Non-natural amino acids as modulating agents of the conformational space of model glycopeptides

DOI: 10.1002/chem.200800460

Non-natural Amino Acids as Modulating Agents ofthe Conof rmational

Space ofModel Glycopeptides

Alberto Fernµndez-Tejada, Francisco Corzana,* Jesﬄs H. Busto, Gonzalo JimØnez-OsØs,

JesﬄsM. Peregrina,* and Alberto Avenoza^[a]

Abstract: The synthesis and conforma-

tional analysis in aqueous solution of

different a-methyl-a-amino acid di-

O-glucosylation process largely deter-

mines the conformational preference of

the backbone in these structures. On

the other hand, atypical conformations

of the glycosidic linkage were experi-

mentally determined. Therefore, when

a methyl group was located at the Cb

atom with an R configuration, the gly-

cosidic linkage was rather rigid. Never-

theless, when the S configuration was

displayed, a significant degree of flexi-

bility was observed for the glycosidic

linkage, thus showing both alternate

and eclipsed conformations of the y_s

dihedral angle. In addition, some deriv-

atives exhibited an unusual value for

the f_sangle, which was far from a

value of À608 expected for a conven-

tional b-O-glycosidic linkage. In this

sense, the different conformations ex-

hibited by these molecules could be a

useful tool in obtaining systems with

conformational preferences “à la

carte”.

ACHTREUNGamides, derived from serine, threonine,

b-hydroxycyclobutane-a-amino acids,

and their corresponding model b-O-

glucopeptides, are reported. The study

reveals that the presence of an a-

methyl group forces the model pep-

tides to adopt helix-like conformations.

These folded conformations are espe-

cially significant for cyclobutane deriv-

atives. Interestingly, this feature was

also observed in the corresponding

model glucopeptides, thus indicating

that the a-methyl group and not the b-

Keywords: amino acids · conforma-

tion analysis

·

glycopeptides

·

molecular

dynamics

·

NMR

spectroscopy

Introduction

in protein function.^[3]In contrast, some specific types of O-

glycosylation, such as the b-O-linked attachment of d-glu-

cose (Glc) to Ser/Thr (O-glucosylation), are less frequent

and have been found in the epidermal growth factor (EGF)

domains of different serum proteins^[4]and Notch receptor.^[5]

The role of the Glc moiety in these systems is unknown and

remains controversial. However, it is essential to know the

influence that the carbohydrate moiety has on the confor-

mational equilibrium of the peptide backbone and vice

versa. In this context, we have recently reported that the b-

O-glucosylation of model peptides derived from Ser and Thr

allows the backbone to expand its conformational space,

thus exhibiting not only extended but also folded conforma-

tions.^[6]

On the other hand, one attractive approach to the de-

tailed study of the influence that distinct structural elements

have on the conformational preferences of a molecule is to

design novel glycopeptides in which natural amino acids

have been replaced with non-natural amino acids. In addi-

tion, the resulting novel glycopeptides could stabilize one

conformation (i.e., the bioactive conformation) or could ex-

hibit conformers that have rarely been observed in the natu-

Glycoproteins and glycopeptides are involved in fundamen-

tal biological processes in which the carbohydrate and the

sequence of the amino acids frequently have different and

complex biological functions.^[1]

The most common O-glycosylations involve the a-O-gly-

cosidic linkage of N-acetylgalactosamine (GalNAc) attached

to a rich domain of serine (Ser) and/or threonine (Thr) resi-

dues (mucin-type)^[2]or the b-O-glycosidic linkage of N-ace-

tylglucosamine (GlcNAc) linked to a Ser/Thr residue of cy-

toplasmic and nuclear proteins, which play a regulatory role

[a] A. Fernµndez-Tejada, Dr. F. Corzana, Dr. J. H. Busto,

Dr. G. JimØnez-OsØs, Dr. J.M. Peregrina, Dr. A. Avenoza

Departamento de Química

Universidad de La Rioja

UA-CSIC 26006 LogroÇo (Spain)

Fax : (+34)941-299-621

E-mail: francisco.corzana@unirioja.es

jesusmanuel.peregrina@unirioja.es

Supporting information for this article is available on the WWW

under http://www.chemeurj.org/ or from the author.

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Chem. Eur. J. 2008, 14, 7042 – 7058

FULL PAPER

ral derivatives, thus modifying the binding to the target mol-

ecules. In this sense, although the synthesis of non-proteino-

genic a-amino acids has received enormous attention, par-

ticularly a,a-disubstituted a-amino acids,^[7]the field of modi-

fied glycosyl-a-amino acids remains relatively unexplored.

Most of the modifications are centered around the saccha-

ride moiety^[8]and rarely on the peptide moiety.^[9]As a con-

sequence, the glycosylation of novel b-hydroxy-a,a-disubsti-

tuted amino acids^[10]could be a useful tool in the design of

molecules with a novel conformational behavior regarding

the natural compounds.

Scheme 1. Design of novel b-O-glucopeptides derived from non-natural

amino acids.

On this basis, we report herein the synthesis and the con-

formational analysis in aqueous solution of different model

peptides derived from non-natural amino acids and the cor-

responding model b-O-glucopeptides (Scheme 1). In these

models, the amino and carboxylic functional groups were

transformed into amides to simulate the peptide backbone.

The main goal of this study was to investigate in detail the

effects that the substituents at the Ca and/or Cb atoms have

on the peptide backbone, the lateral chain, and the glycosi-

dic linkage conformational preferences.

The study involved the use of distance information based

on NOE interactions and coupling constants, which were in-

terpreted with the assistance of molecular-dynamics (MD)

simulations. It is important to note that not only was the

synthesis of the non-natural target compounds difficult, as

the a-amino acid derivatives bear quaternary carbon atoms,

but their structural analysis was also extremely complicated.

Indeed, the absence of the Ha atom in the a-substituted a-

amino acid derivatives implicates the lack of some NOE in-

teraction signals and coupling constants, which are pivotal

keys in eliciting the conformations. Moreover, the computa-

tional studies of these systems are not trivial. Indeed, the

currently used all-atom force fields that predict reasonable

conformational dynamics for larger peptides or proteins fail

to reproduce the measured conformational distributions for

di- and tripeptide systems.^[11]In addition, the use of general

force fields, which have not been tested for all types of or-

ganic molecules, is required for the study of non-natural

amino acids.

Abstract in Spanish: En este artículo se presenta la síntesis y

el anµlisis conformacional en disolución acuosa de diferentes

diamidas de a-metil-a-aminoµcidos derivados de Ser, Thr y

b-hidroxiciclobutan-a-aminoµcidos, así como de sus corres-

pondientes b-O-glucopØptidos modelo. El estudio reveló que

la presencia de un grupo metilo en posición a del aminoµci-

do fuerza al pØptido modelo a adoptar conformaciones ple-

gadas de tipo hØlice. Estas conformaciones plegadas son es-

pecialmente significativas para los derivados que incorporan

la estructura de ciclobutano. Esta característica tambiØn fue

observada para los correspondientes glucopØptidos modelo,

lo cual indica que es el grupo metilo en posición a y no la b-

O-glucosilación lo que determina en gran medida las prefe-

rencias conformacionales de la cadena peptídica en estas es-

tructuras. Por otro lado, se observaron experimentalmente

conformaciones atípicas del enlace glicosídico. Así, cuando

un grupo metilo estµ localizado en el Cb con configuración

R, el enlace glicosídico es bastante rígido. Sin embargo,

cuando dicho carbono muestra configuración S se observa

un alto grado de flexibilidad en el enlace glicosídico, mos-

trando el µngulo diedro y_stanto conformaciones alternadas

como eclipsadas. Ademµs, algunos derivados exhibieron valo-

res inusuales del µngulo diedro f_s, los cuales se apartan bas-

tante del valor esperado de À608 para un enlace b-O-glicosí-

dico convencional. En este sentido, las diferentes conforma-

ciones exploradas por estas molØculas podrían ser utilizadas

como herramientas ﬄtiles para obtener sistemas con preferen-

cias conformacionales “à la carte”.

Results and Discussion

Synthesis: Scheme 2 shows all the model peptides and glyco-

peptides studied herein. Compounds Ac-l-Ser-NHMe, Ac-l-

(b-O-d-Glc)Ser-NHMe (referred to as Ac-l-Ser*-NHMe),

Ac-l-Thr-NHMe, and Ac-l-(b-O-d-Glc)Thr-NHMe (re-

ferred to as Ac-l-Thr*-NHMe) were previously synthesized

and studied by us.^[6]The a-methylated compound was de-

rived from a-methylserine (MeSer), the a,b-dimethylated

compounds from a-methylthreonine (MeThr), and the cy-

clobutane derivatives from 2-hydroxy-1-aminocyclobutane-

carboxylic acid (c₄Ser).

The synthesis of the model peptides was carried out as de-

scribed in the Supporting Information. The corresponding

model glycopeptides were obtained under the modified con-

ditions of the Koenigs–Knorr glycosylation.^[12]Scheme 3

shows the synthesis of Ac-(S)-MeSer^*-NHMe, Ac-(R,R)-

MeThr^*-NHMe, Ac-(S,S)-c₄Ser^*-NHMe, and Ac-(R,R)-

c₄Ser^*-NHMe as representative examples of non-natural

model glycopeptides. The syntheses of the rest of the model

glycopeptides are described in the Supporting Information.

Glycopeptide Ac-(S)-MeSer^*-NHMe was synthesized

from derivative 1, which has been previously glycosylated

by our group^[13]to give 2. Further deprotection of the car-

boxylic acid and subsequent reaction with methylamine led

to 4. Finally, the transformation of the azide group into the

amine group with H₂and Pd/C as a catalyst, followed by

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J.M. Peregrina, F. Corzana et al.

Scheme 2. Model peptides and glycopeptides studied herein.

