Synthesis and use of N-Fmoc-l-fluoroalanine

Article abstract of DOI:10.1016/j.tetlet.2015.01.117

We report a practical synthesis of N-Fmoc protected l-fluoroalanine 6 from l-serine. The key step involves a deoxofluorination reaction which was best achieved using XtalFluor-E in the presence of triethylamine trihydrofluoride. We also report the use of 6 in solid-phase peptide synthesis for the preparation of a model tripeptide demonstrating the possibility of incorporating a fluorinated probe in a peptide without elimination. Furthermore, cleavage of the dipeptide permitted to verify that 6 has a high enantiopurity.

Full text of DOI:10.1016/j.tetlet.2015.01.117

Tetrahedron Letters 56 (2015) 1244–1246
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis and use of N-Fmoc-L-fluoroalanine
⇑
Claudia Carpentier, Raphaël Godbout, François Otis, Normand Voyer
Département de Chimie and PROTEO, Université Laval, Pavillon Alexandre-Vachon, Faculté des sciences et de génie, 1045 avenue de la Médecine, Québec G1V 0A6, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
We report a practical synthesis of N-Fmoc protected L-fluoroalanine 6 from L-serine. The key step involves
Received 1 December 2014
Revised 15 January 2015
Accepted 19 January 2015
Available online 30 January 2015
a deoxofluorination reaction which was best achieved using XtalFluor-E in the presence of triethylamine
trihydrofluoride. We also report the use of 6 in solid-phase peptide synthesis for the preparation of a
model tripeptide demonstrating the possibility of incorporating a fluorinated probe in a peptide without
elimination. Furthermore, cleavage of the dipeptide permitted to verify that 6 has a high enantiopurity.
Ó 2015 Elsevier Ltd. All rights reserved.
Keywords:
Fluorinated amino acid
Fmoc-L-fluoroalanine
Fluoroalanine
Solid-phase synthesis
Introduction
OTBS
COOH
OH
a,b
Incorporation of fluorine atoms at different positions on
bioactive molecules has been shown to improve significantly their
properties, including potency.1 This phenomenon has been
thoroughly described and reviewed in the literature.2–4 Likewise,
due to the strong NMR signal of 19F, introducing such atoms into
peptides and proteins is a useful strategy that enables specific
biophysical studies.5–8 Particularly, solution and solid state NMR
of fluorinated peptides and proteins can provide unique informa-
tion on their interactions with bilayer membranes not possible to
obtain otherwise. Hence, developing fluorinated amino acids as
biomolecular probes is of great interest.
CbzHN
CbzN
H2N
COOH
2
1
c
F
OTBS
O
d
O
O
CbzN
O
4
3
e
F
F
f
Toward that goal, elegant approaches have been reported.9–14
Of special interest is the work of Hoveyda and Pineault who
H2N
COOH
FmocHN
COOH
5
6
reported the synthesis of enantiopure
L
-fluoroalanine from
Scheme 1. Synthesis of Fmoc-fluoroalanine 6. (a) CbzCl, pH 8–10, H2O/acetone, 3 h;
(b) TBSCl, DMAP, TEA, CH2Cl2; (c) para-formaldehyde, p-TsOH, toluene; (d) TEA-
3HF, Xtalfluor-E, CH2Cl2, 24 h; (e) BCl3, CH2Cl2, 25 °C, 30 min; (f) Fmoc-OSu, Na2CO3,
dioxane/H2O.
L
-serine.15 They also reported the introduction of
L-fluoroalanine
in a dipeptide, though with modest yields and in solution phase
synthesis. Therefore, there is a need to develop enantiopure
monofluorinated alanine under a form that is compatible with
solid-phase peptide synthesis.16 Hereafter, we described such a
fluorenylmethyloxycarbonyl (Fmoc) derivative and its use in the
preparation of a model tripeptide by solid-phase peptide synthesis.
L
-Serine was first protected efficiently to N-benzyloxycarbonyl-
L-serine using benzylchloroformate in a water/acetone mixture
(15/2). The side chain alcohol was then protected as a dimethyl-
t-butylsilyl ether with the appropriate silyl chloride in dry dichlo-
romethane to yield 51% of the desired protected compound 2
having a free carboxylate. The work-up has been investigated in
order to optimize the yield. Compound 2 was then treated with
paraformaldehyde and a catalytic amount of p-toluenesulfonic acid
in toluene in a Dean Stark apparatus. This reaction produced
efficiently oxazolidinone 3 in quantitative yield.
Synthesis of N-Fmoc-L-fluoroalanine 6
Based on literature precedents, we focus our synthetic efforts
exploiting -serine as starting material. Our synthetic strategy is
L
illustrated in Scheme 1.
For the desilylation-deoxofluorination of 3, several reagents
and conditions were investigated. A combination of HF-pyridine
⇑
Corresponding author. Tel.: +1 418 656 2131x3613; fax: +1 418 656 7916.
E-mail address: normand.voyer@chm.ulaval.ca (N. Voyer).
http://dx.doi.org/10.1016/j.tetlet.2015.01.117
0040-4039/Ó 2015 Elsevier Ltd. All rights reserved.

