New family of base- and nucleophile-sensitive amino-protecting groups. A Michael-acceptor-based deblocking process. Practical utilization of the 1,1-dioxobenzo[b]thiophene-2-ylmethyloxycarbonyl (Bsmoc) group

Full text of DOI:10.1021/ja9713690

J. Am. Chem. Soc. 1997, 119, 9915-9916
9915
substrates were in some cases subject to premature or competi-
tive deblocking.4,5 All such problems were eliminated by
switching to a Michael acceptor bearing a â-substitutent such
as one based on alcohols 4 or 5. While the â-substitutent
protects the system from premature deblocking, reactivity is still
many times greater than for the Fmoc system and, in addition,
the presence of the styrene chromophore [for 9 (MeOH), λmax
310.5 nm (ꢀ 3048)] allows for facile UV detection, tracking,
and quantitation.6,7 Alcohol 4 was obtained by hydrolysis of
the allyl bromide, whereas 5 was obtained from commercially
available benzo[b]thiophene by hydroxymethylation (n-BuLi/
(CH2O)n; 80%) followed by peracid oxidation (80%). Because
of its desirable properties and greater availability, most work
to date has concentrated on the 1,1-dioxobenzo[b]thiophene-2-
ylmethyloxycarbonyl (Bsmoc) group derived from 5. Acylation
of amino acids could be effected by the Bolin method8 via
chloroformate 6a or via the N-hydroxysuccinimide ester 6b.9
New Family of Base- and Nucleophile-Sensitive
Amino-Protecting Groups. A
Michael-Acceptor-Based Deblocking Process.
Practical Utilization of the
1,1-Dioxobenzo[b]thiophene-2-ylmethyloxycarbonyl
(Bsmoc)† Group
Louis A. Carpino,* Michael Philbin, Mohamed Ismail,
George A. Truran, E. M. E. Mansour,‡ Shin Iguchi,
Dumitru Ionescu,§ Ayman El-Faham,‡ Christoph Riemer,
Ralf Warrass, and Manfred S. Weiss
Department of Chemistry, UniVersity of Massachusetts
Amherst, Massachusetts 01003
ReceiVed April 30, 1997
Base-sensitive amino-protecting groups such as Fmoc and
related functions owe their reactivity to facile â-elimination
processes. A recent attempt to apply Fmoc protection to an
inverse Merrifield synthesis of peptide segments foundered when
resins bearing cyclic secondary amino functions were found to
be inefficient scavenging agents for the â-elimination byproduct
dibenzofulvene (DBF).1 These disappointing results triggered
a search for a fundamentally different type of amino-protecting
group in which the deblocking event is simultaneously a
scavenging event. Appropriate positioning of a urethane
function relative to a Michael acceptor accomplished the desired
result (eq 1).2
NMR studies in CDCl3 clearly reveal the high reactivity of
the Bsmoc residue and provide evidence for a reactive inter-
mediate generated by initial Michael-like attack by piperidine.
Thus treatment of 7 with 2 equiv of piperidine is followed by
(1)
(2)
As expected for a Michael-type addition process, significant
rate variation accompanied changes in the nature of the electron-
withdrawing group (EWG), the presence or absence of substit-
uents at the â-position, and the chemical nature and/or steric
constraints built into the deblocking agent. For model substrates
1 (R1 ) R2 ) R3 ) R4 ) H; R ) -C6H4Cl-p) the following
order of reactivity toward a number of cyclic secondary amines
(piperidine, morpholine, etc.) was established for various
EWGs: C6H5SO2 > Me3CSO2 > EtOCO > C6H5SO > p-O2-
NC6H4-. In view of its lack of significant alternate reactivity,
its position at the head of this series, and the ready availability
of key precursors, the sulfone unit, built into an allylic alcohol
3, was selected for initial evaluation.3 Although 3 could be
complete liberation of p-chloroaniline (PCA) within 3-5 min
1
and concomitant formation of a labile intermediate whose H
NMR spectrum is consistent with structure 8. Adduct 8 decays
over the next 8-10 min to give the final stable deblocking
product 9.