Scheme 3. Synthetic route to model glycopeptides Ac-(S)-MeSer^*-NHMe, Ac-(R,R)-MeThr^*-NHMe, Ac-(S,S)-c₄Ser^*-NHMe, and Ac-(R,R)-c₄Ser^*-NHMe.

a) H₂, Pd/C, AcOEt, 258C 16 h (90%); b) MeNH₂·HCl, TBTU, DIEA, CH₃CN 258C 16 h (84%); c) i. H₂, Pd/C MeOH 258C 15 h; ii. Ac₂O, pyridine,

258C 4 h (77%); d) MeONa/MeOH 258C 3 h (90%); e) described in the Supporting Information; f) MeONa/MeOH, 258C, 3 h (87%); g) LiOH·H₂O,

MeOH/H₂O (3:1), 258C, 12 h (97%); h) MeNH₂·HCl, TBTU, DIEA, MeCN, 258C, 10 h (92%); i) H₂, Pd/C, MeOH, 1 atm, 258C, 12 h (99%);

j) i. 2,3,4,6-tetra-O-benzoyl-a-d-glucopyranosyl bromide, AgTfO, CH₂Cl₂, À30–258C, 4- MS, 15 h (30%; 10+11); ii. column chromatography (dichloro-

methane/MeOH 9.5:0.5); k) MeONa/MeOH, 258C, 3 h (92% for Ac-(S,S)-c₄Ser^*-NHMe and 85% for Ac-(R,R)-c₄Ser^*-NHMe). MS=molecular sieves.

acetylation and subsequent deprotection gave glycopeptide

On the other hand, the model glycopeptide Ac-(R,R)-

MeThr^*-NHMe was obtained from the model peptide Ac-

(R,R)-MeThr-NHMe, which was prepared according to the

Ac-(S)-MeSer^*-NHMe.

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Conformational Analysis of Model b-O-Glucopeptides

FULL PAPER

procedure described in the Supporting Information from the

amino acid (R,R)-MeThr, the synthesis of which has been

previously reported.^[10f]The treatment of model peptide Ac-

(R,R)-MeThr-NHMe with 2,3,4,6-tetra-O-benzoyl-a-d-gluco-

pyranosyl bromide in the presence of silver trifluorometh-

ly, the deprotection of ester moiety in 14 with LiOH·H₂O

gave (R,R)-8, which was converted into Ac-(R,R)-c₄Ser^*-

NHMe following the reactions given above.

Conformational study of model peptides: The torsional

angles and the labeling of the atoms used herein for the

compounds are shown in Figure 1. In a first step, full assign-

ment of the protons in all of the compounds was carried out

using COSY and HSQC experiments. Then, selective 1D

NOESY experiments in D₂O (see the Supporting Informa-

ACHTREUNGanesulfonyl (AgOTf) gave derivative 6, which was further

deprotected to afford the model glycopeptide Ac-(R,R)-

MeThr^*-NHMe.

In the case of the cyclobutane derivatives Ac-(S,S)-c₄Ser^*-

NHMe and Ac-(R,R)-c₄Ser^*-NHMe, the synthesis started

from a racemic mixture of compound 7, which was recently

synthesized by our group.^[10j]Later, the hydrolysis of the

methyl ester with LiOH·H₂O gave the corresponding acid 8

in an almost quantitative manner (97%). Further treatment

with MeNH₂·HCl and N,N,N’,N’-tetramethyl-O-(benzotria-

zol-1-yl)uronium tetrafluoroborate (TBTU) as a coupling

agent in the presence of diisopropylethylamine (DIPEA) as

a base afforded diamide 9. Final hydrogenolysis with H₂and

Pd/C quantitatively removed the benzyl group, thus leading

to the desired Ac-c₄Ser-NHMe as a racemic mixture. The

treatment of Ac-c₄Ser-NHMe with 2,3,4,6-tetra-O-benzoyl-

a-d-glucopyranosyl bromide in the presence of AgOTf ex-

clusively gave b-anomers 10 and 11, which were separated

by column chromatography on silica gel, in a moderate

yield. Finally, the deprotection of the hydroxy groups of the

carbohydrate moiety with NaOMe gave the corresponding

model glycopeptides Ac-(S,S)-c₄Ser^*-NHMe and Ac-(R,R)-

c₄Ser^*-NHMe.

To unambiguously confirm the absolute configuration of

compounds Ac-(S,S)-c₄Ser^*-NHMe and Ac-(R,R)-c₄Ser^*-

NHMe, acid derivative 8 was treated with ethyl-3-(3-dime-

thylamino)propylcarbodiimide hydrochloride salt (ED-

CI·HCl) as a coupling agent and 4-dimethylaminopyridine

(DMAP) as a base to give 12 in an 87% yield (Scheme 4).

Oxazolone 12 was then transformed into esters 13 and 14 by

reaction with (S)-(À)-1-(2-naphtyl)ethanol in the presence

of KOtBu as a base. The ester compounds were separated

by column chromatography on silica gel, thus obtaining 14

in a pure form (6% yield from 12). We were able to obtain

single crystals of 14 by slow evaporation of a solution of this

compound in hexane and dichloromethane. The new stereo-

genic centers were found to have a R,R configuration. Final-

Figure 1. Molecular structures of Ac-(S,S)-MeThr-NHMe, Ac-(S,S)-

MeThr*-NHMe, Ac-(S,S)-c₄Ser-NHMe, and Ac-(S,S)-c₄Ser*-NHMe

showing the definitions of the torsional angles and the atom labeling.

The same definitions were used for all the molecules studied herein.

Scheme 4. Determination of the absolute configuration of glycopeptides Ac-(S,S)-c₄Ser^*-NHMe and Ac-(R,R)-c₄Ser^*-NHMe. a) EDCI·HCl, DMAP,

CH₂Cl₂, 258C 2 h (87%); b) i. (S)-(À)-1-(2-naphtyl)ethanol, tBuOK, THF, 258C, 12 h (70%); ii. column chromatography (hexane/ethyl acetate, 4:6) to

obtain 14 in a pure form; c) LiOH·H₂O, MeOH/H₂O (3:1), 258C, 4 d (96%).

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7045

J.M. Peregrina, F. Corzana et al.

tion) and 2D NOESY experiments (see Figure 2 and the

Supporting Information) in H₂O/D₂O (9:1) were carried out

As a first step in deducing the conformational behavior of

these molecules, relaxed potential-energy maps were calcu-

lated for all the compounds using the AMBER94,

CHARMM, and MM+ force fields as described in the Ex-

perimental Section. These surfaces just provide a first esti-

mation of the conformational regions that are energetically

accessible for each compound (see Figure 3a and the Sup-

porting Information). As shown in Figure 3a, the potential-

for all the compounds. In addition, ³J(H_a,H_b) and ³J-

ACHTREUNG

Although the model peptides studied herein are too short

to adopt a defined secondary structure, the NOESY data

can still be used to assess “conformational preferences”.

This behavior is based on the observation of key sequential

À

NH NH (i,i+1) and Ha NH (i,i+1) connectivities. Conse-

quently, the observation of strong Ha-NH (i,i+1) NOE in-

À

teractions along with weak or absent NH NH (i,i+1) NOE

interactions suggests a conformational preference for ex-

tended conformations. On the contrary, the observation of

À

weak–medium Ha NH (i,i +1) NOE interactions and

À

strong NH NH (i,i+1) NOE interactions suggests a confor-

mational preference for helical dihedral space.^[14]

Figure 2 (upper panel) shows the amide region of the 2D

NOESY spectra of model peptides Ac-allo-l-Thr-NHMe,

Ac-(S)-MeSer-NHMe, and Ac-c₄Ser-NHMe as representa-

tive cases. As reported for peptides derived from natural Ser

and Thr^[6](i.e., Ac-l-Ser-NHMe and Ac-l-Thr-NHMe, re-

spectively), the 2D NOESY spectrum of the (S)-allo-threo-

nine derivative (compound Ac-allo-l-Thr-NHMe) displayed

Figure 3. a) Potential-energy surfaces calculated with the CHARMM

force field for Ac-(S)-MeSer-NHMe. b) f_p/y_pdistribution obtained from

10 ns unrestrained MD simulations in explicit water (298 K, 1 atm, TIP3P

water molecules, boundary conditions, and Ewald sums for the treatment

of electrostatic interactions) for Ac-(S)-MeSer-NHMe.

À

a strong Ha NH1 NOE interaction and the absence of the

À

NH1 NH2 NOE interaction, which suggests a conforma-

tional preference for extended-like conformations.^[14]On the

À

contrary, the medium NH1 NH2 NOE interaction observed

energy surface calculated with, for example, the CHARMM

force field for Ac-(S)-MeSer-NHMe shows the presence of

four minima, referred to as a_D(with f_pclose to À608 and y_p

around À608), a_L(characterized by f_pclose to 608 and y_p

around 608), polyproline type II (PPII; with f_pclose to À608

and y_paround 1808), and an

in derivatives Ac-(S)-MeSer-NHMe and Ac-c₄Ser-NHMe

could indicate the presence of an significant population of

the helix-like conformations (see the Supporting Informa-

tion).

unusual conformation for natu-

ral amino acids (f_pclose to 608

and y_paround À1808), denoted

as F* according to the defini-

tion used previously.^[15]

The next and most important

step in the conformational anal-

ysis of the molecules was to get

an ensemble that could repro-

duce our experimental NMR

spectroscopic data. As stated

above, the unrestrained MD

simulations carried out on small

systems normally fail to repro-

duce the conformational behav-

ior of the peptide backbone. In

particular, the AMBER94 force

field used in our MD simula-

tions shows a clear tendency to

overestimate the helix-like con-

formations (see Figure 3b and

Figure 2. Section of the 800 ms 2D NOESY spectrum (400 MHz) in H₂O/D₂O (9:1) at 258C of different pep-

tides (upper panel) and glycopeptides (lower panel) studied herein. For the a-methylated and cyclobutane de-

rivatives, the amide–amide cross-peaks region is shown. The diagonal peaks are negative. The NOE interac-

tions are represented as positive cross-peaks.

the Supporting Information).

Consequently, the relative pop-

ulation of conformers obtained

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Conformational Analysis of Model b-O-Glucopeptides

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from the unrestrained MD simulations of the peptide back-

bone is not in good agreement with the experimental NMR

spectroscopic data. On the other hand, it is important to

note that the direct interpretation of NOE interaction data

in flexible molecules in terms of a single structure may lead

to the generation of high-energy virtual conformations.^[16]

Therefore, and following our previously reported proto-

col,^[6,17]we combine herein the NMR spectroscopic data

with time-averaged restrained MD (MD-tar) simulations

with the objective of obtaining a distribution of low-energy

conformers (according to the AMBER force field) able to

quantitatively reproduce the NMR spectroscopic data. This

procedure overcomes the limitations inherent in both tech-

niques (NMR spectroscopic analysis and MD simulations)

and provides a simple and robust way to consider the flexi-

bility of the molecule in the interpretation of the NMR

spectroscopic data.

