, 0.23 mmol) was dissolved in 95% EtOH and
p-toluenesulfonic acid monohydrate (11 mg, 0.05 mmol, 0.25
equiv) was added. The solution was stirred and heated at reflux until
TLC (hexanes–EtOAc, 1:1) indicated the complete loss of the start-
ing material (Rf 0.75) and formation of the desired product alcohol
(Rf 0.44) (5 h). The reaction was cooled to r.t. and the solvent was
removed in vacuo. The resulting colorless oil was immediately dis-
solved in wet MeCN (1.4 mL, 0.75 vol% H2O) and cooled to 0 °C
in an ice-water bath. This solution was treated with a stock solution
containing H5IO6 (132 mg, 0.58 mmol, 2.5 equiv) and CrO3 (0.25
mg, 2.50 mol, 1.1 mol%) in wet MeCN (1.4 mL, 0.75 vol% H2O)
added dropwise over 40 min. After complete addition, the reaction
mixture was stirred for an additional 20 min. Any active oxidant re-
maining in the heterogeneous reaction mixture was quenched with
the addition of i-PrOH (1 mL) and stirring for 10 min until a light
green color was observed. All solvents were removed in vacuo to
leave a crude solid residue. This residue was dissolved in half-sat.
aq NaCl and this solution was extracted with CH2Cl2. The combined
extracts were dried (Na2SO4) and condensed in vacuo to leave the
crude carboxylic acid product 14 as a colorless oil. This oil was dis-
solved in MeOH (5 mL) and the solution was cooled to 0 °C in an
ice-water bath before 30% aq NH4OH (1.5 mL) was added. The re-
action mixture was stirred for 1 h and then the flask was warmed to
r.t. The solvents were removed in vacuo and the remaining residue
was redissolved in MeOH and the solution was acidified (pH 2.0)
with 1 N KHSO4. Removal of the solvents again in vacuo left the
crude product as a white solid residue which was purified by silica
gel column chromatography eluting with a solution consisting of
62.5% EtOAc, 30% hexanes, and 7.5% AcOH. The product-con-
taining fractions were combined and the solvent was removed in
vacuo. Removal of the final traces of AcOH was facilitated by re-
peatedly dissolving the product in 1:5 EtOAc–cyclohexane and
evaporating in vacuo. The desired product 15 was obtained as a
white solid (40 mg, 54% yield in 3 steps from 13); Rf 0.38 (EtOAc–
hexanes–AcOH, 62.5:30.0:7.5); mp 146–149 °C; [ ]D23 –14.8 (c =
0.75, MeOH).
(23.7).
CI-HRMS (NH3): m/z calcd for C18H24F2NO6 (MH+) 388.1572,
found 388.1556.
Anal. Calcd for C18H23F2NO6: C, 55.81; H, 5.98; N, 3.62. Found: C,
55.38; H, 5.99; N, 3.67.
(4S)-4-(2-Ethoxycarbonyl-2,2-difluoroethyl)-2,2,-dimethyl-
oxazolidine-3-carboxylic Acid Benzyl Ester (13)
The diastereomeric alcohols 11 (500 mg, 1.29 mmol) and 1,1 -thio-
carbonyldiimidazole (346 mg, 1.94 mmol, 1.5 equiv) were dis-
solved in THF and stirred at 30–40 °C for 48 h. The THF was
removed in vacuo and the resulting residue was dissolved in a min-
imum of EtOAc and purified by silica gel column chromatography
(hexanes–EtOAc, 1:1) to give the desired intermediate 12 as a col-
orless oil containing an inseparable mixture of diastereomers (548
mg, 85%). The intermediate 12 derived from only the lower Rf di-
astereomer of 11 was used for characterization; Rf 0.52 (hexanes–
EtOAc, 1:1).
1H NMR (DMSO-d6, 300 MHz, 80 °C): = 1.18 (t, 3 H, J = 7.0 Hz),
1.23 (s, 3 H), 1.41 (s, 3 H), 4.18 (dd, 1 H, J = 9.9, 7.0 Hz), 4.27 (m,
2 H), 4.38 (d, 1 H, J = 9.9 Hz), 4.61 (d, 1 H, J = 6.5 Hz), 5.18 (s, 2
H), 6.69 (dd, 1 H, J = 15.2, 10.7 Hz), 7.12 (m, 1 H), 7.33–7.41 (m,
5 H), 7.77 (m, 1 H), 8.44 (m, 1 H).
19F NMR (DMSO-d6, 282.38 MHz, 80 °C): = –38.7 (d, 2JF-F = 266
Hz), –35.6 (dd, 2JF-F = 266 Hz, 3JH-F = 85 Hz).
FAB-MS (NBA with Na+): m/z (%) = 498.1 (MH+, 21.6), 91.0
(100.0).
FAB-HRMS (with Na+): m/z calcd for C22H26F2N3O6S (MH+)
498.1510, found 498.1521.