(3) Alcohol 3 was obtained by hydrolysis of the corresponding bromide2c
in wet methanol in the presence of NaOCHO according to a reported method
(Harris, M.; Bull, M. J. Synth. Commun. 1985, 15, 1225).
(4) Potential problems associated with premature deblocking of the Fmoc
group have been evaluated previously. See: Bodanszky, M.; Deshmane,
S. S.; Martinez, J. J. Org. Chem. 1979, 44, 1622.
(5) For example, in the acylation of H-Phe-OCMe3 with Bspoc-Phe-Cl,
the desired dipeptide (94%) was accompanied by the ester (2%) formed by
competing attack at the olefinic residue. No comparable premature
deblocking occurred in the case of the Bsmoc group.
(6) Comparison of deblocking rates under identical conditions by NMR
analysis in CDCl3 for Bspoc-PCA and Bsmoc-PCA using 3 equiv of amine
showed the following relative rates (time in minutes for complete deblock-
ing/scavenging). Piperidine: Bspoc, <5 min; Bsmoc, <5 min. Morpho-
line: Bspoc, <5 min; Bsmoc, 45 min. cis-2,6-Dimethylpiperidine: Bspoc,
45 min; Bsmoc, no reaction. Comparison of the deblocking of Bsmoc-
PCA with that of Fmoc-PCA showed the former underwent complete
deblocking/scavenging with 2 equiv of piperidine in CDCl3 within 40 min,
whereas the latter required over 300 min for deblocking and no scavenging
had occurred at this point.
(7) For a series of four 2-phenylsulfonylpropenoxycarbonyl systems i
differently substituted at the â-position complete deblocking by morpholine
in CDCl3 solution (0.02 M) gave the results indicated (time in minutes for
complete reaction).
converted to the appropriate protected 2-tert-butylsulfonyl-2-
propenoxycarbonyl (Bspoc-) amino acids which were success-
fully used in the assembly of model peptides, the protected
† Common name: benzo[b]thiophenesulfone-2-methyloxycarbonyl.
‡ On leave of absence from the Department of Chemistry, Faculty of
Science, Alexandria University, Alexandria, Egypt.
§ Fulbright Scholar. On leave of absence from the Department of Organic
Chemistry, Faculty of Chemistry, University of Bucharest, 70346 Bucharest,
Romania.
(1) More appropriate were indene-derived Fmoc-analogous functions, yet
even in these cases, small amounts of the analogous fulvene byproducts
remained unscavenged. See: Carpino, L. A.; Cohen, B. J.; Lin, Y.-Z.;
Stephens, K. E., Jr.; Triolo, S. A. J. Org. Chem. 1990, 55, 251.
(2) Previously, related structural elements have been built into substrates
studied in connection with protein cross-linking and so-called multiple
coupling agents. See: (a) Mitra, S.; Lawton, R. G. J. Am. Chem. Soc. 1979,
101, 3097. (b) Seebach, D.; Knochel, P. HelV. Chim. Acta 1984, 67, 261.
(c) Knochel, P.; Normant, J. F. Tetrahedron Lett. 1985, 425.
(8) Bolin, D. R.; Syturu, I.; Humeic, F.; Meienhofer, J. Int. J. Pept.
Protein Res. 1983, 33, 353.
(9) Compare: Paquet, A. Can. J. Chem. 1982, 60, 976.
S0002-7863(97)01369-3 CCC: $14.00 © 1997 American Chemical Society

9916 J. Am. Chem. Soc., Vol. 119, No. 41, 1997
Communications to the Editor
Most common amino acids have been converted to their
Bsmoc derivatives10 and used in peptide assembly. Generally
the corresponding acid fluorides or in situ activation via
ammonium (guanidinium, formamidinium) or phosphonium salts
were used for the coupling step. Both solution and solid phase
syntheses were executed. Because of its hydrophilic character,
the Bsmoc residue made possible a unique simplification of the
recently described technique for the rapid solution synthesis of
short peptide segments based on Fmoc chemistry.11 With the
latter, the deblocking/scavenging step is carried out with
4-(aminomethyl)piperidine or preferably tris(2-aminoethyl)amine
(TAEA) with buffer extractions at pH 5.5 being used to remove
the byproduct Fm adduct. By substituting the Bsmoc for the
Fmoc group, it is possible to dispense with the phosphate buffer
and simply wash with water or saturated sodium chloride
solution since the TAEA adduct 10 is highly soluble in water.
coupling was favored by about 13% in the Bsmoc case (ratio
16/des-Aib-16 of 54/46 vs 41/59 for the Fmoc case).
