Letters in Peptide Science, 1 (1994) 197-205
`ESCOM
`
`LIPS 026
`
`197
`
`Sequence dependence of aspartimide formation during
`9-fluorenylmethoxycarbonyl solid-phase peptide synthesis
`
`Janelle L Lauer°, Cynthia G. Fields° and Gregg B. Fields''*
`
`"Department of Laboratory Medicine and Pathology, bBiomedical Engineering Center and "Department of Biochemistry,
`University of Minnesota, Box 107, Minneapolis, MN 55455, U.S.A.
`
`Received 31 January 1995
`Accepted 23 February 1995
`
`Key words: Aspartimide; Fmoc solid-phase peptide synthesis; Piperidides; Side reactions
`
`SUMMARY
`
`We have examined the sequence dependence of aspartimide formation during Fmoc-based solid-phase synthesis
`of the peptide Val-Lys-Asp-X-Tyr-Ile. The extent of aspartimide formation and subsequent conversion to the a-
`or P-piperidide was characterized and quantitated by analytical reversed-phase high-performance liquid chromatog-
`raphy and fast atom bombardment mass spectrometry. Aspartimide formation occurred for X = Arg(Pmc),
`Asn(Trt), Asp(OtBu), Cys(Acm), Gly, Ser, Thr and Thr(tBu). No single approach was found that could inhibit this
`side reaction for all sequences. The most effective combinations, in general, for minimization of aspartimide
`formation were (i) tert-butyl side-chain protection of aspartate, piperidine for removal of the Fmoc group, and
`either 1-hydroxybenzotriazole or 2,4-dinitrophenol as an additive to the piperidine solution; or (ii) 1-adamantyl
`side-chain protection of aspartate and 1,8-diazabicyclo[5.4.0]undec-7-ene for removal of the Fmoc group.
`
`INTRODUCTION
`
`Successful solid-phase syntheses are dependent
`upon highly efficient coupling/deprotection cycles
`and minimization of deleterious side reactions.
`The cyclization of aspartate to form aspartimide
`has long been recognized as a substantial side
`reaction occurring during both synthesis and
`storage of peptides [1-3]. Aspartimide formation
`during peptide synthesis can be either acid or base
`catalyzed, with the kinetics of ring closure de-
`pending upon the nature and strength of the acid
`or base, the structure of the aspartate side-chain
`protecting group, and the aspartate carboxyl
`
`*To whom correspondence should be addressed.
`
`0929-5666/$ 10.00
`
`1995 ESCOM Science Publishers B.V.
`
`neighboring residue. Extensive studies have been
`published of aspartimide formation during tert-
`butyloxycarbonyl (Boc)-based peptide synthesis
`which have focused on protecting group strategies
`[4,5], sequence dependence [6], and the nature and
`strength of the acid or tertiary base [5,7]. These
`studies found that aspartimide formation can be
`adequately suppressed by additives such as 1-
`hydroxybenzotriazole (HOBt) or 2,4-dinitrophenol
`(Dnp) during base neutralization [8] or by using
`2-adamantyl (2-Ada) or cyclohexyl side-chain
`protection of aspartate instead of benzyl (Bzl)
`protection [4,5].
`It had been assumed that for 9-fluorenyl-
`
`MYLAN EXHIBIT - 1024
`Mylan Pharmaceuticals, Inc. v. Bausch Health Ireland, Ltd. - IPR2022-00722
`
`