Compound 12 (431 mg, 0.87 mmol) and triethylsilane (7 mL) were
added to a flask equipped with a condenser and drying tube and di-
oxane was added slowly until the solution was clear and homoge-
neous. The reaction mixture was stirred and heated at reflux
temperature while portions of solid benzoyl peroxide (10 mg) were
added at 15 min intervals until complete consumption of the starting
material was observed by TLC. The reaction was cooled to r.t. and
repeatedly diluted with EtOAc and concentrated in vacuo to remove
the triethylsilane and obtain the crude product. This crude residue
was purified by silica gel column chromatography (hexanes–
EtOAc, 4:1) and the desired product 13, was obtained as a colorless
oil (200 mg, 62% yield from 11); Rf 0.75 (hexanes–EtOAc, 1:1);
[ ]D23 +19.0 (c = 0.95, MeOH).
1H NMR (CD3OD, 300 MHz): = 2.50–2.76 (m, 2 H), 4.45 (dd, 1
H, J = 9.7, 3.3 Hz), 5.09 (s, 2 H), 7.33 (m, 1 H).
13C NMR (CD3OD, 75.5 MHz): = 36.50 (t, JC-F = 23.7 Hz),
2
1
50.22, 67.68, 119.74 (d, JC-F = 252 Hz), 128.72, 128.95, 129.44,
138.13, 158.32, 168.28 (t, 2JC-F = 29 Hz), 174.25.
19F NMR (CD3OD, 282.38MHz, 80 °C): = –31.31 (dt, 2JF-F = 257,
3JH-F = 16 Hz), –30.21 (dt, 2JF-F = 257, 3JH-F = 16 Hz).
DCI-MS (NH3): m/z (%) = 334.0 ([M + NH]+, 6.6), 169.1 (65.4),
139.0 (139.0), 106.0 (63.8), 88.0 (74.3).
DCI-HRMS (NH3): m/z calcd for C13H18F2N3O5 ([M + NH4]+)
334.1214, found 334.1218.
1H NMR (DMSO-d6, 300 MHz, 75 °C): = 1.26 (t, 3 H, J = 7.1 Hz),
1.45 (s, 3 H), 1.52 (s, 3 H), 2.30–2.51 (m, 2 H), 3.82 (dd, 1 H,
J = 9.2, 1.5 Hz), 4.04 (dd, 1 H, J = 9.2, 5.5 Hz), 4.20 (m, 1 H), 4.27
(q, 2 H, J = 7.1 Hz), 5.12 (s, 2 H), 7.37 (m, 5 H).
N-Cbz-(L)-Glutamic Acid- -tert-butyl Ester -(2-Trimethyl-
silyl)ethyl Ester (17)
13C NMR (DMSO-d6, 75.47 MHz, 75 °C): = 14.11, 24.11, 27.24,
N-Cbz-(L)-glutamic acid- -tert-butyl ester dicyclohexylammonium
salt (16; 800 mg, 1.54 mmol) and 2-(trimethylsilyl)ethanol (260 mg,
2.2 mmol, 1.4 equiv) were dissolved in CH2Cl2. DMAP (188 mg,
1.54 mmol, 1.0 equiv) and then 1-ethyl-3-(3 -dimethylaminopro-
pyl)carbodiimide hydrochloride (EDC, 383 mg, 2.0 mmol, 1.3
equiv) were added to the solution and the mixture was stirred at r.t.
for 36 h. The mixture was diluted with CH2Cl2 and the CH2Cl2 layer
was washed with 0.5 N aq HCl, H2O, and brine. The organic layer
was dried (Na2SO4) and condensed in vacuo to give the crude prod-
uct, which was purified by silica gel column chromatography (hex-
anes–EtOAc, 2:1). The desired product 17, was obtained as a
colorless oil (580 mg, 86%); Rf 0.45 (hexanes– EtOAc, 2:1).
37.74 (m), 52.21, 63.58, 66.81, 67.67, 93.79, 115.74 (t, 1JC-F = 250
2
Hz), 128.16, 128.41, 128.90, 137.07, 152.08, 163.47 (t, JC-F = 32
Hz).
19F NMR (DMSO-d6, 282.38 MHz, 75 °C): = –26.4 (d, 2JF-F = 263
Hz), –28.6 (dt, 2JF-F = 262 Hz, 3JH-F = 19 Hz).
FAB-MS (NBA): m/z (%) = 372.2 (MH+, 33.6), 328.2 (23.4), 91.0
(100.0).
FAB-HRMS (NBA): m/z calcd for C18H24F2NO5 (MH+) 372.1622,
found 372.1633.
Anal. Calcd for C18H23F2NO5 0.7H2O: C, 56.33; H, 6.40; N, 3.65.
Found: C, 56.33; H, 6.00; N, 3.52.