NOE interaction experiments mentioned above, the f_p/y_p

dihedral values (backbone) of Ac-allo-l-Thr-NHMe were

characteristic of extended conformations, such as PPII and

the b sheet, and only

a small number of conformers

(ca. 11%) showed f_p/y_pdihedral values that correspond to

an a-helical conformation. This result is very similar to that

previously found for Ac-l-Ser-NHMe and Ac-l-Thr-NHMe

derivatives.^[6]

On the other hand, the model peptides derived from a,a-

disubstituted amino acids (i.e., Ac-(S)-MeSer-NHMe, Ac-

(S,S)-MeThr-NHMe, and Ac-c₄Ser-NHMe) showed a signifi-

cant population of conformers with f_p/y_pdihedral values

characteristic of helix-like conformations. Figure 5 indicates

Consequently, proton–proton distances were experimen-

tally determined from the corresponding NOE interaction

build-up curves^[18](see the Supporting Information) and dis-

tances involving NH protons were semiquantitatively deter-

mined by integrating the volume of the corresponding cross

3

peaks. These data together with the J coupling constants^[19]

were used as restraints in MD-tar analysis.^[20]The distribu-

tion for the peptide backbone f_p/y_pof the model peptides

obtained from the MD-tar simulations is shown in Figure 4.

It is important to note that the MD-tar simulation of Ac-

(S)-MeSer-NHMe now explores the four conformations

shown in Figure 3a, with relative populations in accordance

with the experimental NMR spectroscopic data (see Fig-

ure 4b and the Supporting Information). According to the

Figure 5. Total helix-like population (dark gray: a_d; light gray: a_l) ob-

tained for the model peptides from the MD-tar simulations.

the total number of a helix-like conformations a_d+a_lpres-

ent in the model peptides in aqueous solution. In the case of

Ac-(S)-MeSer-NHMe and Ac-(S,S)-MeThr-NHMe, the a_d

and a_lconformers coexist with extended conformations,

such as PPII and F*. It is important to mention that these

four major conformers found for Ac-(S)-MeSer-NHMe lie

at the local minima previously calculated for model peptide

Ac-(S)-MeSer-NHMe.^[21]The cyclobutane ring significantly

promoted the helix-like structures (a_dꢀ64%), in good ac-

cordance with the results previously described by others.^[22]

Additionally, the MD simulations carried out on the cyclo-

butane derivative suggest the existence of approximately

17% of the g-turn conformation (g_D).

In this case, the same conformational preference for the

backbone of Ac-c₄Ser-NHMe was observed in the solid state

with f_p=À84.48 and y_p=À7.88 (Figure 6). Moreover, the

molecule adopts only one ring-puckering conformation in

the solid state, with the pucker angle q close to À23.98 and

with the substituent at the Cb atom in an equatorial posi-

tion, which is similar to that previously described by us for

other cyclobutane derivatives.^[23]In the same way, a single

ring-puckering conformation was found in aqueous solution,

as deduced from the MD-tar simulations, with q=À20.38.

As we have reported,^[23]the existence of a single ring-puck-

ering conformation is entirely compatible with the experi-

Figure 4. Distribution of the peptide backbone (f_p/y_p) of the model pep-

tides obtained from the MD-tar simulations: a) Ac-allo-l-Thr-NHMe,

b) Ac-(S)-MeSer-NHMe, c) Ac-(S,S)-MeThr-NHMe, and d) Ac-c₄Ser-

NHMe. For Ac-c₄Ser-NHMe, the conformation of the backbone in the

solid state is shown as a grey circle.

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J.M. Peregrina, F. Corzana et al.

trast, the a-methylated derivative Ac-(S)-MeSer-NHMe ex-

hibited a rather flexible behavior for the c¹angle. On the

other hand, for the a- and b-methylated compound Ac-

(S,S)-MeThr-NHMe the lateral chain is rather rigid and dis-

plays only the anti conformation. Finally, the cyclobutane

derivative Ac-c₄Ser-NHMe presented a rigid lateral chain as

a result of the restriction imposed by the four-membered

ring. Indeed, the c¹angle has a value of around 1058

throughout the MD simulation.

Conformational study of model glycopeptides: As a first

step and following the protocol previously described for the

model peptides, the relaxed potential-energy maps were cal-

culated for all the glycopeptides using the AMBER94,

CHARMM, and MM+ force fields. Figure 8a shows the po-

tential-energy map calculated for Ac-(S,S)-MeThr*-NHMe

with the CHARMM force field. Alternatively, and to ex-

plore the conformational behavior of these molecules,

100 ns unrestrained MD simulations in a vacuum at 400 K

were carried out for all the glycopeptides (see Figure 8b and

the Supporting Information). In this case, the high tempera-

ture avoids kinetically trapping the molecules in some local

minima (Figure 8b). As can be seen for glycopeptide Ac-

(S,S)-MeThr*-NHMe, this simulation explores all the

minima found by the CHARMM force field (compare Fig-

ure 8a and b). As in the case of the peptides, the unrestrain-

Figure 6. Some geometrical features of the crystal structure of Ac-c₄Ser-

NHMe and the pucker angle q.

À

mentally determined distance Hb Hda of 2.7 (experimen-

tally determined from the NOE interaction build-up

curves).

As far as the lateral chain is concerned, its conformational

behavior is characterized by the c¹torsional angle, which

can adopt three lowest-energy staggered rotamers, denoted

as gauche(À) or g(À)

A

G

and anti or t(c¹ꢀÀ1808; Figure 7a).

Figure 7. a) More stable conformers of the lateral chain c¹. b) c¹distribu-

tions obtained from the MD-tar simulations for the different model pep-

tides. Angle c¹is around 1058 for Ac-c₄Ser-NHMe.

Figure 7b shows the population of the c¹angle obtained

from the MD-tar simulations for the model peptides. A

number of points warrant particular attention. Model pep-

tides derived from the natural l-Ser and l-Thr showed a

conformational preference for the c¹angle close to 608. Fur-

thermore, and as a result of the steric requirements of the b-

methyl group of Thr, the population of gauche(+) is slightly

higher in Ac-l-Thr-NHMe (ca. 72%). Markedly, a similar

result was obtained for derivative Ac-allo-l-Thr-NHMe, in

which the Cb atom has the opposite configuration. In con-

Figure 8. a) Potential-energy surfaces calculated with the CHARMM

force field for Ac-(S,S)-MeThr*-NHMe. b) f_p/y_pdistribution obtained

from 100 ns unrestrained MD simulations in a vacuum at 400 K for Ac-

(S,S)-MeThr*-NHMe. c) f_p/y_pdistribution obtained from 10 ns unre-

strained MD simulations in explicit water (298 K, 1 atm, TIP3P water

molecules, boundary conditions, and Ewald sums for the treatment of

electrostatic interactions) for Ac-(S,S)-MeThr*-NHMe. d) f_p/y_pdistribu-

tion obtained from the MD-tar simulations for Ac-(S,S)-MeThr*-NHMe.

7048

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Conformational Analysis of Model b-O-Glucopeptides

FULL PAPER

ed MD simulation carried out in explicit water at 298 K

failed to reproduce the conformational behavior of the pep-

tide backbone. Thus, for Ac-(S,S)-MeThr*-NHMe only one

of the minimum was populated, namely the a_lconformer

(Figure 8c). In contrast, the inclusion of the NMR spectro-

On the other hand, and as previously observed for Ac-

c₄Ser-NHMe, only one ring-puckering conformation of the

cyclobutane ring was observed in model glycopeptides Ac-

(S,S)-c₄Ser*-NHMe and Ac-(R,R)-c₄Ser*-NHMe (q=À31.6

and

3

scopic data (proton–proton distances and J coupling con-

30.08, respectively). This fact is in good agreement with the

À

stants) as restraints in the MD-tar simulations gave a f_p/y_p

distribution that can quantitatively reproduce the experi-

mental data (see Figure 8d and the Supporting Informa-

tion).

The 2D NOESY pattern spectra corresponding to the

model glycopeptides is, in all cases, rather similar to those

obtained for their parent peptides (see the lower panel of

Figure 2 and the Supporting Information). Consequently, a

f_p/y_pdistribution comparable to the distribution obtained

for the peptides could be expected. That is, the b-O-glucosy-

lation does not have a significant effect upon the conforma-

tion of the peptide backbone in the case of non-natural

amino acids.

Hb Hda distances experimentally determined for these

compounds (see the Supporting Information).^[23]Moreover,

and following the same behavior observed for the model

peptide Ac-c₄Ser-NHMe, the glycopeptides present the sub-

stituent at the Cb atom in an equatorial position.

Figure 10 shows the conformational preference of the lat-

eral chain for the model glycopeptides. Concerning the gly-

copeptides derived from the natural amino acids we previ-

The percentages of helix-like conformation for model gly-

copeptides Ac-d-Ser*-NHMe and Ac-allo-l-Thr*-NHMe

were calculated to be 12 and 11%, respectively (Figure 9).

Figure 10. c¹distributions obtained from the MD-tar simulations for the

different model glycopeptides. Angle c¹is around 105 and À1058 for Ac-

(S,S)-c₄Ser*-NHMe and Ac-(R,R)-c₄Ser*-NHMe, respectively.

Figure 9. Total helix-like population (dark gray: a_d, light gray: a_l) ob-

tained for the model glycopeptides from the MD-tar simulations.

ously described,^[6]whereas the c¹dihedral angle in glycopep-

tide Ac-l-Ser*-NHMe has a similar distribution to the

parent peptide Ac-l-Ser-NHMe, glycopeptide Ac-l-Thr*-

NHMe differs significantly from Ac-l-Thr-NHMe. Indeed,

the lateral chain in Ac-l-Thr*-NHMe mainly adopts the

gauche(+) and anti conformations (44 and 47%, respective-

ly), whereas the gauche(+) conformer is mainly populated

(72%) in the model peptide Ac-l-Thr-NHMe. A similar fea-

ture was observed when Ac-(S)-MeSer-NHMe is compared

to Ac-(S)-MeSer*-NHMe. On the other hand, b-O-glucosy-

lation does not significantly affect the lateral chain of the

Ac-allo-l-Thr-NHMe model peptide. Concerning the a-

methylthreonine derivatives, whereas the lateral chain of

model peptide Ac-(S,S)-MeThr-NHMe is totally rigid, with

an c¹angle of around 1808, the corresponding model glyco-

peptides Ac-(S,S)-MeThr*-NHMe and Ac-(R,R)-MeThr*-

NHMe exhibit the three possible conformers but also show

a clear preference for the anti conformations. Markedly, the

rotation around the c¹angle in the glycopeptide Ac-d-Ser*-

These results are similar to those obtained for non-glycosy-

lated Ac-d-Ser-NHMe and Ac-allo-l-Thr-NHMe (Figure 5)

and differ from those previously described for the natural

amino acids Ac-l-Ser-NHMe and Ac-l-Thr-NHMe, for

which an important increment of the folded conformations

were experimentally and theoretically observed for the gly-

cosylated derivatives (Ac-l-Ser*-NHMe and Ac-l-Thr*-

NHMe) when compared to the model peptides.^[6]On the

other hand, although the backbone of Ac-(S,S)-c₄Ser*-

NHMe shows mainly a a_dhelical conformation, the back-

bone of the diastereoisomer Ac-(R,R)-c₄Ser*-NHMe adopts

the contrary helix (a_lhelix), which is in accordance with the

opposite configuration that both compounds present at the

Ca atom. The same feature can be found in derivatives Ac-

(S,S)-MeThr*-NHMe (a_dhelix) and Ac-(R,R)-MeThr*-

NHMe (a_lhelix).