`

`198
`
`methoxycarbonyl (Fmoc)-based solid-phase syn-
`thesis, tert-butyl (tBu) side-chain protection of
`aspartate inhibits aspartimide formation. How-
`ever, Nicolas et al. [9] found substantial asparti-
`mide formation (see Scheme 1) resulting from
`piperidine treatment of the resin-bound sequence
`Val-Lys(Boc)-Asp(OtBu)-Gly-Tyr(tBu)-Ile. Sub-
`sequently, other sequences have been found to be
`susceptible to aspartimide formation during Fmoc
`solid-phase synthesis [10-13]. As shown in Scheme
`1, aspartimide-containing peptides can be con-
`verted to the a- or (3-piperidide by continued
`exposure to piperidine during solid-phase syn-
`thesis [11-14]. Alternatively, aqueous conditions
`could result in ring opening to either the a- or 13-
`peptide. Ring opening to either the piperidide or
`
`peptide is accompanied by aspartate racemization
`[15]. Although comprehensive studies have not
`been performed, aspartimide and piperidide for-
`mation have been shown to be dependent upon
`the peptide sequence, solvent polarity, and con-
`formation of the peptide chain [11,13,16,17]. We
`have further examined the sequence dependence
`of aspartimide formation using the model peptide
`Val-Lys-Asp-X-Tyr-Ile, based on the initial stud-
`ies of Nicolas et al. [9]. In addition, we have
`compared the effect of different bases [piperidine
`versus 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)],
`additives (HOBt and Dnp), and aspartate side-
`chain protecting groups [tBu and 1-adamantyl (1-
`Ada)] [4,18] on aspartimide and subsequent piper-
`idide formation.
`
`sfl)
`—NH— CH —C —NH—
`
`H27
`
`Asp
`
`C — OtBu
`II
`0
`
`base
`
`/ 0
`—NH—CH--C/ Aspartimide
`\
`/
`piperidine
`N-...,
`ItiC
`' N7
`II
`0
`
`aqueous base
`
`V
`—NH—CH —C—NH-
`
`0-Piperidide
`
`\ /
`
`H2C
`I
`C--
`I
`i
`
`+
`
`o
`II
`—NII ?H—C —N
`
`a-Piperidide
`
`—NH—
`
`H2C,
`
`II
`
`O
`II
`—NH—CH—C —NH—
`
`0
`II
`—NH—CH—C —0-
`
`+
`
`a -Peptide
`--O--
`
`H27
`
`II
`o
`Scheme 1. Formation and ring opening reactions of aspartimide.
`
`I
`H27
`
`II
`
`P-Peptide
`
`—Nil—
`
`