Synthesis 2002, No. 17, 2616–2626 ISSN 0039-7881 © Thieme Stuttgart · New York

2624
D. W. Konas et al.
SPECIAL TOPIC
1H NMR (CDCl3, 300 MHz): = 0.03 (s, 9 H), 0.97 (m, 2 H), 1.46
(s, 9 H), 1.81–2.41 (m, 4 H), 4.16 (m, 2 H), 4.29 (m, 1 H), 5.10 (s,
2 H), 5.43 (d, 1 H), 7.34 (m, 5 H).
13C NMR (CDCl3, 75.5 MHz): = –1.34, 17.42, 28.09, 28.11,
30.53, 54.00, 63.04, 67.07, 82.54, 128.25, 128.29, 128.67, 136.43,
156.07, 171.14, 173.06.
alcohol product 21, was obtained as a colorless oil (19 mg, 65%);
Rf 0.41 (hexanes–EtOAc, 1:1).
1H NMR (CDCl3, 300 MHz): = 0.02 (s, 9 H), 0.97 (m, 2 H), 1.46
(s, 9 H), 1.94–2.52 (m, 6 H), 2.95 (br, 1 H), 3.68 (m, 2 H), 3.96 (m,
1 H), 4.17 (m, 2 H), 4.45 (m, 1 H), 5.07 (dd, 2 H, J = 18, 12 Hz),
5.49 (d, 1 H, J = 8.3 Hz), 7.27–7.35 (m, 5 H).
FAB-MS (NBA with Na+) m/z (%) = 460.1 (MNa+, 13.8), 354.0
(10.3), 310.1 (18.82), 91.0 (100.0).
13C NMR (CDCl3, 75.5 MHz): = –1.30, 17.47, 27.04, 28.14,
2
30.47, 35.26 (t, JC-F = 23 Hz), 48.28, 52.74, 63.45, 64.89, 67.09,
1
83.43, 117.33 (t, JC-F = 253 Hz), 128.26, 128.34, 128.74, 136.52,
FAB-HRMS (NBA with Na+): m/z calcd for C22H35NNaO6Si
156.42, 164.60 (t, 2JC-F = 29 Hz), 170.11, 173.26.
(MNa+) 460.2131, found 460.2120.
19F NMR (CDCl3, 282.38 MHz): = –27.1 (dt, 2JF-F = 261 Hz,
Anal. Calcd for C22H35NO6Si: C, 60.38; H, 8.06; N, 3.20. Found: C,
60.17; H, 7.96; N, 3.02.
3JH-F = 16 Hz), –28.6 (dt, 2JF-F = 261 Hz, 3JH-F = 17 Hz).
N-Cbz-(2S)-4,4-Difluoroglutamic Acid- -tert-butyl Ester- -
glutamic Acid- -tert-butyl- -(2-trimethylsilyl)ethyl Ester (22)
Compound 21 (48 mg, 0.08 mmol) was dissolved in wet MeCN (1.0
mL, 0.75 vol% H2O) and cooled to 0 °C in an ice-water bath. A
stock solution containing H5IO6 (46 mg, 0.20 mmol, 2.5 equiv) and
CrO3 (0.09 mg, 1.1 mol%) in wet MeCN (1.0 mL, 0.75 vol% H2O)
was slowly added dropwise over 40 min. After complete addition
the mixture was stirred for an additional 1.5 h before being
quenched with aq phosphate buffer [prepared by dissolving
Na2HPO4 (600 mg) in H2O (10 mL)]. The solution was extracted
with EtOAc. The combined extracts were dried (Na2SO2), filtered,
and evaporated in vacuo to leave the crude carboxylic acid interme-
diate. The carboxylic acid (48 mg, 0.08 mmol) was dissolved in
CH2Cl2 and cooled to 0 °C in an ice-water bath. A solution of N,N -
dicyclohexyl-O-tert-butylisourea (56 mg, 0.2 mmol, 2.5 equiv) in
CH2Cl2 was added and after 15 min the ice-water bath was removed
and the mixture stirred for 24 h. An additional portion of N,N -dicy-
clohexyl-O-tert-butylisourea43 (56 mg, 0.2 mmol, 2.5 equiv) in
CH2Cl2 was added and the mixture stirred for an additional 12 h.
The reaction was diluted with CH2Cl2, filtered, and the filtrate was
condensed to give the crude product as a light yellow oil. The crude
product was purified by two sequential silica gel columns: 1st (hex-
anes–EtOAc, 2:1), 2nd (CHCl3–EtOAc, 95:5) to give the desired
product, 22, as a colorless oil (47 mg, 89% yield in two steps from
21); Rf 0.49 (CHCl3–EtOAc, 95:5).