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7049

J.M. Peregrina, F. Corzana et al.

NHMe is somehow restricted, with a clear preference for

the anti conformers (close to 70%). Finally, the cyclobutane

derivatives Ac-(S,S)-c₄Ser*-NHMe and Ac-(R,R)-c₄Ser*-

NHMe display, as expected, only one conformer of the later-

al chain (Figures 7 and 10).

The conformation of the glycosidic linkage, characterized

by the f_sand y_storsional angles, is mainly determined by

the exo-anomeric effect,^[24]which fixes the value of f_s

around À608 in all the derivatives. On the other hand the

glycosidic linkage of Ac-d-Ser*-NHMe and Ac-(S)-MeSer*-

NHMe showed a conformational behavior similar to that

observed for Ac-l-Ser*-NHMe (Figure 11), thus indicating

that neither the stereochemistry at the Ca atom nor the

presence of a methyl group at the Ca atom significantly

affect the conformation of the glycosidic linkage in the

serine derivatives.

On the contrary, the presence of the methyl group at the

Cb atom notably affects the conformation of this linkage. In

this sense, we recently reported that the methyl group of

Thr forces the y_sangle to adopt a value close to 1208 for

glycopeptides with N-acetylgalactosamine. Therefore, the

À

Hb Cb and O1s C1s bonds are in an eclipsed conforma-

tion.^[17b]The typical alternate conformation of the y_sangle

(denoted herein as the anti conformation) is drastically de-

stabilized in Ac-l-Thr*-NHMe by steric effects between the

H1s atom and the b-methyl group (Figure 12). As a result,

this conformation is not populated throughout the MD tra-

jectory.

Consequently, the eclipsed conformer (centered around

y_s=1208 and denoted as E in Figures 12 and 13) is mainly

populated (65%) in Ac-l-Thr*-NHMe. Additionally, a dif-

ferent alternate conformation of the y_sangle is present (de-

noted as g(+) in Figures 12 and 13), with y_sclose to 608.

The g(+) conformation of the y_sangle is mainly populated

when the c¹angle has a value close to 1808 (Figure 14a). In

contrast, the eclipsed conformers show largely a c¹value of

around 608.

To determine whether this unexpected result comes from

artifacts in the MD simulations (Figure 14), relaxed poten-

tial-energy surface (PES) scans at the B3LYP/6-31GACHTREUNG(d,p)

level were performed on the reduced model of Ac-l-Thr*-

NHMe around the y_sdihedral angle considering the two

Figure 11. Glycosidic linkage (f_s/y_s) distributions obtained from the MD-

tar simulations for the model glycopeptides Ac-d-Ser*-NHMe and Ac-

(S)-MeSer*-NHMe.

À

Figure 12. Newman projections of the Cb O1s bond of Ac-l-Thr*-NHMe and Ac-allo-l-Thr*-NHMe, together with representative 3D structures of each

conformation.

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Chem. Eur. J. 2008, 14, 7042 – 7058

Conformational Analysis of Model b-O-Glucopeptides

FULL PAPER

major conformations of the c¹torsional angle, that is the

anti and g(+) conformations (Figure 15a). As a result, the

alternate and eclipsed conformations of y_s(g(+) and E, re-

Figure 15. Relaxed PES scan at the B3LYP/6-31GACHTRE(UNG d,p) level performed

with the reduced models of a) Ac-l-Thr*-NHMe and b) Ac-allo-l-Thr*-

NHMe around the y_sdihedral angle. For the reduced model of Ac-l-

Thr*-NHMe, the two major conformations of the lateral chain (c¹) were

considered: g(+) and anti. On the other hand, for the reduced model of

Ac-allo-l-Thr*-NHMe only the major conformation of the lateral chain

(c¹close to 608) was considered in the calculations.

spectively) of the reduced model were found as minima-

energy rotamers. Interestingly, the g(+) conformation of the

y_sangle is stabilized by a hydrogen bond between the O5s

and NH1 atoms (Figure 14). In this sense, the atypical

values for f_s(around À1008) presented in this conformation

could contribute to optimization of the hydrogen bond. On

the other hand, and considering the important role that

water molecules play in the conformational preferences of

these systems, it is important to mention that this hydrogen-

bonding interaction was also found (44% of the total trajec-

tory time) in the unrestrained MD simulations carried out

on Ac-l-Thr*-NHMe with explicit water molecules (see Fig-

ure 14b and the Supporting Information).

Figure 13. Glycosidic linkage (f_s/y_s) distributions obtained from the MD-

tar simulations for the model glycopeptides Ac-l-Thr*-NHMe, Ac-allo-l-

Thr*-NHMe, Ac-(S,S)-MeThr*-NHMe, Ac-(R,R)-MeThr*-NHMe, Ac-

(S,S)-c₄Ser*-NHMe, and Ac-(R,R)-c₄Ser*-NHMe. The g(+) conformation

of f_s(O5s-C1s-O1s-Cb=608) is also known as anti-f (corresponding to

1808 when the f_sis defined as H1s-C1s-O1s-Cb).

Some important conclusions

can be drawn from the analysis

of the glycosidic linkage of the

b-methylated compounds with

an S configuration at the Cb

atom

(i.e.,

Ac-allo-l-Thr*-

NHMe,

Ac-(S,S)-MeThr*-

NHMe, and Ac-(S,S)-c₄Ser*-

NHMe). This configuration of

the b-methyl group significantly

increases the flexibility of the

glycosidic linkage (Figure 13).

Indeed, the y_sangle has values

from À160 to À608 for the acy-

clic derivatives Ac-allo-l-Thr*-

NHMe and Ac-(S,S)-MeThr*-

NHMe. This feature is in good

agreement with the relaxed

Figure 14. a) Time series monitoring y_sand c¹dihedral angles during 80 ns MD-tar simulations; b) time series

monitoring the distance O5s···NH1; c) 3D structures of conformers E and g(+) of y_sin Ac-l-Thr*-NHMe. PES scan at the B3LYP/6-31G-

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7051

J.M. Peregrina, F. Corzana et al.

A

population of approximately 12% for this unusual confor-

mation. Although the existence of this higher-energy confor-

mation has been experimentally confirmed for some di- and

trisaccharides^[26]and for oligosaccharides bound to pro-

teins,^[27]to the best of our knowledge this case is the first

time that an anti-f conformation has been experimentally

observed in model glycopeptides. The exceptionally large

proportion of anti-f conformations found for glycopeptide

Ac-(S,S)-c₄Ser*-NHMe was experimentally detected by a

Thr*-NHMe along the y_sdihedral angle, in which a plateau

of less 1 Kcalmol^À1is observed between these angle values

(Figure 15b).

For compounds Ac-allo-l-Thr*-NHMe and Ac-(S,S)-

MeThr*-NHMe, the eclipsed conformation of the y_sangle

(denoted as E’ in Figure 13) is the most populated. Interest-

ingly, an alternate conformation anti, with an y_svalue of

around À1808 is also present. This conformation shows that

the f_svalue is close to À1208 to avoid the steric contact be-

tween the O5s atom and the b-methyl group. Additionally,

the g(À) conformation of the y_sangle could be stabilized by

an intramolecular hydrogen bond between the NH2 atom

and the O5s and O6s atoms (Figure 16a). Notably, although

both the steric repulsion between the b-methyl group and

the carbohydrate moiety and the possible hydrogen bonding

must play important roles in the unexpected flexibility of

the y_sangle, the influence of water molecules could also be

significant. Indeed, the conformation of Ac-(S,S)-c₄Ser*-

NHMe centered at f_sand y_svalues of around À608 could

be explained in terms of the existence of bridging water

molecules between the O2s atom and the carbonyl O1 atom

of the peptide backbone, as can be deduced from the corre-

sponding two-dimensional radial-pair distribution function^[25]

obtained from the unrestrained MD simulation (see Fig-

ure 16b and the Supporting Information).

On the other hand, the glycosidic linkage of Ac-(S,S)-

c₄Ser*-NHMe shows a larger degree of mobility not only

around y_sbut also, and more importantly, around the f_s

angle. Whereas the most populated conformations exhibit a

typical value for the f_sdihedral angle around À608, there is

also a significant population with the f_svalue close to 608.

This conformation, which is in accordance with the exo-

anomeric effect, is usually referred as an anti-f conforma-

tion (the f_svalues are close to 1808 when defined as H1s-

C1s-O1s-Cb; Figure 13). The MD-tar simulations suggest a

À

medium NOE Hb H2s interaction, exclusive of this confor-

À

mational region. That is, the existence of a short Hb H2s

average distance (estimated to be 2.9 from the NOE in-

teraction build-up curves) can never be explained without

À

assuming the existence of the anti-f population (the Hb

H2s distance in a syn conformation is longer than 4.5 ;

Figure 17).

This relatively large proportion of the anti-f conformers

in aqueous solution could be stabilized by the formation of

intramolecular hydrogen bonds. Indeed, there is a strong

correlation between the anti-f conformation and the exis-

tence of two simultaneous hydrogen bonds, between the

NH1 and O5s atoms and the NH1 and O6s atoms. The be-

havior of the glycosidic linkage of Ac-(S,S)-c₄Ser*-NHMe

was also observed in the unrestrained MD simulations car-

ried out in explicit water (Figure 18). Finally, the anti-f con-

formation was also experimentally detected for Ac-allo-l-

Thr-NHMe and Ac-(S,S)-MeThr*-NHMe (Figure 13), that

is, for compounds with an S,S configuration at the Ca and

Cb atoms. However, the population of this conformation is

less than 4% in both cases (Figure 13), which is in accord-

À

ance with the distance of H2s Hb deduced from the NOE

interaction build-up curves (3.7 and 3.4 for Ac-allo-l-

Thr*-NHMe and Ac-(S,S)-MeThr*-NHMe, respectively).