`

`METHODS
`
`All standard peptide synthesis chemicals were
`analytical reagent grade or better and purchased
`from Applied Biosystems, Inc. (Foster City, CA)
`or Fisher (Pittsburgh, PA). DBU was obtained
`from Aldrich (Milwaukee, WI), trifluoroacetic
`acid (TFA) from Applied Biosystems, benzoic
`anhydride from Kodak, Fmoc-Ile-4-hydroxymeth-
`ylphenoxy (HMP) resin (substitution level = 0.46
`mmol/g) and benzotriazole-1-yl-oxy-tris-pyrrolidi-
`nophosphonium hexafluorophosphate (PyBOP)
`from Novabiochem (La Jolla, CA), 2-(1H-benzo-
`triazol-1-y1)-1,1,3,3-tetramethyl uronium hexafluo-
`rophosphate (HBTU) from Richelieu Biotechno-
`logies (St.-Hyacinthe, Quebec), and all Fmoc-
`amino acids from Novabiochem or Millipore
`Corp. (Bedford, MA). All amino acids are of the
`L-configuration where applicable.
`Incorporation of individual amino acids was by
`Fmoc solid-phase methodology on either an Ap-
`plied Biosystems 431A peptide synthesizer or a
`Gilson automated multiple peptide synthesizer
`AMS 422. Cycles for the ABI 431A were as de-
`scribed [19], while cycles for the Gilson AMS 422
`were modified from those described previously
`[20] by using double couplings with sixfold
`excesses of Fmoc-amino acids. All peptides were
`assembled on Fmoc-Ile-HMP resin, capped with
`a 10-fold excess of benzoic anhydride using stan-
`dard coupling cycles, and cleaved and deprotected
`with H2O—TFA (1:19) as described [21]. Peptides
`were purified by preparative reversed-phase high-
`performance liquid chromatography (RP-HPLC)
`on a Rainin AutoPrep system equipped with a
`Vydac C1, column (15-20 µm particle size, 300 A
`pore size, 250 x 22 mm). The elution gradient was
`0-60% B in 60 min at a flow rate of 5.0 ml/min,
`where A was water containing 0.1% TFA and B
`was acetonitrile containing 0.1% TFA. Detection
`was at 229 nm. Analytical RP-HPLC was per-
`formed on a Hewlett-Packard 1090 Liquid Chro-
`matograph equipped with a Dynamax C18 column
`(5 µm particle size, 300 A pore size, 250 x 4.6
`mm). The elution gradient was 0-60% B in 45
`
`199
`
`min at a flow rate of 1.0 ml/min, where A was
`water containing 0.045% TFA and B was aceto-
`nitrile containing 0.035% TFA. Detection was at
`230 nm. Elution peaks were integrated using the
`Hewlett-Packard ChemStation software set at
`peak width 0.100, threshold —2, and area reject
`<10% of the largest peak.
`Edman degradation sequence analysis was
`performed on an Applied Biosystems 477A pro-
`tein sequencer/120A analyzer and fast atom bom-
`bardment mass spectrometry (FABMS) on a VG
`7070E-HF mass spectrometer as described previ-
`ously [19,22]. Sequence analysis of selected pep-
`tide-resins indicated efficient assemblies. Mass
`spectral analysis of the benzoyl-Val-Lys-Asp-X-
`Tyr-Ile peptides (either purified or crude when no
`side products were seen by analytical RP-HPLC)
`gave (theoretical molecular weights are given in
`parentheses): X = Ala, [M+H]+ = 812.4 Da (812.8
`Da); X = aminoisobutyric acid (Aib), [M+H]+ =
`826.4 Da (826.9 Da); X = Arg (from Arg(Pmc)),
`[M+H]+ = 897.4 Da (897.9 Da); X = Asn (from
`Asn(Trt)), [M+H]+ = 855.4 Da (855.8 Da); X =
`Asp (from Asp(OtBu)), [M+H]+ = 856.5 Da (856.8
`Da); X = Cys (from Cys(Trt)), [M+H]+ = 844.5 Da
`(844.9 Da); X = Cys(Acm), [M+H]+ = 915.4 Da
`(916.0 Da); X = Gln (from Gln(Trt)), [M+H]+ =
`869.5 Da (869.9 Da); X = Glu (from Glu(OtBu)),
`[M+H]+ = 870.5 Da (870.9 Da); X = His (from
`His(Trt)), [M+H]+ = 878.5 Da (878.9 Da); X = Ile,
`[M+H]+ = 854.5 Da (854.9 Da); X = Leu, [M+H]+
`= 854.5 Da (854.9 Da); X = Lys (from Lys(Boc)),
`[M+H]+ = 869.5 Da (869.9 Da); X = Met, [M+H]+
`= 872.5 Da (873.0 Da); X = Phe, [M+H]+ = 888.5
`Da (888.9 Da); X = Pro, [M+H]+ = 838.5 Da
`(838.9 Da); X = Ser, [M+H]+ = 828.5 Da (828.8
`Da); X = Ser (from Ser(tBu)), [M+H]+ = 828.5 Da
`(828.8 Da); X = Thr, [M+H]+ = 842.3 Da (842.9
`Da); X = Thr (from Thr(tBu)), [M+H]+ = 842.5
`Da (842.9 Da); X = Trp, [M+H]+ = 927.4 Da
`(928.0 Da); X = Trp (from Trp(Boc)), [M+H]+ =
`927.5 Da (928.0 Da); X = Tyr (from Tyr(tBu)),
`[M+H]+ = 904.6 Da (904.9 Da); and X = Val,
`[M+H]+ = 840.5 Da (840.9 Da). FABMS analysis
`of benzoyl-Val-Lys-Asp-Gly-Tyr-Ile obtained us-
`
`

`

`200
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`300-
`
`7 200-
`CC
`E
`
`100-
`
`140-
`
`1223 -
`
`100-
`
`60:
`
`40-
`
`20-
`
`0 -
`
`15
`
`CD
`Cri
`
`ID
`h
`
`CO
`
`If)
`
`(D
`IT)
`
`•
`
`.
`
`.
`25
`20
`Time (min. )
`
`A
`
`B
`
`C
`
`30
`
`35
`
`Fig. 1. RP-HPLC elution profiles of the cleavage products from (A) peptide-resin la after 16 h piperidine treatment; (B) peptide-
`resin lb after 0 h piperidine treatment; and (C) peptide-resin lb after 16 h piperidine treatment. The peptides eluting at 18.9 min
`in (A) and 18.8 min in (B) are the desired products ([M+H]' = 798.7 and 798.3 Da). The peptide eluting at 20.0 min is the
`aspartimide-containing product ([M+H]' = 780.3 Da). The peptides eluting at 22.3, 23.7, and 25.1 min are L- or D-, a- or [3-
`piperidide-containing products ([M+H]' = 865.4 Da).
`
`