N-Cbz-Difluorooxazolidine- -(L)-glutamic Acid- -tert-butyl
Ester -(2-Trimethylsilyl)ethyl Ester (20)
Compound 13 (250 mg, 0.67 mmol) was dissolved in THF and
cooled to 0 °C in an ice-water bath before 1 N NaOH (870 L, 0.87
mmol, 1.3 equiv) was added. The solution was stirred at 0 °C for 1
h. The solvents were removed in vacuo and the solid residue was re-
dissolved in H2O, frozen in a –78 °C bath, and lyophilized to give
the desired sodium salt 19 as a white hygroscopic solid which was
used without further purification. The differentially protected
glutamate 17 (550 mg, 1.26 mmol) was dissolved in MeOH–i-PrOH
(4:1) and 10% Pd/C (100 mg) was added. The mixture was shaken
in a Parr apparatus with H2 (40 psi) for 90 min. The reaction was di-
luted with an equal volume of EtOAc and filtered through Celite to
remove the catalyst. The solvents were removed in vacuo to give the
desired amine 18 as a colorless oil, which was used without further
purification. CH2Cl2 (5 mL) was added to the sodium salt 19 (0.67
mmol) and DMF was slowly added until the mixture was complete-
ly homogeneous. The amine 18 (303 mg, 1.0 mmol, 1.5 equiv),
HOBt (135 mg, 1.0 mmol, 1.5 equiv), and finally DCC (206 mg, 1.0
mmol, 1.5 equiv) were added to the solution and it was allowed to
stir at r.t. for 48 h. The reaction mixture was diluted with CH2Cl2
and filtered through Celite. The filtrate was concentrated in vacuo
to give the crude product which was purified by two sequential sil-
ica gel columns: 1st (hexanes–EtOAc, 2:1), 2nd (CHCl3–EtOAc,
95:5). The desired amide product 20 was obtained as a colorless oil
(402 mg, 95% yield in two steps from 13); Rf 0.32 (CHCl3–EtOAc,
95:5); [ ]D23 +17.1 (c = 0.70, CHCl3).
1H NMR (CDCl3, 300 MHz): = 0.03 (s, 9 H), 0.97 (m, 2 H), 1.47
(s, 18 H), 2.02–2.74 (m, 6 H), 4.17 (m, 2 H), 4.46 (m, 2 H), 5.10 (dd,
2 H, J = 16, 12 Hz), 5.57 (d, 1 H, J = 8 Hz), 7.21 (d, 1 H, J = 8 Hz),
7.35 (m, 5 H).
1H NMR (DMSO-d6, 500 MHz, 55 °C): = 0.02 (s, 9 H), 0.94 (m,
2 H), 1.40 (s, 9 H), 1.43 (s, 3 H), 1.50 (s, 3 H), 1.96 (m, 1 H), 2.07
(m, 1 H), 2.33 (m, 2 H), 2.36 (t, J = 7.8 Hz), 3.86 (dd, J = 9.2, 1.4
Hz), 4.00 (dd, J = 9.1, 5.5 Hz), 4.13 (m, 2 H), 4.21 (m, 2 H), 5.11 (s,
2 H), 7.30–7.37 (m, 5 H), 8.81 (d, 1 H, J = 7.3 Hz).
13C NMR (DMSO-d6, 125.75 MHz, 60 °C): = –1.89, 16.53, 25.13,
26.42, 27.24, 29.90, 39.62, 51.99, 61.50, 65.81, 66.57, 80.85, 92.69,
116.18 (t, 1JC-F = 254 Hz), 127.16, 127.44, 127.97, 136.20, 151.08,
163.08 (t, 2JC-F = 29 Hz), 169.20, 171.63;
13C NMR (CDCl3, 75.5 MHz): = –1.28, 17.47, 27.16, 28.05,
2
28.16, 29.94, 35.76 (t, JC-F = 23 Hz), 49.94, 52.66, 63.36, 67.20,
1
83.14, 83.34, 116.95 (t, JC-F = 254 Hz), 128.28, 128.34, 128.71,
136.48, 155.86, 163.62 (t, 2JC-F = 29 Hz), 169.64, 169.98, 173.13;
19F NMR (CDCl3, 282.38 MHz): = –28.17 (t, J = 16 Hz).
N-Boc-Abz-4,4-Difluoroglutamic Acid- -tert-butyl- -glutamic
Acid- -tert-butyl- -(2-trimethylsilyl)ethyl Ester (23)
19F NMR (DMSO-d6, 470.56 MHz, 55 °C): = –28.04 (m).
Compound 22 (45 mg, 0.068 mmol) was dissolved in THF and a
suspension of 10% Pd/C (12 mg, catalytic) in THF was added. The
mixture was cooled to 0 °C in an ice-water bath and then vigorously
stirred under an atmosphere of H2 (1 atm) for 2 h. The reaction mix-
ture was diluted with EtOAc, the catalyst was removed by filtration,
and the filtrate was concentrated in vacuo to give the desired inter-
mediate deprotected amine, which was immediately used without
further purification. This amine was dissolved in anhyd DMF and
then N-Boc-ortho-aminobenzoic acid (25 mg, 0.102 mmol, 1.5
equiv), HOBt (14 mg, 0.102 mmol, 1.5 equiv), and DCC (21 mg,
0.102 mmol, 1.5 equiv) were added in that order. The mixture
stirred at r.t. for 4 d. The reaction mixture was diluted with EtOAc,
filtered, and then the solvents were removed in vacuo to give the
crude product. This crude residue was dissolved in a minimum of
EtOAc and purified by silica gel column chromatography (hex-
FAB-MS (NBA with Na+) m/z (%) = 651.2 (MNa+, 39.8), 487.1
(49.5), 91.0 (100.);
FAB-HRMS (NBA with Na+): m/z calcd for C30H46F2N2NaO8Si
(MNa+) 651.2889, found 651.3008.
