The secondary structure of a heptapeptide containing trifluoromethyl-λ6-tetrafluorosulfanyl substituted amino acids

Article abstract of DOI:10.1039/c9ob01797f

Site specific introduction of the polar hydrophobic trifluoromethyl-λ⁶-tetrafluorosulfanyl (CF₃SF₄) group can effectively control the secondary structure of a heptapeptide, the minimum repeat unit of an α-helix. The struct

Full text of DOI:10.1039/c9ob01797f

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The secondary structure of a heptapeptide
containing trifluoromethyl-λ6-tetrafluorosulfanyl
substituted amino acids†
Cite this: DOI: 10.1039/c9ob01797f
Received 14th August 2019,
Accepted 21st August 2019
Akari Ikeda, Aimée Capellan and John T. Welch
*
DOI: 10.1039/c9ob01797f
rsc.li/obc
Site specific introduction of the polar hydrophobic trifluoro- high Hammett substituent values (σp) associated with CF3SF4-
methyl-λ6-tetrafluorosulfanyl (CF3SF4) group can effectively substitution are associated with decreased desolvation energy
control the secondary structure of a heptapeptide, the minimum and enhanced dipolar interactions10 the same properties corre-
repeat unit of an α-helix. The structural influence of CF3SF4-con- lated with polar hydrophobicity.11 The Connolly volume,
taining amino acid on the heptapeptide was established using NMR 138.93 Å3, and surface area,9 156.76 Å2, of the CF3SF4 group
methods.
are also greater than the corresponding values of the penta-
fluorosulfanyl (SF5) group, 102.96 Å3 and 122.71 Å2 respect-
ively. When the CF3SF4 group is introduced to the first and
fifth residues of a heptapeptide, the substituent effects were
predicted to influence the heptapeptide secondary structure.
NMR methods demonstrated that even the sterically demand-
ing CF3SF4-substituted sidechains of both the protected and
deprotected peptides strongly associate in an intramolecular
manner.
Earlier the influence of the racemic pentafluorosulfanylated
amino acid (RS)-1 on the conformation12 of a heptapeptide
substituted with 1 at the first and fifth positions was exam-
ined. NMR solution structure determination methods13
enabled the tentative assignment of four coiled structures that
were tentatively assigned to the four diastereomeric heptapep-
tides that were prepared.12 In marked contrast to readily avail-
able aryl pentafluorosulfanyl compounds,14 aliphatic SF5-con-
taining building blocks or pentafluorosulfanyl halides are
inaccessible. This inaccessibility made the preparation of opti-
cally pure 1, or other aliphatic pentafluorosulfanylated amino
acids, economically challenging.
The reactivity and conformation of amino acids is often influ-
enced by fluorination. The most common fluorinated amino
acids, leucine and valine, typically contain three to six omega
fluorines commonly in trifluoromethyl (CF3) groups. In
addition to well-known lipophobic1 effects or specific fluo-
rine–fluorine interactions,1a,2 CF3 groups may have other struc-
tural influences. In particular, fluorination3 may affect poly-
peptide secondary structure, such as helix-formation.1b,2a,b,3g,4
Incorporation of fluorinated amino acids may result in a
reduced propensity for α-helix formation relative to native
amino acid-containing sequences.5 The accessibility of various
secondary structures4d,6 may be diminished by electrostatic
interactions of fluorinated side chains with the amide back-
bone. However, incorporation of fluorinated analogues of
leucine, isoleucine or valine at the core of coiled-coil heptad
repeats can result in enhancement of the thermal stability of
coiled-coil peptide assemblies.2a,4d,7
The simple and convenient preparation of trans-trifluoro-
methyl-λ6-tetrafluorosulfanyl chloride facilitated the introduc-
tion of the CF3SF4 group8 and hence the preparation and
resolution of CF3SF4-substituted amino acids. The CF3SF4
group is one of the most hydrophobic groups known with an
experimentally determined lipophilicity partition coefficient of
2.13 (ref. 9) (πp), significantly greater that that of the penta-
fluorosulfanyl group (SF5) (1.23)9 or the trifluoromethylsulfanyl
group (SCF3) (1.44). The large lipophilicity increments (πp) and
In contrast the ready availability of CF3SF4Cl made testing the
efficacy of CF3SF4-substitution on controlling secondary
peptide structure much more accessible. (S,E)-N-(tert-
Butoxycarbonyl)-2-amino-6-trifluoromethyl-λ6-tetrafluoro-sulfa-
nylpent-4-enoic acid (Boc-TtsNVa) 2 was prepared by the
Department of Chemistry, University at Albany, SUNY, 1400 Washington Ave.,
Albany, NY 12222, USA. E-mail: jwelch@albany.edu
†Electronic supplementary information (ESI) available: Experimental section
and spectroscopy. See DOI: 10.1039/c9ob01797f
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Scheme 1 Preparation of (S,E)-N-(tert-butoxycarbonyl)-2-amino-6-
trifluoromethyl-λ6-tetrafluorosulfanylpent-4-enoate(Boc-TtsNVa) 2.