C. Carpentier et al. / Tetrahedron Letters 56 (2015) 1244–1246
1245
PS
F
O
O
H
N
FmocHN
b,c
a
O
FmocHN
O
O
O
HO
d
F
OH
O
H
N
b,e
FmocHN
OH
O
7
F
F
O
O
O
O
H
H
d
N
N
FmocHN
FmocHN
OH
N
O
N
H
H
O
O
8
Scheme 2. Synthesis of a di- and a tripeptide using Fmoc-fluoroalanine. (a) Fmoc-Leu-OH, DIC, HOBt, DIEA DMAP, CH2Cl2; (b) piperidine/DMF 20%; (c) 6, HATU, NMM, DMF;
(d) TFA/H2O 95%; (e) Fmoc-Ala-OH, HATU, NMM, DMF.
(Olah’s reagent), Xtalfluor-E, and 1,8-diazabicyclo[5.4.0]undec-7-
ene (DBU) in dry dichloromethane did not yield the compound
sought-after. No fluorination took place as evidenced by the absence
of signal by 19F NMR in the crude product of those reactions. As
HF-pyridine requires notoriously complicated handling precau-
tions, we tried substituting it for tetrabutylammonium fluoride
(TBAF) as the deprotection agent and source of hydrogen fluoride.
Using TBAF in tandem with Xtalfluor-E and DBU still did not yield
any fluorinated compound. The deoxofluorination reagent was next
changed for Deoxo-fluor with TBAF in dichloromethane, but still no
trace of a fluorinated compound was found. The first success
obtained was using a combination of 8 equiv of HF-pyridine, 2 equiv
of triethylamine trihydrofluoride, 1.5 of Xtalfluor-E and 3 in dichlo-
romethane, producing 4 though with a low yield. Substituting
HF-pyridine for TBAF or removing completely the external source
of HF from the reaction did not impact the resulting yield. The best
combination of reagents was found to be 2 equiv of XtalFluor-E and
4 equiv of triethylamine trihydrofluoride in dry dichloromethane,
which lead to the fluorinated oxazolidinone 4 in 59% yield.
Noteworthy of mention, performing the deprotection and the
deoxofluorination steps separately did not allow for the formation
of the desired compound 4 under any condition tried.
Another important transformation was the deprotection and
ring-opening steps to get 5. Again, we investigated unsuccessfully
different strategies, opening the ring first then deprotecting the
Cbz group by catalytic hydrogenation. Ring-opening with 2 N HCl
in dioxane, as proposed by Hoveyda et al., did give the linear com-
pound with yields never exceeding 70%.15 The following deprotec-
tion of the Cbz by catalytic hydrogenation yielded an unidentified
fluorinated compound that could not be isolated. This strategy was
abandoned as these two steps were best achieved with a one pot
reaction using 1 M BCl3 in DCM.17 This quick 30 min reaction gave
a white solid that was used as is for the final step of the synthesis.
The crude product was finally protected with Fmoc-OSu in a
the dipeptide from the resin. ES-MS confirmed the preparation of
the N-Fmoc-protected dipeptide and the purity was estimated to
60% by HPLC. Interestingly, NMR analyses were used to confirm
the structure of dipeptide 7. From those analyses, we could
conclude that 6 has a high enantiopurity, although it is also
possible that a kinetic resolution process leads to 7 with a high
diastereomeric excess. On the other hand, as reported by others,
elimination of HF leading to dehydroalanine was insignificant
under the reaction conditions used.15
To further demonstrate the usefulness of 6, the N-Fmoc-dipep-
tide 7 on the resin was deprotected using two 5 min treatments
with 20% piperidine in DMF and the resulting free dipeptide was
coupled with 2 equiv of N-Fmoc-
ing reagent. Since the ninhydrin test clearly showed an uncom-
pleted coupling, the resin was mixed with 10 equiv of N-Fmoc-
L-Alanine using HATU as activat-
L-
alanine and HATU for another 1 h. This second coupling resulted
in a complete reaction step. This increased difficulty of carrying
out the coupling can be explained by the lower nucleophilicity
of the amino group of the L-fluoroalanine, which is in agreement
with literature precedents.11,18,19 Interestingly, it was not possible
to observe the characteristic signals of dehydroalanine by NMR.
This indicates that HF elimination leading to dehydroalanine, if
any, occurred minimally during deprotection of and coupling to
fluoroalanine. The obtention of tripeptide 8 demonstrates the
possibility of using N-Fmoc-L-fluoroalanine 6 in solid-phase
peptide synthesis.
Conclusion
We reported the first synthesis of N-Fmoc-L-fluoroalanine and its
characterization. All steps have been optimized, especially the
deoxofluorination, the one pot Cbz-group deprotection and ring-
opening, and the Fmoc protection steps. Although HATU and a
higher concentration of reagents were necessary for high yield cou-
mixture of dioxane/water to yield the desired N-Fmoc-L-fluoroala-
pling, N-Fmoc-L-fluoroalanine 6 has been coupled efficiently to a
nine 6, which was fully characterized by 1D- and 2D-NMR
spectroscopy and by mass spectrometry (see also Supporting
information for salient spectroscopic data). The optical rotation
leucine linked to the Wang resin. Analysis of the dipeptide prepared
demonstrated that the coupling proceeded with very low racemiza-
tion and elimination. Addition of an alanine onto the dipeptide leads
to the preparation of a tripeptide by solid-phase peptide synthesis
on a classic Wang resin, demonstrating the usefulness of 6 for the
preparation of fluorinated bioactive peptides.
of N-Fmoc-
L
-fluoroalanine 6 was determined to be À6.7 (c = 1.06,
in methanol).
Work is currently underway to prepare such bioactive peptides
and to use them in biophysical studies with lipid membranes.
Solid-phase synthesis using 6
In order to demonstrate the utility of 6 for solid-phase peptide
synthesis, we coupled it to a L-Leucine attached to Wang Resin
Acknowledgments
(Scheme 2). The coupling was performed using 2 equiv of 6 and
using HATU as a coupling reagent. The coupling lasted 1 h. An
aliquot of the resin was then treated for 1 h with 95% TFA to cleave
This work was supported by the NSERC of Canada and the
FRQNT of Québec. C.C. and R.G. thank PROTEO and the FRQNT for