N-Cbz-(l)-4-Aminobutyryl-3,3-difluoro-(2-hydroxymethyl)-
(L)-glutamic Acid- -tert-butyl Ester -(2-Trimethylsilyl)ethyl
Ester (21)
Compound 20 (31 mg, 0.05 mmol) was dissolved in MeCN and then
H2O (40 L) and TsOH H2O (3 mg, 0.015 mmol, 0.3 equiv) were
added. The solution was stirred at r.t. for 19 h. The solvents were re-
moved in vacuo. The crude product was purified by silica gel col-
umn chromatography (hexanes–EtOAc, 1:1) and the desired
Synthesis 2002, No. 17, 2616–2626 ISSN 0039-7881 © Thieme Stuttgart · New York

SPECIAL TOPIC
4,4-Difluoroglutamyl Peptide Synthesis and Biochemical Studies
2625
anes–EtOAc, 2:1) to give the desired product 23, as a colorless oil
(44 mg, 87% yield in two steps from 22); Rf 0.57 (hexanes–EtOAc,
2:1).
1H NMR (CDCl3, 500 MHz): = 0.02 (s, 9 H), 0.97 (m, 2 H), 1.41
(s, 9 H), 1.49 (s, 9 H), 1.50 (s, 9 H), 2.03 (m, 1 H), 2.19 (m, 1 H),
2.34 (m, 2 H), 2.79 (m, 2 H), 4.15 (m, 2 H), 4.40 (m, 1 H), 4.79 (m,
1 H), 7.00 (t, 1 H, J = 7.5 Hz), 7.14 (d, 1 H, J = 7.6 Hz), 7.29 (d, 1
H, J = 7.5 Hz), 7.43 (t, 1 H, J = 7.3 Hz), 7.51 (d, 1 H, J = 6.9 Hz),
8.36 (d, 1 H, J = 8.4 Hz), 10.07 (s, 1 H).
FAB-HRMS (DTT/DTE w/Na+): m/z calcd for C26H27F2N5NaO12,
662.1522 [M + Na]+, found 662.1507.
A fraction of the crude material (3.2 mg) was purified by ion ex-
change chromatography using Whatman DE52 cellulose in a mini-
column. DE52 (3.0 g) was prepared following the manufacturer’s
directions. The column was packed in a glass pipette to a height of
8 cm and equilibrated in 0.02 M Et3NH2CO3, pH 8, conductivity
1.22 mS. A solution of crude 2 in 0.02 M Et3NH2CO3 was applied
to the column and eluted with a linear gradient from 0.02 M
Et3NH2CO3 (100 mL) to 1.0 M Et3NH2CO3 (pH 8, conductivity 30.0
mS, 100 mL). Fractions containing 2 were identified by UV absor-
bance spectra and were selectively combined based on comparison
of chromatograms (HPLC) obtained with a gradient elution of 15 to
20% solvent A (0.1% AcOH, 0.02% TFA in MeCN) in solvent B
(0.1% AcOH, 0.02% TFA in H2O) over 25 min followed by an in-
crease to 95% solvent A over 15 additional min. The total column
recovery was 90.3% based on UV absorbance. Selected fractions
each containing greater than 90% of the desired product by HPLC
were combined and lyophilized to provide 1.1 mg (34.4%) of the
desired product 2; HPLC: TR = 22.9, 24.8 min.
13C NMR (CDCl3, 125.75 MHz): = –1.34, 17.44, 27.11, 28.04,
28.52, 30.36, 35.37 (t, 2JC-F = 23.7 Hz), 48.66, 52.68, 63.35, 80.37,
1
83.31, 83.38, 116.87 (t, JC-F = 254 Hz), 119.10, 119.87, 121.65,
127.15, 132.96, 140.67, 153.18, 163.78 (t, 2JC-F = 28.6 Hz), 168.74,
169.41, 169.73, 173.06.
19F NMR (CDCl3, 282.38 MHz): = –25.8 (dt, 2JF-F = 264 Hz,
2
2JH-F = 15.2 Hz), –28.4 (dt, 2JF-F = 264 Hz, JH-F = 16.9 Hz).
FAB-MS (3-NBA with Na+): m/z (%) = 766.5 (MNa+, 7.6), 329.1
(21.5), 307.1 (21.4), 176.1 (54.3), 154.1 (100.0), 136.1 (70.8).
FAB-HRMS (3-NBA with Na+): m/z calcd for C35H55F2N3NaO10Si
1H NMR: (D2O, 300 MHz): = 1.19–1.24 [t, 21 H, (CH3CH2)3N],
1.65–2.06 (m, 4 H), 2.68–3.04 (m, 4 H), 3.10–3.18 [q, 13 H,
(CH3CH2)3N], 3.88 (m, 1 H), 4.33–4.37 (m, 1 H), 4.51–4.54 (m, 1
H), 6.78–6.84 (m, 2 H), 7.02–7.05 (d, 1 H), 7.26 (m, 1 H), 7.36–7.45
(m, 2 H), 7.84–7.87 (d, 1 H).