Summary ofthe conofrmational study ofmodel peptides

and glycopeptides: It is well known that the preferred con-

Figure 16. a) Calculated B3LYP/6-31G

A

pair distribution function of O1 and O2s and a representative frame of the 20 ns unrestrained MD simulation of Ac-(S,S)-c₄Ser*-NHMe showing a bridg-

ing water molecule (the distances are given in ngstrçms and dihedral angles in degrees). The maximum density of this shared water site was 2.5 times

greater than the bulk density, with maximum and average residence times of 25.0 and 1.99 ps, respectively. The average distance between O2s and O1

was 5.3 , ranging from 2.5 to 7.2 .

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Conformational Analysis of Model b-O-Glucopeptides

FULL PAPER

formations of the backbone of

model peptide derived from Ser

diamide (Ac-l-Ser-NHMe) in

aqueous solution are extended

conformations.

Additionally,

the lateral chain (c¹dihedral

angle) has a great flexibility,

thus showing the coexistence of

the g(+), g(À), and anti con-

formers, with the former being

the most populated.^[6]

Herein, we have demonstrat-

ed that the incorporation of

substituents at the Ca or Cb

atoms of Ac-l-Ser-NHMe can

modulate both the conforma-

tion of the backbone and lateral

chain. Therefore, whereas the

incorporation of a methyl group

at the Cb position of Ac-l-Ser-

NHMe in S or R configurations

mainly does not affect the back-

bone, this group rigidifies the

lateral chain, which clearly pre-

fers the g(+) conformation

(compare Ac-l-Ser-NHMe with

Ac-l-Thr-NHMe and Ac-allo-l-

Figure 17. a) The 800 ms 2D NOESY spectrum (400 MHz) of Ac-(S,S)-c₄Ser*-NHMe in H₂O/D₂O (9:1) at

258C and pH 4.8. The diagonal peaks are negative. The NOE interactions are represented as positive cross-

peaks. b) NOE interaction build-up curves that correspond to the Hb atom of Ac-(S,S)-c₄Ser*-NHMe (upper

panel) and schematic representation of the characteristic NOE interactions of syn- and anti-f conformations

(lower panel). c) Selective 1D NOESY experiments with the 1D-DPFGSE NOESY pulse sequence, which cor-

responds to the inversion of the Hb atom in derivative Ac-(S,S)-c₄Ser*-NHMe (a 1D spectrum of this molecule

is also shown in the upper part of the figure).

Thr-NHMe in Figures 5 and 7). On the other hand, the in-

corporation of the methyl group at the Ca position of Ac-l-

Ser-NHMe or Ac-l-Thr-NHMe promotes the appearance of

folded conformations of the backbone. Additionally, the

methyl group at the Cb position (MeThr) drastically rigidi-

fies the c¹dihedral angle, thus modulating its values toward

the anti conformation (compare Ac-l-Ser-NHMe and Ac-l-

Thr-NHMe with Ac-(S,S)-MeThr-NHMe in Figure 7). More-

over, the restriction imposed by the incorporation of a cy-

clobutane ring into the structure of Ser largely promotes

helix-like conformations of the backbone and it makes the

g(+) conformation exclusive for the lateral chain (compare

Ac-l-Ser-NHMe with Ac-c₄Ser-NHMe in Figures 5 and 7).

These results are summarized in Table 1.

On the other hand, we have studied how b-O-glucosyla-

tion affects the conformational preference of the backbone

and the lateral chain in the above-discussed model peptides.

As a result, although the incorporation of a b-O-glucose

unit into the non-natural a- or b-substituted b-hydroxy-a-

amino acids does not significantly affect the backbone con-

formation, glycosylation promotes an important increment

of folded conformations in the natural amino acids Ac-l-

Ser-NHMe and Ac-l-Thr-NHMe^[6](compare Figures 5 and

9). Concerning the lateral chain, glycosylation does not

affect the conformational space, except for Ac-l-Thr*-

NHMe and Ac-(S)-MeSer*-NHMe. In the former, glycosyla-

tion makes the c¹dihedral angle more flexible, thus promot-

ing the shift from the g(+) toward the anti conformation.

For Ac-(S)-MeSer*-NHMe, there is a restriction around the

c¹angle, with a clear preference for g(À) conformations

Figure 18. Time series monitoring the f_sdihedral angle and distances

NH1···O5s and NH1···O6s in a 20 ns unrestrained MD simulation in ex-

plicit water for Ac-(S,S)-c₄Ser*-NHMe.

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J.M. Peregrina, F. Corzana et al.

Table 1. Exploration of the conformational space of Ser diamide derivatives that incorporate substituents at

the Ca and/or Cb positions.

Ac-(S,S)-c₄Ser*-NHMe and Ac-

(R,R)-c₄Ser*-NHMe are struc-

turally rigid molecules, whereas

Ac-(R,R)-c₄Ser*-NHMe exhib-

its large conformational stiff-

ness and its diastereoisomer

Ac-(S,S)-c₄Ser*-NHMe displays

a great flexibility towards the

glycosidic linkage and a signifi-

cant rigidity at the amino acid

moiety. Logically, these inher-

ent properties (flexibility and/or

rigidity) must be taken into ac-

count in the analysis of glyco-

peptide structures and in the in-

teraction of these compounds

with other types of molecules

(Table 2).

Entry Group incorporated Position of Configuration Ac-Xxx-NHMe Backbone Lateral chain

in Ser unit

the group Ca,Cb

f_p/y_p

c¹

1

2

3

4

5

6

–

S,–

S,R

S,S

S,–

S,S

l-Ser

folded

A

flexible

anti, g(+), g(À)

À

CH₃

b

l-Thr

very rigid

U

g(+) (72%)

very rigid

g(+) (70%)

flexible

À

CH₃

b

allo-l-Thr

MeSer

MeThr

c₄Ser

U

À

CH₃

a

E

anti, g(+), g(À)

À

CH₃

a,b

very rigid

T

anti (100%)

very rigid

close to g(+) (100%)

À

CH₂CH₂

T

(compare Figures 7 and 10). It is important to note that b-

O-glucosylation of Ac-d-Ser-NHMe principally induces an

anti conformation of the lateral chain.

The most important conclusions are related to the confor-

mation of the glycosidic linkage and particularly with the y_s

dihedral angle, since the f_sdihedral angle adopts a typical

value of À608, g(À) conformation, determined by the exo-

anomeric effect in most of the glycopeptides. Interestingly,

the only remarkable feature concerning the f_sdihedral

angle involves the appearance of a significant amount of the

unusual anti-f conformation, experimentally observed

mainly in the case of Ac-(S,S)-c₄Ser*-NHMe, which is stabi-

lized by intramolecular hydrogen bonding.

Conclusion

In summary, the incorporation of a- and/or b-substituted b-

hydroxy-a-amino acids in model b-O-glucopeptides is a

powerful tool to design and modulate the conformational

space of the different synthesized glycopeptides. In this

sense, the expansion of these novel small systems to larger

glycopeptidic systems will be considered in future to design

derivatives that could stabilize a bioactive conformation or

could show conformers that are rarely observed in the natu-

ral compounds, thus modifying the binding to the target

molecules.

The y_svalues are strongly dependent on the substitution

at the Ca or Cb atoms: 1) The absence of substituents at the

Cb position of a glucosylated b-hydroxy-a-amino acid (i.e.,

Ac-l-Ser*-NHMe, Ac-d-Ser*-NHMe, and Ac-(S)-MeSer*-

NHMe) forces the y_sangle to adopt the usual anti confor-

mation. 2) The presence of a methyl group at the Cb posi-

tion at an R configuration of the amino acid (i.e., Ac-l-

Thr*-NHMe) does not favor the alternate conformations of

y_santi and g(À); therefore, this substitution gives prefer-

ence to the g(+) alternate conformation and, surprisingly, to

the eclipsed (E) conformation (1208). 3) A similar situation

occurs when the methyl group at the Cb position of an

amino acid (i.e., Ac-allo-l-Thr*-NHMe) is placed in the

S configuration; in this case, the anti and g(+) alternate con-

formations are not favored, whereas g(À) alternate and

eclipsed (E’; y_sclose to À1208) conformations of the y_s

angle are preferred. 4) When the Ca and Cb positions of an

amino acid are substituted at R,R configurations, the y_sdi-

hedral angle is rigidified toward the eclipsed conformer in

the case of Ac-(R,R)-MeThr*-NHMe and toward a g(+) al-

ternate conformer in the case of Ac-(R,R)-c₄Ser*-NHMe.

5) On the contrary and unexpectedly, when the Ca and Cb

positions are substituted at S,S configurations (i.e., Ac-(S,S)-

MeThr*-NHMe and Ac-(S,S)-c₄Ser*-NHMe) the y_sdihedral

angle is made more flexible toward several conformations.

Finally, it is important to highlight that structural rigidity

is not a synonym of conformational rigidity. For example,

Experimental Section

General procedures: The solvents were purified according to standard

procedures. Analytical TLC was performed using Polychrom SI F254

plates. Column chromatography was performed on silica gel 60 (230–400

mesh). H and ¹³C NMR spectra were recorded on Bruker ARX 300 and

1

Bruker Avance 400 spectrometers. H and ¹³C NMR spectra were record-

ed in CDCl₃with trimethylsilane (TMS) as the internal standard and in

D₂O at 258C (chemical shifts are reported in ppm on the d scale and the

coupling constants are given in hertz). Melting points were determined

on a Büchi B-545 melting-point apparatus and are uncorrected. Optical

rotations were measured on a Perkin-Elmer 341 polarimeter. Microanaly-

ses were carried out on a CE Instruments EA-1110 analyzer and are in

good agreement with the calculated values. Compound 2 was synthesized

from 1 according to a procedure reported in reference [13] and com-

pound 7 was synthesized according a procedure reported in referen-

ce [10j].

Compound 3: A solution of 2 (150 mg, 0.26 mmol) in ethyl acetate

(10 mL) was hydrogenolyzed by using 10% Pd/C (70 mg) as a catalyst at

258C for 16 h. The catalyst and solvent were removed, and the residue

corresponding to 3 (113 mg, 90%) was used without any purification.