In a few cases among the Bsmoc-protected proteinogenic
amino acids and their acid fluorides, the products were oily or
amorphous materials not obtainable in crystalline form. For
such systems, it appeared generally possible to obtain crystalline
intermediates by substitution of the Bsmoc function by an
equivalent residue of higher molecular weight. Thus neither
Bsmoc-Pro-OH nor Bsmoc-Pro-F could be obtained in solid
form, whereas the related 1,1-dioxonaphtho[1,2-b]thiophene-
2-ylmethyloxycarbonyl (Nsmoc) derivatives 17 are nicely
crystalline. The Bsmoc and Nsmoc derivatives are deblocked
at comparable rates.
In view of the high base-sensitivity of the Bsmoc residue, it
was possible to deblock this system selectively in the presence
of Fmoc or Fm protection. As an example, the tripeptide 18
could be assembled from Bsmoc-Leu-OFm by treatment with
2% TAEA/DCM followed by coupling with Bsmoc-Leu-F and
subsequent repetition of the same procedure. On the other hand,
precisely the opposite selectivity could be achieved upon
deblocking via 10% N-methylcyclohexylamine or diisoprop-
ylamine in DCM. Such selective removal of the Fm residue,
which might at first seem surprising, is illustrative of the
importance of steric factors in additions to a Michael acceptor.16
In addition, the amount of excess TAEA used could be reduced
with consequent reduction in loss of material during aqueous
washings. Coupling could be carried out under two-phase
(DCM/H2O/NaHCO3) or one-phase (DCM/DIEA) conditions.
In the latter case TAEA is added directly to the reaction mixture
once coupling is over (TLC). Ionic and highly polar byproducts
from the coupling step are then washed out along with adduct
10. This technique was followed in the synthesis of several
short peptides including Bsmoc-Tyr(t-Bu)-Ile-Asp(O-t-Bu)-Gly-
O-t-Bu (11) (87%), Bsmoc-Tyr(t-Bu)-Gly-Gly-Phe-Leu-O-t-Bu
(12) (49%), Bsmoc-Phe-Phe-Val-Gly-Leu-Met-OBn (13) (37%),
and Fmoc-Ile-Thr(t-Bu)-Arg(Pbf)-Gln(Trt)-Arg(Pbf)-Tyr(t-Bu)-
ODcpm12 (14) (40.3%) (Dcpm ) dicyclopropylmethyl). Cycle
times were approximately 1 h. Classic methods of solution
synthesis may also benefit from a change to Bsmoc chemistry
since the corresponding Fmoc-based syntheses are sometimes
compromised by the unpredictable tendency of DBF to undergo
polymerization.
Acknowledgment. We are indebted to the National Science
Foundation (NSF CHE-9003192), the National Institutes of Health
(GM-09706), and Research Corporation Technologies for support of
this work. The National Science Foundation is also thanked for grants
used to purchase the high-field NMR spectrometers used in this
research.
Supporting Information Available: Experimental procedures and
characterizing spectral data for the utilization of Bsmoc protection and
HPLC and MS data for Bsmoc-derived peptides (38 pages). See any
current masthead page for ordering and Internet access instructions.