(MNa+) 766.3522, found 766.3526.
N-Boc-Abz-4,4-Difluoroglutamic Acid- -tert-butyl- -glutamic
Acid- -tert-butyl- -3-nitrotyrosine-tert-butyl Ester (24)
Compound 23 (40 mg, 0.054 mmol) was dissolved in THF and
cooled to 0 °C in an ice-water bath. A 1 M aq solution of TBAF (81
L, 1.5 equiv) was added and the reaction mixture was stirred for 2
h. The reaction was quenched with the addition of sat. aq NH4Cl and
then all of the solvents were removed in vacuo. The resulting crude
acid product was partially purified by silica gel column chromatog-
raphy (2% AcOH in CHCl3–EtOAc, 3:1) (Rf 0.38). All product-
containing fractions were combined and concentrated in vacuo to
give a residue corresponding to slightly greater than 100% yield.
This carboxylic acid and L-3-NO2-tyrosine-tert-butyl ester (28 mg,
0.10 mmol, 1.8 equiv) were dissolved in anhyd DMF and HOBt (11
mg, 0.081 mmol, 1.5 equiv) and then DCC (17 mg, 0.081 mmol, 1.5
equiv) were added. The yellow reaction mixture stirred at r.t. for 48
h. The DMF was removed in vacuo and the resulting yellow residue
was dissolved in EtOAc, filtered, and then the filtrate was concen-
trated in vacuo to give the crude product. This crude material was
purified by silica gel column chromatography (hexanes–EtOAc,
1:5) to give the desired product 24, as a light yellow oil (44 mg, 90%
yield in 2 steps from 23); Rf 0.42 (hexanes–EtOAc, 1:5).
The low integration observed for (CH3CH2)3N in the 1H NMR spec-
trum suggests the lyophilized material contains ca. 2 (CH3CH2)3N
per molecule.
19F NMR: (D2O, 282 MHz): = –25.8 (dt), –28.2 (dt), –29.4 (dt), –
32.7 (dt).
LC-MS: TR 34 min; [M + H]+ m/z calcd 640.2, found 640.1.
Fluorogenic Assay
The assay was carried out as previously described15 with the follow-
ing modifications. Concentrations of stock solutions of 1 and 2 were
based on UV absorbance at 285 nm ( = 5080 M–1cm–1) in 0.1 M
NaOH. Histidine-tagged recombinant rat GH, expressed in the same
construct as described for human GH,47 was used in this assay. An
aliquot of GH (10 L, 33 nM) in storage buffer (25 mM NaOAc, 50
mM BME, 1 mM octyl- -glucopyranoside, 1 mM EDTA, and 0.1
M NaCl) was incubated at 37 °C for 30 min and then added to 90
L of assay buffer containing the desired concentration of substrate,
50 mM BME, 50 mM NaOAc, pH 6.0, at 37 °C in a quartz microcu-
vette. Fluorescence was monitored using a Spex Fluoromax-2 with
the excitation wavelength set at 325 nm and emission at 415 nm
with the temperature maintained at 37 °C by a circulating water
bath. Controls with storage buffer replacing GH were used to cor-
rect for the low intrinsic fluorescence of the substrate due to incom-
plete quenching at each concentration. The fluorescence of the
controls did not increase significantly over the time of the assay.
1H NMR (CDCl3, 300 MHz): = 1.42–1.50 (36 H), 2.20–3.10 (8 H),
4.2–4.5 (3 H), 6.50 (1 H), 6.99–7.86 (7 H), 8.33 (1 H), 10.00 (1 H),
10.48 (1 H).
FAB-MS (3-NBA with Na+): m/z (%) = 930.3 (MNa+, 31.4), 640.2
(23.6), 176.0 (22.6), 154.0 (36.7), 120.0 (100.0).
FAB-HRMS (3-NBA with Na+): m/z calcd for C43H59F2N5NaO14
(MNa+) 930.3924, found 930.3942.
LC-MS Identification of Fragments from GH Cleavage of 2
A solution of 100 M (1 mL) 2 in assay buffer was hydrolyzed with
GH (100 nM). The sample was monitored by fluorescence as de-
scribed above and the enzyme-catalyzed hydrolysis of 2 was judged
to be complete when the addition of more GH yielded no further in-
crease in fluorescence. A control with only storage buffer added in
place of GH was subjected to the same conditions. Samples were
centrifuged at 8,000 rpm for 15 min, filtered through a 0.2 m sy-
ringe filter, and lyophilized. The resulting residue was dissolved in
double-distilled H2O (200 L) and injected onto a Vydac C18 col-
umn which was eluted with the gradient described in general proce-
dures. UV absorbance was monitored at 278 nm and mass spectra
of the two product peaks were obtained: TR 4.0, m/z = 303.1 (M +
H)+ and TR 18.5, m/z = 356.1 (M + H)+.