¹H NMR (400 MHz, CDCl₃): d=1.43 (s, 3H), 1.99 (s, 3H), 2.01 (s, 3H),

2.04 (s, 3H), 2.08 (s, 3H), 3.64 (d, J=10.4 Hz, 1H), 3.67–3.73 (m, 1H),

4.07–4.19 (m, 2H), 4.24 (dd, J=4.4, 12.4 Hz, 1H), 4.63 (d, 1H J=

7.8 Hz), 4.97 (dd, J=8.1, 9.0 Hz, 1H), 5.09 (“t”, J=9.6 Hz, 1H),

5.20 ppm (“t”, J=9.4 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃): d=19.8,

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Conformational Analysis of Model b-O-Glucopeptides

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Table 2. Exploration of the conformational space of b-O-Glc-Ser diamide derivatives that incorporate substituents at the Ca and/or Cb positions.

Entry Group incorporated

in Ser

Position of the

group

Configuration

Ca,Cb

Compound Ac-Xxx*-

NHMe

Backbone Lateral chain

f_p/y_p

Glycosidic Glycosidic

linkage f_slinkage y_s

c¹

1

2

3

4

5

6

7

8

–

S,–

S,R

R

l-Ser*

folded

(22%)

folded

(21%)

folded

(12%)

folded

(11%)

folded

(34%)

folded

(46%)

folded

(47%)

folded

(75%)

rigid

g(+) (62%)

flexible

g(+), anti

rigid

anti (66%)

rigid

g(À)

rigid

g(À)

rigid

g(À)

rigid

g(À)

rigid

g(À)

rigid

g(À)

rigid

g(À)

flexible

g(À),

g(+)^[a]

rigid

g(À)

anti with

flexibility

rigid

g(+), E

anti with

flexibility

rigid

g(À), E’

anti with

flexibility

rigid

anti, E’

rigid

À

CH₃

–

b

l-Thr*

–

d-Ser*

À

CH₃

b

S,S

S,–

S,S

R,R

S,S

allo-L-Thr*

MeSer*

MeThr*

c₄Ser*

g(+) (61%)

flexible

À

CH₃

a

g(À), g(+)

À

CH₃

a,b

flexible

anti, g(+)

flexible

anti, g(À)

very rigid

close to g(+)

(100%)

very rigid

close to g(À)

(100%)

À

CH₃

E

À

CH₂CH₂

flexible

anti, g(À)

À

9

CH₂CH₂

a,b

R,R

c₄Ser*

folded

(68%)

rigid

g(+)

[a] g(+) conformation of the f_sangle (O5s-C1s-O1s-Cb=608) is also so-called anti-f (corresponding to 1808 when the f_sangle is defined as H1s-C1s-

O1s-Cb).

20.5, 20.6, 20.7, 21.0, 61.8, 66.0, 68.3, 71.0, 71.8, 72.6, 74.1, 100.8, 169.5,

169.7, 170.3, 170.9, 174.0 ppm.

Ac-(S)-MeSer*-NHMe: A solution of 5 (52 mg, 0.10 mmol) in MeOH

(5 mL) was treated with MeONa/MeOH (0.5m) to pH 9. The reaction

mixture was stirred for 3 h and then neutralized with Dowex 50W-X8, fil-

tered, and concentrated. Purification of the residue with a C₁₈reverse-

phase sep-pak cartridge gave the desired model glycopeptide Ac-(S)-

MeSer*-NHMe (31 mg, 90%). [a]²_D⁹=À23.7 (c=0.6, MeOH); ¹H NMR

(400 MHz, D₂O): d=1.36 (s, 3H), 1.89 (s, 3H), 2.60, (s, 3H), 3.18 (dd, J=

8.1, 9.3 Hz, 1H), 3.23 (“t”, J=9.6 Hz, 1H), 3.30–3.34 (m, 1H), 3.37 (“t”,

J=9.1 Hz, 1H), 3.60 (dd, J=5.6, 12.3 Hz, 1H), 3.71 (d, J=10.2 Hz, 1H),

3.79 (dd, J=1.9, 12.3 Hz, 1H), 4.04 (d, J=10.2 Hz,1H), 4.34 ppm (d, J=

7.9 Hz, 1H); ¹³C NMR (100 MHz, D₂O): d=19.8, 22.2, 26.0, 59.5, 60.7,

69.6, 72.2, 73.0, 75.6, 75.9, 102.7, 173.8, 174.8 ppm; elemental analysis

calcd (%) for C₁₃H₂₄N₂O₈: C 46.42, H 7.19, N 8.33; found: C 46.54, H

7.11, N 8.39.

Compound 4: A solution of 3 (109 mg, 0.23 mmol) in acetonitrile (5 mL)

was treated with diisopropylethylamine (DIPEA; 0.2 mL, 1.5 mmol),

methylamine hydrochloride (29 mg, 0.46 mmol), and TBTU (90 mg,

0.27 mmol) under an inert atmosphere. The reaction mixture was stirred

at 258C for 16 h and then partitioned between water (5 mL) and ethyl

acetate (5 mL). The organic layer was washed with 0.5n HCl (115 mL)

and brine (115 mL). The aqueous phase was extracted with CHCl₃/

iPrOH (3:1, 115 mL). The organic layer was dried over anhydrous

Na₂SO₄, filtered, and evaporated to give a residue that was purified by

column chromatography on silica gel eluting with CH₂Cl₂/MeOH (9:1) to

1

yield 4 as a white oil (94 mg, 84%). H NMR (400 MHz, CDCl₃): d=1.53

(s, 3H), 2.01 (s, 3H), 2.03 (s, 3H), 2.07 (s, 3H), 2.09 (s, 3H), 2.81 (d, J=

4.2 Hz, 3H), 3.68–3.75 (m, 2H), 4.08–4.17 (m, 2H), 4.25 (dd, J=4.7,

12.4 Hz, 1H), 4.56 (d, J=7.9 Hz, 1H), 5.03 (dd, J=7.9, 9.5 Hz, 1H), 5.09

(“t”, J=9.7 Hz, 1H), 5.22 (“t”, J=9.5 Hz, 1H), 6.59–6.67 ppm (m, 1H);

¹³C NMR (100 MHz, CDCl₃): d=19.1, 20.6, 20.6, 20.6, 20.7, 26.4, 61.8,

66.4, 68.3, 71.2, 71.9, 72.6, 73.8, 100.9, 169.4, 169.4, 170.2, 170.2,

170.7 ppm; elemental analysis calcd (%) for C₁₉H₂₈N₄O₁₁: C 46.72, H

5.78, N 11.47; found: C 46.59, H 5.84, N 11.32.

Ac-(R,R)-MeThr*-NHMe: A solution of 6 (52 mg, 0.06 mmol) in MeOH

(5 mL) was treated with MeONa/MeOH (0.5m) to pH 9. The reaction

mixture was stirred for 3 h at 258C and then neutralized with Dowex 50-

X8, filtered, and concentrated. Purification of the residue with C₁₈re-

verse-phase sep-pak cartridge gave Ac-(R,R)-MeThr*-NHMe as a color-

less oil (21 mg, 87%). [a]_D²⁵=+4.2 (c=0.97, H₂O); ¹H NMR (400 MHz,

D₂O): d=1.19 (d, J=6.4 Hz, 3H), 1.45 (s, 3H), 1.99 (s, 3H), 2.72 (s, 3H),

3.29–3.36 (m, 1H), 3.43–3.55 (m, 3H), 3.82 (dd, J=4.5, 12.1 Hz, 1H),

3.94–3.99 (m, 1H), 4.12 (q, J=6.4 Hz, 1H), 4.51 ppm (d, J=7.9 Hz, 1H);

¹³C NMR (100 MHz, D₂O): d=14.1, 18.3, 22.4, 26.0, 60.5, 62.2, 69.4, 72.7,

75.6, 75.9, 78.0, 100.6, 173.4, 173.6 ppm; elemental analysis calcd (%) for

C₁₄H₂₆N₂O₈: C 47.99, H 7.48, N 8.00. found: C 48.11, H 7.52, N 7.94.

Compound 5: A solution of methylamide 4 (94 mg, 0.19 mmol) in MeOH

(10 mL) was hydrogenated using 10% Pd/C (40 mg) as a catalyst at 258C

for 15 h. The catalyst and solvent were removed, and the residue was

used without any purification. The crude product was dissolved in pyri-

dine (3 mL) and acetic anhydride (1 mL) was added. The resulting mix-

ture was stirred for 4 h at 258C. The solvent was evaporated and the

crude produce purified by column chromatography on silica gel eluting

with CH₂Cl₂/MeOH (95:5) to yield 5 (74 mg, 77%). ¹H NMR (400 MHz,

CDCl₃): d=1.56 (s, 3H), 2.00 (s, 3H), 2.01 (s, 3H), 2.03 (s, 3H), 2.06 (s,

3H), 2.11 (s, 3H), 2.80 (d, J=4.8 Hz, 3H), 3.71 (ddd, J=2.3, 4.8, 10.0 Hz,

1H), 3.86 (d, J=9.9 Hz, 1H), 4.12 (dd, J=2.2, 12.4 Hz, 1H), 4.29–4.36

(m, 2H), 4.52 (d, J=8.0 Hz, 1H), 5.00 (dd, J=8.0, 9.7 Hz, 1H), 5.07 (“t”,

J=9.7 Hz, 1H), 5.22 (“t”, J=9.5 Hz, 1H), 6.65 (brs 1H), 6.75–6.81 ppm

(m, 1H); ¹³C NMR (100 MHz, CDCl₃): d=20.3, 20.5, 20.6, 24.1, 25.3,

26.5, 59.5, 61.6, 68.2, 71.2, 71.5, 71.9, 72.3, 100.5, 169.4, 169.6, 170.0, 170.4,

170.6, 172.9 ppm; elemental analysis calcd (%) for C₂₁H₃₂N₂O₁₂: C 50.00,

H 6.39, N 5.55; found: C 49.89, H 6.32, N 5.59.

Compound 8: LiOH·H₂O (265 mg, 6.3 mmol) was added to a solution of

7 (350 mg, 1.3 mmol) in H₂O/MeOH (1:3, 8 mL) at 258C. After the reac-

tion mixture was stirred at 258C for 12 h, the MeOH was evaporated,

and the reaction mixture diluted with H₂O (20 mL) and ethyl acetate

(25 mL). The aqueous phase was acidified with 2n HCl and extracted

with CHCl₃/iPrOH (3:1, 315 mL). The combined organic phases were

dried over Na₂SO₄and the solvent was removed at reduced pressure to

give the corresponding amino acid derivative 8 as a white solid (320 mg,

97%), which was used without further purification in the next step.