`

`201
`
`ing Asp(O-1-Ada) gave [M+H]+ = 798.5 Da (798.8
`Da).
`
`RESULTS
`
`side-chain protected
`The conversion of
`aspartate to aspartimide can occur by repetitive
`base treatments throughout the synthesis of a
`peptide. We chose the sequence Val-Lys-Asp-Gly-
`Tyr-Ile to study aspartimide formation because of
`its susceptibility to base-catalyzed cyclization of
`the aspartate residue during synthesis [9]. Initially,
`to compare the extent of aspartimide formation,
`peptide-resins la and lb were synthesized, where
`la contained aspartate with tBu side-chain (13-
`carboxyl) protection and lb contained aspartate
`with tBu a-carboxyl protection and no side-chain
`
`protection. During assembly of la and lb, Fmoc
`removal for each amino acid utilized a 9 min
`treatment with piperidine and 0.1 M HOBt in
`NMP (1:4). Thus, the aspartate residue was ex-
`posed to the piperidine solution for 27 min. The
`peptide-resins were subsequently treated with
`piperidine—DMF (1:4) for 0, 2, 4, 6 and 16 h,
`after which they were cleaved and side-chain de-
`protected for 1 h, and the products were analyzed
`by RP-HPLC. After 0 h treatment, the cleavage
`product from la contained only the desired mate-
`rial, eluting at 18.9 min ([M+H]+ = 798.7 Da,
`theoretical 798.8 Da). A 16 h treatment of la re-
`sulted in a product containing 39.3% of the de-
`sired material (elution time = 18.9 min), and sev-
`eral later eluting peptides (Fig. 1A). The peptide
`eluting at 20.0 min corresponded to an aspart-
`
`TABLE 1
`SEQUENCE DEPENDENCE FOR PIPERIDINE-CATALYZED ASPARTIMIDE FORMATION OF BENZOYL-Val-
`Lys(Boc)-Asp(OtBu)-X-Tyr(tBu)-Ile-HMP RESIN
`
`X
`
`Percentage desired product
`
`t = 0 h
`
`t =- 2 h
`
`t = 4 h
`
`t=6h
`
`t= 16 h
`
`Ala
`Aib
`Arg(Pmc)
`Asn(Trt)
`Asp(OtBu)
`Cys(Acm)
`Cys(Trt)
`Gln(Trt)
`Glu(OtBu)
`Gly
`His(Trt)
`Ile
`Leu
`Lys(Boc)
`Met
`Phe
`Pro
`Ser
`Ser(tBu)
`Thr
`Thr(tBu)
`Trp
`Trp(Boc)
`Tyr(tBu)
`Val
`
`100
`100
`82.4
`77.2
`88.5
`52.6
`100
`100
`100
`39.3
`100
`100
`100
`100
`100
`100
`100
`32.7
`100
`12.5
`67.1
`100
`100
`100
`100
`" The product contained —10% of a t-butylated peptide with the desired sequence ([M+Hr = 900.6 Da, theoretical 901.0 Da).
`b ND = not determined.
`
`100
`100
`100
`100
`100
`100
`100"
`100
`100
`100
`100
`100
`100
`100
`100
`100
`100
`100
`100
`81.0
`100
`100
`100
`100
`100
`
`100
`100
`92.9
`100
`100
`100
`100
`100
`100
`ND''
`100
`100
`100
`100
`100
`100
`100
`82.9
`100
`62.5
`100
`100
`100
`100
`100
`
`100
`100
`94.2
`100
`100
`92.0
`100
`100
`100
`ND
`100
`100
`100
`100
`100
`100
`100
`62.7
`100
`44.4
`100
`100
`100
`100
`100
`
`100
`100
`88.8
`100
`96.1
`88.3
`100
`100
`100
`ND
`100
`100
`100
`100
`100
`100
`100
`53.9
`100
`36.1
`100
`100
`100
`100
`100
`
`