N-Abz-4,4-Difluoroglutamyl- -glutamyl- -3-nitrotyrosine (2)
Compound 24 was dissolved in CH2Cl2 (1.5 mL) and cooled to 0 °C
in an ice-water bath. An equal volume of anhyd TFA was added
dropwise. The mixture was allowed to warm to r.t. and stirred for a
total of 24 h. The volatile components of the mixture were removed
in vacuo to leave a yellow-brown residue which was repeatedly tri-
turated with CH2Cl2 and concentrated. The resulting yellow solid
was kept under high vacuum for 24 h to give the crude solid product
2.
1H NMR (D2O, 300 MHz): = 1.66 (m, 1 H), 1.86 (m, 1 H), 2.10
(m, 2 H), 2.69–3.15 (m, 4 H), 4.00 (m, 1 H), 4.70 (m, 1 H), 6.95–
7.82 (m, 7 H).
FAB-MS (3-NBA w/Na+): m/z (%) = 662 (M + Na+, 1.6).
Synthesis 2002, No. 17, 2616–2626 ISSN 0039-7881 © Thieme Stuttgart · New York

2626
D. W. Konas et al.
SPECIAL TOPIC
(21) Leanna, M. R.; Sowin, T. J.; Morton, H. E. Tetrahedron Lett.
1992, 33, 5029.
Acknowledgments
This research was supported by a grant from the National Cancer In-
stitute (CA28097). We thank Mr. Scott Harrison for technical assi-
stance with synthetic chemistry and thank the American Chemical
Society, Division of Fluorine Chemistry, for a Moissan Summer
Undergraduate Research Fellowship in Fluorine Chemistry awar-
ded to Mr. Harrison, Ms. Joyce Reese for assistance in the careful
preparation of this manuscript, and Dr. Thomas J. Ryan for ge-
nerously providing purified recombinant -glutamyl hydrolase.
(22) Jurczak, J.; Gryko, D.; Kobrzycka, E.; Gruza, H.;
Prokopowicz, P. Tetrahedron 1998, 54, 6051.
(23) Hallinan, E. A.; Fried, J. Tetrahedron Lett. 1984, 25, 2301.
(24) Barton, D. H. R.; McCombie, S. W. J. Chem. Soc., Perk.
Trans. 1 1975, 1574.
(25) Chatgilialoglu, C.; Ferreri, C. Res. Chem. Intermed. 1993,
19, 755.
(26) Studer, A.; Amrein, S. Synthesis 2002, 835.
(27) Barton, D. H. R.; Jaszberenyi, J. C. Tetrahedron Lett. 1989,
30, 2619.
(28) Barton, D. H. R.; Jang, D. O.; Jaszberenyi, J. C. Tetrahedron
Lett. 1990, 31, 4681.
(29) Barton, D. H. R.; Jang, D. O.; Jaszberenyi, J. C. Tetrahedron
Lett. 1991, 32, 7187.
(30) Barton, D. H. R.; Jang, D. O.; Jaszberenyi, J. C. Tetrahedron
Lett. 1992, 33, 2311.
(31) Barton, D. H. R.; Jang, D. O.; Jaszberenyi, J. C. Tetrahedron
1993, 49, 7193.
(32) Barton, D. H. R.; Jang, D. O.; Jaszberenyi, J. C. J. Org.
Chem. 1993, 58, 6838.
(33) Barton, D. H. R.; Jacob, M. Tetrahedron Lett. 1998, 39,
1331.
(34) Jang, D. O.; Cho, D. H.; Barton, D. H. R. Synlett 1998, 39.
(35) Zalkin, H. Adv. Enzymol. Relat. Areas Mol. Biol. 1993, 66,
203.
(36) Zalkin, H.; Smith, J. L. Adv. Enzymol. Relat. Areas Mol.
Biol. 1998, 72, 87.
References
(1) Current Address: Department of Immunology NB-30, The
Cleveland Clinic Foundation, 9500 Euclid Avenue,
Cleveland, OH, 44195.
(2) Welch, J. T.; Eswarakrishnan, S. Fluorine in Bioorganic
Chemistry; Wiley: New York, 1991.
(3) Organofluorine Compounds in Medicinal Chemistry and
Biomedical Applications; Filler, R.; Kobayashi, Y.;
Yagupolskii, L. M., Eds.; Elsevier: Amsterdam, 1993.
(4) Tsukamoto, T.; Coward, J. K.; McGuire, J. J. In Biomedical
Frontiers of Fluorine Chemistry; Ojima, I.; McCarthy, J. R.;
Welch, J. T., Eds.; American Chemical Society: Washington
D.C., 1996, 118.
(5) McGuire, J. J.; Hart, B. P.; Haile, W. H.; Rhee, M.; Galivan,
J.; Coward, J. K. Arch. Biochem. Biophys. 1995, 321, 319.