¹H NMR (400 MHz, CD₃OD): d=1.60 (“q”, J=10.5 Hz, 1H), 1.96 (s,

3H), 2.12–2.25 (m, 2H), 2.70–2.78 (m, 1H), 4.30 (“t”, J=8.4 Hz, 1H),

4.48 (d, J=11.1 Hz, 1H), 4.66 (d, J=11.1 Hz, 1H), 7.26–7.32 ppm (m,

5H); ¹³C NMR (100 MHz, CD₃OD): d=22.2, 25.6, 25.7, 67.0, 72.8, 78.8,

128.7, 128.9, 129.3, 139.3, 172.8, 173.9 ppm.

Chem. Eur. J. 2008, 14, 7042 – 7058

ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

7055

J.M. Peregrina, F. Corzana et al.

Compound 9: A solution of acid 8 (620 mg, 2.4 mmol) in acetonitrile

(30 mL) was treated with DIPEA (1.56 mL, 9.4 mmol), methylamine hy-

drochloride (318 mg, 4.7 mmol), and TBTU (908 mg, 2.8 mmol) under an

inert atmosphere. The reaction mixture was stirred at 258C for 10 h, then

partitioned between brine (20 mL) and ethyl acetate (12 mL). The organ-

ic layer was washed with 0.1n HCl (215 mL) and 5% NaHCO₃(2

15 mL). The aqueous layer was then extracted with CHCl₃/iPrOH (4:1,

320 mL). Finally, the combined organic layers were dried over Na₂SO₄,

filtered, and evaporated to give a residue that was purified by column

chromatography on silica gel eluting with ethyl acetate/MeOH (95:5) to

(m, 1H), 2.60–2.71 (m, 1H), 2.77 (s, 3H), 3.28 (“t”, J=8.5 Hz, 1H), 3.34–

3.40 (m, 1H), 3.44–3.53 (m, 2H), 3.72 (dd, J=6.7, 12.2 Hz, 1H), 3.92–

4.00 (m, 1H), 4.53 (d, J=7.9 Hz, 1H), 4.66 ppm (“t”, J=8.8 Hz, 1H);

¹³C NMR (100 MHz, D₂O): d=21.8, 24.4, 25.1, 26.1, 60.9, 65.4, 69.8, 72.7,

75.4, 75.5, 76.1, 100.8, 172.3, 173.7 ppm; elemental analysis calcd (%) for

C₁₄H₂₄N₂O₈: C 48.27, H 6.94, N 8.04; found: C 48.17, H 6.88, N 8.10.

Ac-(R,R)-c₄Ser*-NHMe: A solution of 11 (60 mg, 0.08 mmol) in MeOH

(5 mL) was treated with MeONa/MeOH (0.5m) to pH 9. The reaction

mixture was stirred for 3 h at 258C and then neutralized with Dowex 50-

X8, filtered, and concentrated. Purification of the residue with C₁₈re-

verse-phase sep-pak cartridge gave Ac-(R,R)-c₄Ser*-NHMe as a colorless

oil (23 mg, 85%). [a]²_D⁵=À16.1 (c=1.17, H₂O); ¹H NMR (400 MHz,

D₂O): d=1.56–1.68 (m, 1H), 2.00 (s, 3H), 2.07–2.15 (m, 1H), 2.26–2.39

(m, 1H), 2.66–2.79 (m, 4H), 3.23 (“t”, J=8.5 Hz, 1H), 3.35–3.41 (m,

1H), 3.44–3.56 (m, 2H), 3.78 (dd, J=6.0, 12.0 Hz, 1H), 3.99 (dd, J=2.0,

12.0 Hz, 1H), 4.40 (“t”, J=8.9 Hz, 1H), 4.47 ppm (d, J=7.8 Hz, 1H);

¹³C NMR (100 MHz, D₂O): d=21.8, 24.1, 24.7, 26.1, 60.7, 66.0, 69.7, 73.0,

75.5, 75.5, 77.6, 101.3, 172.6, 173.5 ppm; elemental analysis calcd (%) for

C₁₄H₂₄N₂O₈: C 48.27, H 6.94, N 8.04; found: C 48.40, H 7.00, N 7.98.

give 9, as

a

white solid (600 mg, 92%). M.p. 172–1748C; ¹H NMR

(400 MHz, CDCl₃): d=1.82–1.95 (m, 4H), 1.99–2.09 (m, 1H), 2.11–2.28

(m, 2H), 2.78 (d, J=4.7 Hz, 3H), 4.49 (d, J=11.5 Hz, 1H), 4.56–4.69 (m,

2H), 6.39 (brs, 1H), 7.22–7.33 (m, 5H), 7.40 ppm (m, 1H); ¹³C NMR

(100 MHz, CDCl₃): d=23.8, 24.1, 24.5, 26.4, 64.7, 72.2, 78.1, 128.0, 128.0,

128.5, 137.6, 170.1, 172.3 ppm; elemental analysis calcd (%) for

C₁₅H₂₀N₂O₃: C 65.20, H 7.30, N 10.14; found: C 65.33, H 7.25, N 10.20.

Ac-c₄Ser-NHMe: A solution of 9 (600 mg, 2.2 mmol) in MeOH (20 mL)

was hydrogenolyzed using 10% Pd/C (100 mg) as a catalyst at 258C for

12 h. The catalyst and solvent were removed and further purification of

the residue with C₁₈reverse-phase sep-pak cartridge gave c₄Ser as a

white solid (400 mg, 99%). ¹H NMR (400 MHz, D₂O): d=1.51–1.67 (m,

1H), 1.85–2.01 (m, 4H), 2.18–2.35 (m, 1H), 2.48–2.63 (m, 1H), 2.73 (s,

3H), 4.34 ppm (“t”, J=8.7 Hz, 1H); ¹³C NMR (100 MHz, D₂O): d=21.8,

23.8, 26.0, 26.1, 65.9, 71.2, 172.8, 173.9 ppm; elemental analysis calcd (%)

for C₈H₁₄N₂O₃: C 51.60, H 7.58, N 15.04; found: C 51.71, H 7.62, N 15.10.

Compound 12: EDCI·HCl (80 mg, 0.4 mmol) and DMAP (9 mg,

0.08 mmol) were added to a suspension of acid 8 (100 mg, 0.38 mmol) in

dichloromethane (5 mL) in an inert atmosphere. The reaction mixture

was stirred for 2 h at 258C and then concentrated and purified by column

chromatography silica gel eluting with hexane/ethyl acetate (2:3) to give

1

12 as an oil (80 mg, 87%). H NMR (400 MHz, CDCl₃): d=1.95–2.12 (m,

4H), 2.12–2.32 (m, 2H), 2.33–2.53 (m, 1H), 4.26–4.46 (m, 2H), 4.48–4.62

(m, 1H), 7.23–7.42 ppm (m, 5H); ¹³C NMR (100 MHz, CDCl₃): d=14.8,

22.8, 24.1, 71.7, 73.6, 78.0, 127.6, 127.7, 128.1, 137.2, 162.3, 176.6 ppm; ele-

mental analysis calcd (%) for C₁₄H₁₅NO₃: C 68.56, H 6.16, N 5.71; found:

C 68.68, H 6.21, N 5.67.

Compounds 10 and 11: Silver triflate (262 mg, 1.0 mmol) was added to a

suspension of c₄Ser (115 mg, 0.6 mmol) and powdered molecular sieves

(4 , 50 mg) in dichloromethane (4 mL) in an inert atmosphere. The re-

action mixture was stirred at À308C and then 2,3,4,6-tetra-O-benzoyl-a-

d-glucopyranosyl bromide (570 mg, 0.86 mmol) in dichloromethane

(4 mL) was added. The reaction mixture was stirred at this temperature

for 1 h, warmed to 258C, and stirred for additional 14 h. The crude prod-

uct was filtered, concentrated, and purified by column chromatography

on silica gel eluting with CH₂Cl₂/MeOH (95:5) to give a mixture of 10

and 11 as a white solid (140 mg, 30%). Additional column chromatogra-

phy eluting with CH₂Cl₂/MeOH (95:5) allowed us to obtain 11 (55 mg)

and 10 (34 mg) in a pure form.

Compounds 13 and 14: tBuOK (0.3 mL, 0.3 mmol) was added to a solu-

tion of (S)-(À)-1-(2-naphtyl)ethanol (100 mg, 0.6 mmol) in THF (3 mL)

in an inert atmosphere. After the reaction mixture was stirred for 5 min

at 258C, a solution of oxazolone 12 (72 mg, 0.3 mmol) in THF (3 mL)

was added. The reaction mixture was further stirred for 12 h at 258C and

then was concentrated and purified by column chromatography on silica

gel eluting with hexane/ethyl acetate (2:3) to give a mixture of 13 and 14

(85 mg, 70%). Further purification by column chromatography on silica

gel eluting with hexane/ethyl acetate (2:3) allowed pure 14 (7 mg, 6%) to

be obtained with an additional mixture fraction with 13 and 14 in a ratio

of 1:4 (17 mg, 14%).

1

10: M.p. 100–1028C; [a]²_D⁵=+22.4 (c=1.12, MeOH); H NMR (400 MHz,

CDCl₃): d=1.71–1.84 (m, 1H), 1.89 (s, 3H), 1.97–2.09 (m, 1H), 2.21–2.35

(m, 2H), 2.42 (d, J=4.5 Hz, 3H), 4.11–4.20 (m, 1H), 4.49–4.65 (m, 2H),

4.73–4.83 (m, 1H), 5.20 (d, J=8.0 Hz, 1H), 5.55 (dd, J=8.3, 9.5 Hz, 1H),

5.65 (“t”, J=9.7 Hz, 1H), 5.94 (“t”, J=9.7 Hz, 1H), 6.37 (brs, 1H), 6.85–

6.99 (m, 1H), 7.22–7.31 (m, 2H), 7.32–7.47 (m, 7H), 7.47–7.61 (m, 3H),

7.77–7.85 (m, 2H), 7.88–7.94 (m, 2H), 7.96–8.07 ppm (m, 4H); ¹³C NMR

(100 MHz, CDCl₃): d=23.4, 24.3, 26.0, 26.1, 63.0, 64.7, 71.7, 72.4, 72.7,

76.9, 77.2, 100.2, 128.3, 128.4, 128.7, 128.8, 129.7, 133.2, 134.4, 134.5,

165.2, 165.5, 165.6, 166.1, 169.9, 171.4 ppm; elemental analysis calcd (%)

for C₄₂H₄₀N₂O₁₂: C 65.96, H 5.27, N 3.66; found: C 65.82, H 5.33, N 3.60.