`

`202
`
`imide-containing product based on FABMS
`([M+H]+ = 780.3 Da, theoretical 780.8 Da). Pep-
`tides eluting at 22.3, 23.7 and 25.1 min corre-
`sponded to piperidide-containing products based
`on FABMS ([M+H]+ = 865.4 Da, theoretical 865.8
`Da). After 0 h treatment, the cleavage product
`from lb contained 75.3% of the desired material,
`eluting at 18.8 min ([M+H]+ = 798.3 Da, theoreti-
`cal 798.8 Da) and 24.7% of the aspartimide-con-
`taining peptide, eluting at 20.0 min (Fig. 1B).
`Thus, aspartimide formation occurred more rapid-
`ly for the fl-Asp peptide compared to the a-Asp
`peptide. After a 16 h treatment only the piperi-
`dide-containing products of lb were seen, eluting
`at 22.3, 23.7 and 25.1 min (Fig. 1C).
`
`la benzoyl-Val-Lys(Boc)-Asp(OtBu)-Gly-Tyr-
`(tBu)-Ile-HMP resin
`
`lb benzoyl-Val-Ly s(B oc)-13-Asp (a- OtBu)-Gly-
`Tyr(tBu)-Ile-HMP resin
`
`To examine the sequence dependence of aspart-
`imide formation, 25 different residues were sub-
`stituted for glycine in the Val-Lys-Asp-Gly-Tyr-Ile
`sequence. During assembly of the 25 peptide-
`resins, the aspartate residue was exposed to piper-
`idine and 0.1 M HOBt in DMF (1:4) for 1.85 h.
`Peptide-resins were subsequently treated with pi-
`peridine—DMF (1:4) for 0, 2, 4, 6 and 16 h, after
`which they were cleaved and side-chain depro-
`tected for 1 h, and the products were analyzed by
`RP-HPLC (Table 1). The area of the desired
`
`product peak was compared to those of later
`eluting product peaks corresponding to asparti-
`mide- and piperidide-containing peptides (vide
`supra). With no additional piperidine treatment,
`cyclization of aspartate was seen for the sequence
`containing X = Thr. Additional piperidine treat-
`ment resulted in aspartimide formation for se-
`quences containing X = Arg(Pmc) or Ser after 2
`h, Cys(Acm) after 4 h, Asp(OtBu) after 6 h, and
`Asn(Trt) or Thr(tBu) after 16 h. The extent of
`aspartimide formation increased very slowly over
`time for X = Arg(Pmc), Asp(OtBu), or Cys(Acm),
`but very rapidly for X = Ser or Thr. For all of
`these aspartimide-forming sequences, the use of
`DBU-piperidine-DMF (1:1:48) instead of piper-
`idine—DMF (1:4) increased the rate of side reac-
`tion (not shown).
`Inhibition of aspartimide formation was studied
`with HOBt or Dnp as additives to the piperidine
`solution. All sequences from Table 1 that gave as-
`partimide formation were exposed to a 16 h pi-
`peridine—DMF (1:4) treatment in the presence of
`0.1 M potential inhibitor. In most cases, either
`HOBt or Dnp could inhibit, but not always elim-
`inate, aspartate cyclization (Table 2). For the se-
`quences containing X = Cys(Acm), Thr or Thr-
`(tBu), the use of Dnp as an additive significantly
`increased the yield of desired product from 53 to
`76%, 12 to 55%, and 67 to 100%, respectively.
`The synthesis of la was repeated using Fmoc-
`Asp(O-1-Ada) to evaluate the influence of the
`aspartate side-chain protecting group on asparti-
`mide formation. Treatment of benzoyl-Val-Lys-
`
`TABLE 2
`INHIBITION OF PIPERIDINE-CATALYZED ASPARTIMIDE FORMATION OF BENZOYL-Val-Lys(Boc)-Asp(OtBu)-
`X-Tyr(tBu)-Ile-HMP RESIN
`
`Percentage desired product following 16 h piperidine treatment
`
`Arg(Pmc)
`Asn(Trt)
`Asp(OtBu)
`Cys(Acm)
`Ser
`Thr
`Thr(tBu)
`
`No additive
`
`82.4
`77.2
`88.5
`52.6
`32.7
`12.5
`67.1
`
`HOBt
`
`85.2
`84.8
`92.5
`68.6
`45.9
`28.0
`100
`
`Dnp
`
`95.2
`81 .2
`100
`76.3
`41.1
`54.9
`100
`
`