(6) Hart, B. P.; Haile, W. H.; Licato, N. J.; Bolanowska, W. E.;
McGuire, J. J.; Coward, J. K. J. Med. Chem. 1996, 39, 56.
(7) Tsukamoto, T.; Kitazume, T.; McGuire, J. J.; Coward, J. K.
J. Med. Chem. 1996, 39, 66.
(37) Huang, X. Y.; Holden, H. M.; Raushel, F. M. Ann. Rev.
Biochem. 2001, 70, 149.
(38) Tsukamoto, T.; Coward, J. K. J. Org. Chem. 1996, 61, 2497.
(39) (a) Meffre, P.; Dave, R. H.; Leroy, J.; Badet, B. Tetrahedron
Lett. 2001, 42, 8625. (b) Note added in proof: A recently
published correction to Ref. 39a indicates that the reported
synthesis of L-4,4-difluoroglutamine is in error; the product
isolated was L-4,4-difluoroglutamic acid, see: Meffre , P.;
Dave, R. H.; Leroy, J.; Badet, B. Tetrahedron Lett. 2002, 43,
6279. Thus, removal of the Cbz group in 15 (Scheme 3) by
catalytic hydrogenolysis in modest yield (ca 50%) as
reported for the racemic material in Ref. 38 represents the
only tenable route to the synthesis of L-4,4-
difluoroglutamine reported to date.
(40) Ding, Y.; Wang, J. Q.; Abboud, K. A.; Xu, Y. L.; Dolbier,
W. R.; Richards, N. G. J. J. Org. Chem. 2001, 66, 6381.
(41) Zhao, M.; Li, J.; Song, Z.; Desmond, R.; Tschaen, D. M.;
Grabowski, E. J. J.; Reider, P. J. Tetrahedron Lett. 1998, 39,
5323.
(42) Kitagawa, O.; Hashimoto, A.; Kobayashi, Y.; Taguchi, T.
Chem. Lett. 1990, 1307.
(43) Mathias, L. J. Synthesis 1979, 561.
(44) Yao, R.; Schneider, E.; Ryan, T. J.; Galivan, J. Proc. Natl.
Acad. Sci. USA 1996, 93, 10134.
(45) Licato, N. J.; Coward, J. K.; Nimec, Z.; Galivan, J.;
Bolanowska, W. E.; McGuire, J. J. J. Med. Chem. 1990, 33,
1022.
(8) McGuire, J. J.; Hart, B. P.; Haile, W. H.; Magee, K. J.; Rhee,
M.; Bolanowska, W. E.; Russell, C.; Galivan, J.; Paul, B.;
Coward, J. K. Biochem. Pharmacol. 1996, 52, 1295.
(9) Fluorine-Containing Amino Acids, Synthesis and
Properties; Kukhar, V. P.; Soloshonok, V. A., Eds.; Wiley:
Chichester, 1995.
(10) Sutherland, A.; Willis, C. L. Nat. Prod. Rep. 2000, 17, 621.
(11) Asymmetric Fluoroorganic Chemistry: Synthesis,
Applications, and Future Directions; Ramachandran, P. V.,
Ed.; American Chemical Society: Washington D.C., 2000.
(12) Enantiocontrolled Synthesis of Fluoroorganic Compounds:
Stereochemical Challenges and Biomedical Targets;
Soloshonok, V. A., Ed.; Wiley: Chichester, 1999.
(13) Konas, D. W.; Coward, J. K. Org. Lett. 1999, 1, 2105.
(14) Konas, D. W.; Coward, J. K. J. Org. Chem. 2001, 66, 8831.
(15) J. J. Pankuch, unpublished results; for a closely related
FRET peptide substrate, see: Pankuch, J. J.; Coward, J. K.
Bioorg. Med. Chem. Lett. 2001, 11, 1561.
(16) McGuire, J. J.; Coward, J. K. In Folates and Pterins, Vol. 1;
Blakley, R. L.; Benkovic, S. J., Eds.; Wiley: New York,
1984, 135.
(17) Liang, X.; Andersch, J.; Bols, M. J. Chem. Soc., Perkin
Trans. 1 2001, 2136.
(18) Kim, K. S.; Qian, L. Tetrahedron Lett. 1993, 34, 7195.
(19) Otaka, A.; Miyoshi, K.; Burke, T. R.; Roller, P. P.; Kubota,
H.; Tamamura, H.; Fujii, N. Tetrahedron Lett. 1995, 36, 927.
(20) Delle Monache, G.; Di Giovanni, M. C.; Maggio, F.; Misiti,
D.; Zappia, G. Synthesis 1995, 1155.
(46) Pankuch, J.; Hui, M. (University of Michigan, Ann Arbor),
unpublished results.
(47) Chave, K. J.; Auger, I. E.; Galivan, J.; Ryan, T. J. J. Biol.
Chem. 2000, 275, 40365.
Synthesis 2002, No. 17, 2616–2626 ISSN 0039-7881 © Thieme Stuttgart · New York