addition of CF3SF4Cl8 to methyl (S)-N-(tert-butoxycarbonyl)-2-
aminopent-4-enoate 3. Dehydro chlorination and saponifica-
tion formed 2 (Scheme 1). Incorporation of TtsNVa at the first
and fifth position of a heptapeptide such as 5 (TtsNVa-Glu-Ser-
Lys-TtsNVa-Lys-Glu or TtsNV-E-S-K-TtsNV-K-E) (Fig. 1) was
anticipated to promote a helical conformation.
As illustrated in a helix wheel depiction, the CF3SF4-con-
taining amino acids would be adjacent on peptide folding
(Fig. 2).
C- and N-terminal, tri- and tetrapeptide, fragments were
prepared as shown (Scheme 2). Prior to purification, 8 was
formed in 84% ee as determined by the method of Mosher,15
i.e., preparation of a α-methoxy-α-trifluoromethylphenyl-acet-
amide. The minor racemization that occurred on peptide coup-
ling was easily overcome by chromatographic purification of 7.
Addition of 2 to 9 proceeded efficiently and 10 was likewise
purified by chromatography. The coupling of 8 with 11 failed
under the conditions used for condensation of 2 with 6 or 2
with 9 with only unreacted 8 and 11 recovered. Apparently sol-
vation of the CF3SF4-containing peptides strongly influenced
the success of the coupling reaction. The optimum reaction
conditions required dichloromethane as solvent to disrupt
aggregation of the reactant peptides (Scheme 3).
Scheme 2 Preparation of N- and C-terminal fragments, 8 and 11, by
the addition of (TtsNVa) 2 to the corresponding di- and tripeptide
fragments.
Scheme 3 Condensation of N- or C-terminal fragments to form the
targeted heptapeptide 5.
Fig. 1 The CF3SF4-containing heptapeptide
5 (TtsNVa-Glu-Ser-Lys-
TtsNVa-Lys-Glu or TtsNV-E-S-K-TtsNV-K-E).
The influence of the intermolecular CF3SF4-group inter-
actions on the coupling reaction suggests that CF3SF4-substitu-
ents in both the protected and deprotected peptides will affect
the secondary structure of both peptides 12 and 5. The three
dimensional structure of the protected peptide 12 was deter-
mined by 1H-NMR spectroscopy. One dimensional 1H and
19F-NMR spectra and two dimensional HSQC, 1H,1H ROESY,
and 1H,1H TOCSY spectra16 of the peptide were acquired in
CDCl3. The TOCSY data were used to assign the 1H resonances
using heteronuclear coupling of protons to the fluorines of
Fig. 2 A helix wheel representation of the heptapeptide target showing
the CF3SF4 groups in close proximity.
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CF3SF4-group as a point of origin. The ROESY spectrum was
used to determine the NOE (nuclear Overhauser effect) cross-
peak assignments and to confirm unambiguously the distance
relationships between the individual protons (see ESI S2-4-2c,
pS24. See S2-4-1d, p S21† for 12).