`

`TABLE 3
`ASPARTIMIDE FORMATION OF BENZOYL-Val-Lys(Boc)-Asp(O-1-Ada)-Gly-Tyr(tBu)-Ile-HMP RESIN
`
`203
`
`Base
`
`Piperidine
`Piperidine
`Piperidine
`Piperidine
`Piperidine
`Piperidine
`Piperidine
`DBU
`DBU
`DBU
`DBU
`DBU
`DBU
`DBU
`
`Additive
`
`Time (h)
`
`Percentage desired product
`
`None
`None
`None
`None
`None
`HOBt
`Dnp
`None
`None
`None
`None
`None
`HOBt
`Dnp
`
`0
`2
`4
`6
`16
`16
`16
`0
`2
`4
`6
`16
`16
`16
`
`100
`0
`0
`0
`0
`69.3
`86.9
`100
`83.1
`85.0
`73.3
`11.2
`11.6
`12.4
`
`(Boc)-Asp(O-1-Ada)-Gly-Tyr(tBu)-Ile-HMP resin
`(1c) with piperidine—DMF (1:4) resulted in higher
`levels of side products than when Asp(OtBu) was
`used for the same sequence. For example, a 16 h
`treatment of lc yielded none of the desired prod-
`uct (Table 3), whereas 39.3% of the desired prod-
`uct was obtained following identical treatment of
`la. Both HOBt and Dnp were effective inhibitors
`of aspartimide formation of lc (Table 3). Treat-
`ment of lc with DBU was far less detrimental
`than piperidine. All time points beyond 0 h re-
`sulted in more desired product following DBU
`treatment than piperidine. Additives such as
`HOBt or Dnp had little effect on inhibition of
`DBU-catalyzed aspartimide formation (Table 3).
`
`DISCUSSION AND CONCLUSIONS
`
`The formation of aspartimide in aspartate-
`containing peptides has been documented for only
`a few sequences assembled by Fmoc chemistry
`[9-13,16,17,23]. A comprehensive study was clear-
`ly needed to determine if this side reaction is a
`significant problem during Fmoc solid-phase
`chemistry. We utilized the sequence Val-Lys-Asp-
`X-Tyr-Ile as a model for aspartimide formation.
`For X = Gly, there was no detectable conversion
`to aspartimide following peptide assembly, but
`60.7% conversion after an additional 16 h expo-
`
`sure to piperidine—DMF (1:4). Assuming a linear
`relationship, this corresponds to 0.63% conversion
`per 10 min treatment. Nicolas et al. [9] and
`Quibell et al. [23] found 0.46 and 0.5% conver-
`sion, respectively, per 10 min piperidine—DMF
`(1:4) treatment of resin-bound, side-chain pro-
`tected Val-Lys-Asp-Gly-Tyr-Ile. Our results with
`respect to sequence dependence show that aspart-
`imide formation occurs for X = Thr > Ser > Arg-
`(Pmc) = Cys(Acm) > Asp(OtBu) = Asn(Trt) =
`Thr(tBu), where threonine and serine overwhelm-
`ingly gave the most rapid side-product formation.
`It is somewhat surprising that ring closure occurs
`readily in the presence of a bulky side chain [2]
`such as Arg(Pmc). A prior study of base-catalyzed
`aspartimide formation of Boc-Asp(OBz1)-X-B-
`naphthylamide indicated that conversion was
`most rapid for the residues X = Ser > Thr > Gly
`= Asn = Gln [6]. Thus, results of our study are
`consistent with this prior study. We did not exam-
`ine X = Asn or Gln, as there does not appear to
`be much effect of Trt side-chain protection of a
`neighboring asparagine or glutamine on inhibition
`or enhancement of aspartimide formation [17].
`Prior studies of aspartimide formation of Asp-
`(OtBu)-X sequences during Fmoc chemistry found
`significant conversion when X = Gly, Ala, Asn,
`Asn(Trt), Gln, and Gln(Trt) [9-13,16,17]. While
`we did see aspartimide formation for X = Gly and
`
`