Bsmoc amino acids were also used in standard solid-phase
syntheses, using acid fluoride, HBTU,13 or HATU13 coupling
techniques. Examples include the acyl carrier protein fragment
65-74 (ACP decapeptide) magainin-II amide and alamethicin
amide. The deblocking step was routinely carried out with 2,
3, or 5% piperidine rather than the 20% piperidine commonly
used for Fmoc removal, thereby allowing reduction or elimina-
tion of base-catalyzed side reactions. Thus, in the assembly of
Bz-Val-Lys(BOC)-â-Asp(R-O-t-Bu)-Gly-Tyr(t-Bu)-Ile-OH (15)
according to the normal Fmoc protocol (20% piperidine/7 min),
succinimide formation occurred due to the presence of the
sensitive Asp-Gly sequence to the extent of 11.6%.14 Under
normal Bsmoc conditions (2% piperidine/7 min) 4.8% of the
imide was observed. An additional advantage of Bsmoc over
Fmoc assembly was noted in the case of pentapeptide H-Tyr-
Aib-Aib-Phe-Leu-OH (16),15 where the difficult Aib-Aib
JA9713690
(13) HATU ) N-[[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-
yl]methylene]-N-methylmethanaminium hexafluorophosphate N-oxide. HBTU
) N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmetha-
naminium hexafluorophosphate N-oxide. For a description of the current
view of the structure of these coupling reagents, see: Carpino, L. A.; El-
Faham, A.; Albericio, F. J. Org. Chem. 1995, 60, 3561.
(14) The high sensitivity of model hexapeptide 15 toward base-catalyzed
cyclization has been described previously: Lauer, J.; Fields, C. G.; Fields,
G. B. Lett. Pept. Sci. 1994, 1, 197.
(15) Carpino, L. A.; El-Faham, A.; Minor, C. A.; Albericio, F. J. Chem.
Soc., Chem. Commun. 1994, 201.
(16) The sensitivity of the Bspoc and Bsmoc residues was also examined
under conditions commonly used for the deblocking of other standard amino
protecting groups. Stability of the Bspoc group was shown toward either
neat TFA or saturated HCl in HOAc for periods up to 24 h at room
temperature. Degradation however occurred in saturated HBr in HOAc. In
dimethylamine-free DMF no reaction occurred for the Bsmoc residue up
to at least 24 h. Under the conditions of catalytic hydrogenolysis (H2/Pd-
C) the Bspoc function is, as expected, subject to rapid deblocking. Both
tert-butyl 2-propenyl sulfone and tert-butyl isopropyl sulfone were observed
as byproducts, the latter on extended treatment. For Bsmoc derivatives no
reaction has yet been observed on attempted catalytic hydrogenolysis.17
Bsmoc derivatives were also found to be stable toward tertiary amines
(pyridine, DIEA) as well as HOBt/DIEA (1:1) for at least 24 h. On the
other hand, base-catalysed mercaptan deblocking of the Bsmoc group occurs
readily. Thus treatment of the stable solution of 7 and 1 equiv of
R-toluenethiol in MeOH-d4 with 5-10 mol % of DIEA causes rapid
deblocking.
(10) Bsmoc amino acids are available from Oryza Laboratories, Inc.,
Chelmsford, MA 01824.
(11) For a review and earlier references, see: Carpino, L. A.; Beyermann,
M.; Wenschuh, H.; Bienert, M. Acc. Chem. Res. 1996, 29, 268.
(12) For the corresponding Fmoc-based synthesis (20.9%), see: Carpino,
L. A.; Chao, H.-G.; Ghassemi, S.; Mansour, E. M. E.; Riemer, C.; Warrass,
R.; Sadat-Aalaee, D.; Truran, G. A.; Imazumi, H.; El-Faham, A.; Ionescu,
D.; Ismail, M.; Kowaleski, T. L.; Han, C.-H.; Wenschuh, H.; Beyermann,
M.; Bienert, M.; Shroff, H.; Albericio, F.; Triolo, S. A.; Sole, N. A.; Kates,
S. A. J. Org. Chem. 1995, 60, 7718. The reported yields are calculated on
the basis of the fully protected hexapeptide. In both syntheses the final
step involved coupling of Fmoc-Ile-F so that the same product would result.
The increased yield in the Bsmoc case is attributed mainly to the need to
use less TAEA which results in less loss of the growing peptide into the
aqueous phase during the extraction process. Yields reported for peptides
11-13 may not be representative since these syntheses were carried out
with a greater excess of TAEA than needed.
(17) Negative reactions under conditions of catalytic hydrogenolysis must
be interpreted with caution. See: Carpino, L. A.; Tunga, A. J. Org. Chem.
1986, 51, 1930.