1

14: M.p. 133–1358C; [a]²_D⁵=À81.4 (c=1.00, MeOH); H NMR (400 MHz,

CDCl₃): d=1.58 (d, J=6.6 Hz, 3H), 1.86 (s, 3H), 2.17–2.33 (m, 3H),

2.33–2.52 (m, 1H), 4.33–4.47 (m, 2H), 4.56–4.71 (m, 1H), 6.04–6.30 (m,

2H), 7.12–7.25 (m, 5H), 7.36–7.50 (m, 3H), 7.70–7.82 ppm (m, 4H);

¹³C NMR (100 MHz, CDCl₃): d=21.9, 22.6, 23.8, 24.6, 66.4, 71.6, 74.2,

77.2, 124.0, 125.0, 126.1, 126.2, 127.6, 127.8, 127.9, 128.0, 128.3, 128.4,

133.0, 133.1, 137.7, 138.4, 169.8, 170.7 ppm; elemental analysis calcd (%)

for C₂₆H₂₇NO₄: C 74.80, H 6.52, N 3.35; found: C 74.69, H 6.46, N 3.40.

1

11: M.p. 128–1308C; [a]²_D⁵=+11.0 (c=1.07, MeOH); H NMR (400 MHz,

Compound (R,R)-8: LiOH·H₂O (126 mg, 3.0 mmol) was added to a solu-

tion of a mixture of 13 and 14 in a ratio of 1:4 (84 mg, 0.2 mmol) in H₂O/

MeOH (1:3, 4 mL) at 258C. After the reaction mixture was stirred at

258C for four days, the MeOH was evaporated, and the reaction mixture

diluted with H₂O (3 mL) and ethyl acetate (5 mL). The aqueous phase

was acidified with 2n HCl and extracted with CHCl₃/iPrOH (3:1, 4

5 mL). The combined organic phases were dried over Na₂SO₄and the

solvent was removed at reduced pressure to give a white solid corre-

sponding to a mixture of enantiomers (S,S)- and (R,R)-8 in a ratio of 1:4,

respectively (51 mg, 96%). The spectroscopic data are identical to those

obtained for racemic compound 8.

CDCl₃): d=1.87–1.93 (m, 1H), 1.98 (s, 3H), 2.03–2.22 (m, 2H), 2.34–2.45

(m, 1H), 2.64 (d, J=4.7 Hz, 3H), 4.12–4.19 (m, 1H), 4.36 (dd, J=4.2,

12.3 Hz, 1H), 4.84 (dd, J=2.6, 12.3 Hz, 1H), 4.92 (d, J=8.0 Hz, 1H),

4.97 (“t”, J=8.6 Hz, 1H), 5.43 (dd, J=8.1, 9.7 Hz, 1H), 5.71 (“t”, J=

9.8 Hz, 1H), 5.90 (“t”, J=9.7 Hz, 1H), 6.58 (brs, 1H), 6.63–6.69 (m, 1H),

7.24–7.63 (m, 12H), 7.79–7.86 (m, 2H), 7.89–7.96 (m, 4H), 8.05–8.11 ppm

(m, 2H); ¹³C NMR (100 MHz, CDCl₃): d=23.6, 23.7, 24.4, 26.4, 62.2,

65.6, 69.1, 71.8, 72.5, 72.8, 76.8, 77.2, 100.4, 128.3, 128.5, 128.6, 128.7,

129.7, 133.3, 133.5, 133.6, 165.1, 165.3, 165.7, 166.3, 170.2, 170.8 ppm; ele-

mental analysis calcd (%) for C₄₂H₄₀N₂O₁₂: C 65.96, H 5.27, N 3.66;

found: C 66.10, H 5.21, N 3.71.

Two-dimensional NMR experiments: NMR spectroscopic experiments

were recorded on a Bruker Avance 400 spectrometer at 293 K. Magni-

tude-mode ge-2D COSY spectra were recorded with gradients using the

cosygpqf pulse program with a 908 pulse width. Phase-sensitive ge-2D

HSQC spectra were recorded using a z-filter and selection before t1 to

remove the decoupling during acquisition by use of the invigpndph pulse

program with CNST2 (JHC)=145. Two-dimensional NOESY experi-

ments were made using phase-sensitive ge-2D NOESY with WATER-

Ac-(S,S)-c₄Ser*-NHMe: A solution of 10 (38 mg, 0.05 mmol) in MeOH

(5 mL) was treated with MeONa/MeOH (0.5m) to pH 9. The reaction

mixture was stirred for 3 h at 258C and was then neutralized with Dowex

50-X8, filtered, and concentrated. Purification of the residue with a C₁₈

reverse-phase sep-pak cartridge gave Ac-(S,S)-c₄Ser*-NHMe as a color-

1

less oil in (16 mg, 92%). [a]_D²⁵=À7.2 (c=1.24, H₂O); H NMR (400 MHz,

D₂O): d=1.59–1.73 (m, 1H), 2.01 (s, 3H), 2.06–2.19 (m, 1H), 2.28–2.39

7056

www.chemeurj.org

ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2008, 14, 7042 – 7058

Conformational Analysis of Model b-O-Glucopeptides

FULL PAPER

GATE for spectra in H₂O/D₂O (9:1). Selective ge-1D NOESY experi-

ments were carried out using the 1D-DPFGE NOE pulse sequence.

NOE interaction intensities were normalized with respect to the diagonal

peak at zero mixing time. Experimental NOE interactions were fitted to

tions, 3272 unique, full-matrix least-squares (SHELXL97),^[38]R₁=0.0590,

wR₂=0.1102, (R₁=0.0933, wR₂=0.1246 all data), goodness of fit=1.073,

residual electron density=0.278–0.215 e^À3; the hydrogen atoms were

fitted at theoretical positions.

a double exponential function, f(t)=p₀ACTHERNGU )ACHTREUNG

(e^{Àp t}

(1Àe^{Àp t}) with p₀, p₁, and p₂

1

2

as adjustable parameters.^[18]The initial slope was determined from the

first derivative at time t=0, f’(0)=p₀p₂. From the initial slopes, interpro-

ton distances were obtained by employing the isolated spin-pair approxi-

mation. 1D NOESY (D₂O) and 2D NOESY (H₂O/D₂O, 9:1) spectra for

model peptides and glycopeptides and the representative NOE interac-

tion build-up curves are available in the Supporting Information.

Acknowledgements

Computational details

We thank the Ministerio de Educación y Ciencia and FEDER (project

CTQ2006–05825/BQU, Ramón y Cajal contracts for F.C., J.H.B., and a

pre-doctoral grant for A.F.-T.). G.J.-O. thanks the Consolider Project (In-

genio 2010) for financial support, We also thank CESGA for computer

support.

Molecular mechanics calculations: Relaxed potential-energy maps were

calculated by performing relaxed 2D-PES scans of the f_pand y_pdihe-

drals. A grid of 400 conformers was constructed for each structure by

fully scanning these torsions with a step size of 188. The AMBER94,^[28]

CHARMM,^[29]and MM+^[30]force fields were used in the minimizations.

In the case of AMBER94 and CHARMM, a distance-independent di-

ACHTREUNG

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a root-mean square gradient of

0.01 kcal^À1mol^À1or 2000 cycles as the convergence criteria. The rela-

tive energy levels were plotted from 0.0 to 1.6 kcalmol^À1with a contour

spacing of 0.4 kcalmol^À1

.

Molecular dynamics simulations: MD-tar simulations were performed

with AMBER^[31]6.0 (AMBER94), which was implemented with

GLYCAM04 parameters^[32]to accurately simulate the conformational be-

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>

average was used for the dis-

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tances and a linear average was used for the coupling constants. Final tra-

jectories were run using an exponential decay constant of 8000 ps and a

simulation length of 80 ns with a dielectric constant e=80. The distances

derived experimentally and from MD simulations calculated for all the

peptides and glycopeptides and the f_p/y_pdistributions obtained from the

MD-tar simulations for the glycopeptides and MD simulations in explicit

water for Ac-l-Thr*-NHMe and Ac-(S,S)-c₄Ser*-NHMe are available in

the Supporting Information.

Density functional theory (DFT) calculations: All calculations were car-

ried out using the B3LYP hybrid functional^[34]with the Gaussian 03 pack-

age.^[35]The 6-31G

ACHTREUNG(d,p) basis set was used in the full optimizations and for

the relaxed PES scans. In these calculations, a step size of 58 was used.

Basis set superposition error corrections were not considered in this

study. Frequency analyses were carried out at the same level used in the

geometry optimizations, and the nature of the stationary points was de-

termined in each case according to the appropriate number of negative

eigenvalues of the Hessian matrix. Scaled frequencies were not consid-

ered since significant errors in the calculated thermodynamical properties

are not found at this theoretical level.^[36]B3LYP/6-31G

ACHTRE(UNG d,p) coordinates

of the optimized g(À) conformer of Ac-allo-l-Thr*-NHMe are available

in the Supporting Information.

X-ray diffraction analysis^[37]

Crystal data for Ac-c₄Ser-NHMe: C₈H₁₄N₂O₃, M_w=186.21, colorless prism

of 0.250.250.15 mm, T=173 K, monoclinic, space group P2₁/c, Z=4,

a=8.0203(4), b=17.5720(9), c=6.8646(3) , b=92.123(3)8, V=

966.78(8) ³, 1_calcd=1.279 gcm^À3, F

ACHTRE(UNG 000)=400, l=0.71073 (Mo_Ka), m=

0.098 mm^À1, Nonius kappa CCD diffractometer, q range=2.54–27.918,

7579 collected reflections, 2275 unique, full-matrix least-squares

(SHELXL97),^[38]R₁=0.0672, wR₂=0.1969, (R₁=0.1195, wR₂=0.2294 all

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Crystal data for 14: C₂₆H₂₇NO₄, M_w=417.51, colorless prism of 0.40

0.200.04 mm, T=173 K, orthorhombic, space group P2₁2₁2₁, Z=4, a=

5.8415(2), b=15.3797(7), c=24.8256(12) , V=2230.3 (2) ³, 1_calcd

=

1.243 gcm^À3, F

ACHTREUNG

(000)=888, l=0.71073 (Mo_Ka), m=0.083 mm^À1, Nonius

kappa CCD diffractometer, q range=1.56–23.508, 7469 collected reflec-

Chem. Eur. J. 2008, 14, 7042 – 7058

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Received: March 13, 2008

Revised: April 23, 2008

Published online: July 4, 2008

7058

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Chem. Eur. J. 2008, 14, 7042 – 7058

Article Doi

Non-natural amino acids as modulating agents of the conformational space of model glycopeptides

DOI: 10.1002/chem.200800460

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Article abstract of DOI:10.1002/chem.200800460

Full text of DOI:10.1002/chem.200800460

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