`

`204
`
`Asn(Trt), we did not for X = Ala and Gln(Trt).
`Other researchers have shown that not all Asp-
`(OtBu)-Gly are susceptible to cyclization [24].
`These results lend further support to the notion
`that aspartimide formation is both sequence and
`conformation dependent [11,17].
`When DBU was used instead of piperidine, the
`rate of aspartimide formation increased for all
`sequences examined. This is consistent with prior
`results with an Asp(OtBu)-Asn(Trt) sequence [10].
`Previously, DBU has been shown to remove the
`Fmoc group more rapidly than piperidine [24].
`Fmoc group removal requires a base to abstract
`the acidic proton at the 9-position of the fluorene
`ring system, leading to fl-elimination. If DBU
`more rapidly removes this proton than piperidine,
`then the increased rate of aspartimide formation
`may be the result of the rate of abstraction of the
`NH proton leading to cyclization.
`A sufficient number of aspartimide-susceptible
`sequences have now been identified to warrant the
`development of general methods for inhibiting
`aspartate cyclization during Fmoc solid-phase
`synthesis. We have considered the (i) time of base
`treatment; (ii) use of additives during Fmoc re-
`moval; and (iii) protecting group strategy. In-
`creased exposure to piperidine or DBU usually
`results in increased aspartimide and/or piperidide
`formation of susceptible sequences, as found pre-
`viously [12,16]. Obviously, minimizing the length
`of base treatment of susceptible sequences will, in
`general, reduce aspartimide formation. Additives
`such as HOBt or Dnp were effective inhibitors
`when Asp(OtBu) or Asp(O-1-Ada) was used in
`combination with piperidine, similar to their ef-
`fects on base-catalyzed aspartimide formation of
`Boc-Asp(OBz1)-Gly-0-naphthylamide
`[8] and
`Fmoc-Leu-Thr(tBu)-Glu(OtBu)-Asp(OtBu)-Asn-
`(Trt)-Val-Lys(Boc)-HMP resin [16,17]. The use of
`HOBt as an additive to piperidine solutions does
`not appear to have a detrimental effect on Fmoc
`removal [11,25]. However, neither HOBt nor Dnp
`was an effective inhibitor of aspartimide forma-
`tion during DBU treatment of the Asp(O-1-Ada)
`sequence studied here.
`
`The Ada group has been reported to suppress
`acid-catalyzed aspartimide formation better than
`the Bzl and cyclohexyl groups [4,18]. It is believed
`that the relative bulkiness of the Ada group is
`responsible for this inhibitory effect [4]. We found
`that the 1-Ada group was (i) more effective at
`suppressing DBU-catalyzed aspartimide formation
`than the piperidine-catalyzed side reaction; and
`(ii) less effective at suppressing piperidine-cata-
`lyzed aspartimide formation than tBu group side-
`chain protection of aspartate. Based on Ada steric
`effects alone, result (i) is anticipated. However,
`steric effects do not explain result (ii). It is possible
`that Ada or tBu provides minimal steric hindrance
`to piperidine abstraction of the NH proton.
`Other approaches for potential inhibition of
`aspartimide formation can also be considered.
`Since the base-catalyzed reaction requires abstrac-
`tion of the NH proton [2], there should be no
`cyclization when the carboxyl neighbor to as-
`partate is an N-alkyl amino acid. This concept is
`supported by our results, showing no aspartimide
`formation for the sequence containing an N-alkyl
`amino acid (X = Pro). In a similar fashion, a
`recent study showed minimization of aspartimide
`formation using the 2-hydroxy-4-methoxybenzyl
`(Hmb) N-protecting group prior to the incorpor-
`ation of an aspartate residue [23]. An alternative
`method for inhibiting aspartimide formation
`would thus be solid-phase incorporation of an N-
`alkyl amino acid prior to Fmoc-Asp(OtBu). Based
`on our study, the most effective combinations for
`minimization of aspartimide formation were (i)
`tBu side-chain protection of aspartate, piperidine
`for removal of the Fmoc group, and either HOBt
`or Dnp as an additive to the piperidine solution;
`or (ii) 1-Ada side-chain protection of aspartate
`and DBU for removal of the Fmoc group.
`
`ACKNOWLEDGEMENTS
`
`We acknowledge support of this work by the
`NIH (DK 44494 and AR 01929), a McKnight-
`Land Grant Professorship, and the Millipore
`Corporation.
`
`

`

`205
`
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