CARA (computer aided resonance assignment, http://cara.
nmr.ch/doku.php) was used to generate CYANA input from the
ROESY data.13,17 As 12 is composed of protected natural
amino acids and the synthetic amino acid 2, for the structure
fitting, new residue types were constructed in CARA, for
inclusion of the additional spins of the protecting groups and
of 2. This additional information enabled more accurate struc-
ture determination, such as sidechain orientation, than was
previously possible when racemic pentafluorosulfanylated
amino acids were employed.12 Structural calculations were
carried out with the integrated autoassignment module
CANDID13,17,18 using distance restraints derived from the
NOESY spectra and seven pairs of backbone torsion angle con-
straints derived from TALOS.19
The chemical shift identification tolerance was set to
0.020 ppm in both proton dimensions. Initial NOE cross-peaks
were manually assigned in the NMR view of CARA. A total of
300 structures were generated per CANDID cycle with 10 000
torsion angle dynamics (TAD) steps in the CYANA annealing
protocol. A total of 265 manually and CANDID-assigned NOE
cross-peaks were used in structural determination. The 20
lowest-energy structures in the final CANDID round were
retained as an ensemble representation of the derived structure.
The most stable of the final 20 structures determined from
the CDCl3 solution of 12 illustrates the close proximity of the
CF3SF4-containing side chains of the TtsNVa residues (Fig. 3).
While it was anticipated that the profound hydrophobic charac-
ter of the CF3SF4 group9 could drive interfunctional group inter-
actions in polar or aqueous media, the fluorous interactions20 of
those CF3SF4-containing side chains of TtsNVa suprisingly drove
the conformation of the protected peptide 12 in CDCl3. It is pro-
posed that the head-to-tail alignment of the sidechains is pro-
moted by CF3SF4 group induced dipolar interactions, an effect
that would be maximized in the relatively non-polar solvent
system. This conformational control is in marked contrast to the
Fig. 4 The NMR derived structure of the protected peptide 5 in DMSO-
d6. The lowest energy conformer of the best 20 structures is depicted
with the atoms of the TtsNVa side chain represented as van der Waals
spheres.
earlier experiments with SF5-containing heptapeptides12 where
CDCl3 solvation led to a loss of secondary structure.
Following deprotection, the low-energy structure of 5 was
determined in DMSO-d6 using the same NMR methods
employed with 12. A total of 235 manually and CANDID-
assigned NOE cross-peaks were used in structural determi-
nation (see ESI S2-4-2c, pS24† for 5). The TtsNVa side chains
of 5 remained in close proximity but had a slightly more
distant head-to-head relationship as opposed to the head-to-
tail organization of 12. The reduction in the number of detect-
able NOE cross peaks was consistent with 5 assuming a less
ordered conformation than 12. Apparently deprotection and
the more polar environment mitigated the intramolecular pro-
pensity of the CF3SF4 groups to associate (Fig. 4).
In summary, with only seven amino acid residues, the intra-
molecular influence of the CF3SF4 group is insufficient to
provide the necessary driving force for a well-defined helical
structure. Nonetheless the CF3SF4-containing side chains of
TtsNVa effectively associate in both polar aprotic and non-
polar solvent. Surprisingly the seven fluorines of the CF3SF4
group are sufficient to drive that organization. Selective incor-
poration of TtsNVa into longer peptides would be predicted to
influence both the quaternary and tertiary structure of the pep-
tides, independent of whether the peptides were in a highly
polar aqueous environment or not. Along with the steric, elec-
tronic and dipolar effects, the remarkably potent combination
of lipophobic and hydrophobic interactions that accompany
CF3SF4 introduction can dramatically modify the properties of
those substances bearing this unique group.
Conflicts of interest
There are no conflicts to declare.
Notes and references
Fig. 3 The NMR derived structure of the protected peptide 12 in CDCl3.
The lowest energy conformer of the best 20 structures is depicted with
the atoms of the TtsNVa side chain represented as van der Waals
spheres.
1 (a) A. Niemz and D. A. Tirrell, J. Am. Chem. Soc., 2001, 123,
7407–7413; (b) B. Bilgicer, X. Xing and K. Kumar, J. Am.