1246
C. Carpentier et al. / Tetrahedron Letters 56 (2015) 1244–1246
12. Qui, X.-L.; Meng, W.-D.; Qing, F.-L. Tetrahedron 2004, 60, 6711–6745.
13. Sutherland, A.; Willis, C. L. Nat. Prod. Rep. 2000, 17, 621–631.
14. Simon, J.; Nguyen, T. T.; Chelain, E.; Lensen, N.; Pytkowicz, J.; Chaume, G.;
Brigaud, T. Tetrahedron: Asymmetry 2011, 22, 309–314.
graduate and undergraduate scholarships. The authors are also
thankful to M. Pierre Audet for his help in NMR and MS analyses.
15. Hoveyda, H. R.; Pinault, J.-F. Org. Lett. 2006, 8, 5849–5852.
Supplementary data
16. Typical procedure for 6: Crude compound 5 (0.542 g) was dissolved in 20 mL of
aqueous 10% Na2CO3 and the solution was heated to 40 °C. Afterwards, dioxane
(10 mL) was added. Fmoc-OSu (1 equiv, 1.71 g, 5.07 mmol) was dissolved in
dioxane (10 mL) and added dropwise to the reaction vessel. The resulting
mixture was stirred for 48 h at 40 °C. Then, 150 mL of icy water was added and
the organic phase was discarded. The milky white aqueous layer was washed
with diethyl ether (2 times 10 mL) to revert to a transparent solution and then
cooled down to 0 °C before acidification to pH 0–1 with 1N HCl. The milky
mixture was extracted thrice with EtOAc (75 mL) and the aqueous phase was
discarded. The organic phases were combined, washed with 1N HCl and then
with water, dried with MgSO4 and concentrated under reduced pressure. The
product was then crystallized from a EtOAc–Hexanes mixture to yield, over
two steps, 0.294 g (2.74 mmol, 54%) of a white solid. 1H NMR (500 MHz;
CDCl3), d = 4.24–4.27 (t, 1H, J = 7.0, Fmoc-CH), 4.41–4.48 (q, 2H, J = 8.0, Fmoc-
Supplementary data (these include 1H, 13C, 19F, COSY, HRMS of
compounds 6, 7, and 8 as well as complete characterization of all
prepared compounds.) associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.tetlet.2015.01.
117.
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