This journal is © The Royal Society of Chemistry 2019
Org. Biomol. Chem.

View Article Online
Communication
Organic & Biomolecular Chemistry
Chem. Soc., 2001, 123, 11815–11816; (c) B. Bilgicer and
K. Kumar, Tetrahedron, 2002, 58, 4105–4112.
2 (a) H.-Y. Lee, K.-H. Lee, H. M. Al-Hashimi and
E. N. G. Marsh, J. Am. Chem. Soc., 2006, 128, 337–343;
(b) M. Salwiczek and B. Koksch, ChemBioChem, 2009, 10,
Chem., 1995, 38, 2292–2301; (d) L. R. Pratt, Annu. Rev. Phys.
Chem., 1985, 36, 433–449; (e) A. Ben-Naim, Hydrophobic
Interactions, Plenum Press, 1980; (f) C. Tanford, The
Hydrophobic Effect: Formation of Micelles and Biological
Membranes 2d Ed, J. Wiley, 1980.
2867–2870; (c) B. C. Buer, B. J. Levin and E. N. G. Marsh, 11 J. C. Biffinger, H. W. Kim and S. G. DiMagno,
J. Am. Chem. Soc., 2012, 134, 13027–13034; (d) B. C. Buer, ChemBioChem, 2004, 5, 622–627.
J. L. Meagher, J. A. Stuckey and E. N. G. Marsh, Proc. Natl. 12 D. S. Lim, J.-H. Lin and J. T. Welch, Eur. J. Org. Chem.,
Acad. Sci. U. S. A., 2012, 109, 4810–4815. 2012, 3946–3954.
3 (a) H. Yang, C. Tian, D. Qiu, H. Tian, G. An and G. Li, Org. 13 (a) Automated NMR Structure Calculation With CYANA, ed. P.
Chem. Front., 2019, 6, 2365–2370; (b) L. Zhu, J. Xiong, J. An,
N. Chen, J. Xue and X. Jiang, Org. Biomol. Chem., 2019, 17,
3797–3804; (c) Y.-G. Lou, A.-J. Wang, L. Zhao, L.-F. He,
Güntert, 2004; (b) P. Guntert, in Protein NMR Spectroscopy:
Practical Techniques and Applications, John Wiley & Sons,
Ltd, 2011, pp. 159–192.
X.-F. Li, C.-Y. He and X. Zhang, Chem. Commun., 2019, 55, 14 (a) J. T. Welch, in Fluorine in Pharmaceutical and Medicinal
3705–3708; (d) C. Odar, M. Winkler and B. Wiltschi,
Biotechnol. J., 2015, 10, 427–446; (e) E. N. G. Marsh, Acc.
Chem. Res., 2014, 47, 2878–2886; (f) M. Salwiczek,
E. Nyakatura, U. I. M. Gerling, S. Ye and B. Koksch, Chem.
Soc. Rev., 2012, 41, 2135–2171; (g) U. I. M. Gerling,
M. Salwiczek, C. D. Cadicamo, H. Erdbrink, C. Czekelius,
S. L. Grage, P. Wadhwani, A. S. Ulrich, M. Behrends,
Chemistry: From Biophysical Aspects to Clinical Applications,
ed. V. Gouverneur and K. Muelller, Imperial College Press
Co., 2012, vol. 5, pp. 171–204; (b) C. N. von Hahmann,
P. R. Savoie and J. T. Welch, Curr. Org. Chem., 2015, 19,
1592–1618; (c) P. R. Savoie and J. T. Welch, Chem. Rev.,
2015, 115, 1130–1190; (d) S. Altomonte and M. Zanda,
J. Fluorine Chem., 2012, 143, 57–93.
G. Haufe and B. Koksch, Chem. Sci., 2014, 5, 819–830; 15 T. R. Hoye, C. S. Jeffrey and F. Shao, Nat. Protoc., 2007, 2,
(h) A. A. Berger, J.-S. Voeller, N. Budisa and B. Koksch, Acc.
Chem. Res., 2017, 50, 2093–2103.
4 (a) M. A. Molski, J. L. Goodman, C. J. Craig, H. Meng,
K. Kumar and A. Schepartz, J. Am. Chem. Soc., 2010, 132,
2451.
16 J. Cavanagh, W. Fairbrother, A. G. Palmer, III and
N. Skelton, Protein NMR Spectroscopy: Principles and
Practice, Academic, San Diego, 1995.
3658–3659; (b) L. Merkel and N. Budisa, Org. Biomol. 17 (a) P. Güntert, in BioNMR in Drug Research, Wiley-VCH
Chem., 2012, 10, 7241–7261; (c) E. K. Nyakatura,
O. Reimann, T. Vagt, M. Salwiczek and B. Koksch, RSC
Adv., 2013, 3, 6319–6322; (d) C. Jäckel, M. Salwiczek and
B. Koksch, Angew. Chem., Int. Ed., 2006, 45, 4198–4203.
Verlag GmbH & Co. KGaA, 2003, pp. 39–66; (b) P. Guntert,
Prog. Nucl. Magn. Reson. Spectrosc., 2003, 43, 105–125;
(c) C. Mumenthaler, P. Güntert, W. Braun and K. Wüthrich,
J. Biomol. NMR, 1997, 10, 351–362.
5 H.-P. Chiu, Y. Suzuki, D. Gullickson, R. Ahmad, B. Kokona, 18 T. Ikeya, J.-G. Jee, Y. Shigemitsu, J. Hamatsu, M. Mishima,
R. Fairman and R. P. Cheng, J. Am. Chem. Soc., 2006, 128,
15556–15557.
Y. Ito, M. Kainosho and P. Güntert, J. Biomol. NMR, 2011,
50, 137–146.
6 (a) S. S. Pendley, Y. B. Yu and T. E. Cheatham, 3rd, Proteins, 19 G. Cornilescu, F. Delaglio and A. Bax, J. Biomol. NMR, 1999,
2009, 74, 612–629; (b) C. Jaeckel, W. Seufert, S. Thust and
B. Koksch, ChemBioChem, 2004, 5, 717–720.
13, 289–302.
20 (a) M. Cametti, B. Crousse, P. Metrangolo, R. Milani and
G. Resnati, Chem. Soc. Rev., 2012, 41, 31–42;
(b) D. P. Curran, in Handbook of Fluorous Chemistry, Wiley-
VCH Verlag GmbH & Co. KGaA, 2004, pp. 128–155;
(c) I. T. Horvath, D. P. Curran and J. A. Gladysz, in
Handbook of Fluorous Chemistry, Wiley-VCH Verlag GmbH &
Co. KGaA, 2004, pp. 1–4; (d) K. Olofsson and M. Larhed, in
Handbook of Fluorous Chemistry, Wiley-VCH Verlag GmbH &
Co. KGaA, 2004, pp. 359–365; (e) J.-M. Vincent, Chem.
Commun., 2012, 48, 11382–11391; (f) W. Zhang, in Current
Fluoroorganic Chemistry, ACS Symp. Ser., American
Chemical Society, 2007, vol. 949, pp. 207–220.
7 (a) S. Son, I. C. Tanrikulu and D. A. Tirrell, ChemBioChem,
2006, 7, 1251–1257; (b) B. C. Buer, R. de la Salud-Bea,
H. M. Al Hashimi and E. N. G. Marsh, Biochemistry, 2009,
48, 10810–10817; (c) J. K. Montclare, S. Son, G. A. Clark,
K. Kumar and D. A. Tirrell, ChemBioChem, 2009, 10, 84–86.
8 A. Ikeda, L. Zhong, P. R. Savoie, C. N. von Hahmann,
W. Zheng and J. T. Welch, Eur. J. Org. Chem., 2018, 772–780.
9 P. Kirsch and A. Hahn, Eur. J. Org. Chem., 2006, 1125–1131.
10 (a) K. A. Dill, Biochemistry, 1990, 29, 7133–7155;
(b) P. L. Privalov and S. J. Gill, Adv. Protein Chem., 1988, 39,
191–234; (c) J. Gao, S. Qiao and G. M. Whitesides, J. Med.
Org. Biomol. Chem.
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